Altered Bone Resorption in Type 1 and Type 2 Diabetes

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Altered Bone Resorption in Type 1 and Type 2 Diabetes
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1 online resource (163 p.)
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english
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Catalfamo, Dana
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University of Florida
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Gainesville, Fla.
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Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Medical Sciences, Immunology and Microbiology (IDP)
Committee Chair:
Wallet, Shannon
Committee Members:
Mathews, Clayton Elwood
Grieshaber, Scott Stephen
Holliday, Lexie S

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Subjects / Keywords:
arthritis -- bone -- diabetes -- inflammation -- lipopolysaccharide -- osteoclast -- periodontitis
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
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Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
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Abstract:
Inflammation perturbs bone homeostasis by inducing bone loss.  Thus, inflammatory diseases such as diabetes mellitus are epidemiologically linked to bone pathologies.  However, the mechanisms of bone loss in these inflammatory diseases are complex where disturbances in the control of bone remodeling are involved.  Two major cell types are involved in the regulation of bone remodeling: osteoclasts are responsible for removal of bone and osteoblasts are responsible for formation of new bone.  While decreased function by osteoblasts from hosts with diabetes have been implicated in mechanisms of altered bone homeostasis, osteoclasts and their specific contributions were previously unclear.   Bone marrow-derived osteoclasts were isolated from pre-diabetic T1D-prone non-obese diabetic NOD mice and hyperglycemic T2D-prone C57BL/6-Leprdb3J db/db, along with peripheral blood mononuclear cell-derived osteoclasts from participants with well-controlled type 2 diabetes T2D.  Osteoclasts derived from NOD mice had decreased fusion during differentiation, along with elevated RANK-L-induced enzyme production resulting in enhanced bone resorption.  Conversely, osteoclasts derived from hyperglycemic db/db mice displayed enhanced fusion along with similar RANK-L-induced bone resorption compared to controls.  Osteoclasts prepared from well-controlled T2D participants displayed no difference in fusion along with similar RANK-L-induced bone resorption compared to controls.  All diabetes derived-osteoclasts overcame LPS-induced inhibition whereby diabetes-derived cultures continued to resorb bone in the presence of LPS while control-derived cultures resorbed little to no bone in the presence of LPS.  In addition, diabetes-derived cultures also contained elevated levels of pro-osteoclastogenic mediators compared to controls.  Importantly, the altered LPS-responsiveness observed was not solely due to soluble mediators produced by the cultures, as a pro-inflammatory cocktail nor conditioned supernatants from diabetes-derived cultures could overcome LPS-induced inactivation in control cultures. Together, these data indicate that diabetes-derived osteoclasts are more resorptive in nature through different mechanisms depending on type and state of disease.  In addition, all diabetes-derived osteoclasts are refractory to LPS-induced deactivation whereby enhanced pro-osteoclastic mediator expression is also evident.
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In the series University of Florida Digital Collections.
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Includes vita.
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by Dana Catalfamo.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
Local:
Adviser: Wallet, Shannon.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

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1 ALTERED BONE RESORPTION IN TYPE 1 AND TYPE 2 DIABETES By DANA LYNN CATALFAMO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Dana Lynn Catalfamo

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3 To my mother, father, and brother for being a constant support to me during this challenging endeavor

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4 ACKNOWLEDGMENTS I wish to thank my parents for giving me the raw intellectual capability to pursue my doctorate and for always being supportive during these five challenging years. My close friends were also instrumental in providing moral support and shoulders to lean ( and cry) on even in the most challenging times. I would like to thank my mentor, Dr. Shannon Wallet, for pushing me to my intellectual limits and aiding my development into an independent scientist. Her endless time spent helping me with grant writing, manuscript preparation, experimental design, data analysis, and training me in various lab techniques did not go unnoticed. Dr. Kathleen Neiva, my post doc and good friend, gave me the support I needed and spent countless hours at the micr oscope counting m y osteoclasts. Andrea Knowlton and Heather Brown shared their confocal microscopy expertise and were constant friends. Heather Sorenson bestowed her endless positivity and guidance and was an invaluable resource with the in vivo arthritis model and human osteoclast experiments. Nadia Calderon performed Luminex experiments and human osteoclast optimization as well as shared with me her undying optimism. Scott Harden bequeathed his brilliance in designing software to analyze my scanning electron microscop y data. Dr. Shannon Holliday and Dr. Edgardo Toro provided great help in osteoclast culturing and bone resorption techniques. Dr. Toro was also a great friend and discussed osteoclasts with me for hours. Dr. Holliday offered his expertise in osteoclast biology and contributed to the direction of my project in committee meetings. Dr. Clayton Mathews and Dr. Scott Grieshaber, my other committee members, gave me intellectual support and guidance during this project. Dr. Steve Ghivizzani and Dr.

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5 Rachel W atson were instrumental in helping develop and optimize the in vivo arthritis model. Dr. Ghivizzani also spent valuable time teaching me histology of joints and graciously shared his endless knowledge on arthritis. Finally, I would like to thank Dr. Todd Britten and Dr. Doug Storch for being part of the T2D derived osteoclast project. Dr. Britten worked on the murine experiments while Dr. Storch collaborated with our lab for the human osteoclast studies.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 17 Diabetes and Inflammatory Bone Loss ................................ ................................ ... 17 Bone Remodeling ................................ ................................ ............................. 17 Osteoclasts ................................ ................................ ................................ ....... 19 Effects of Diabetes on Osteoclast Mediated Inflammatory Bone Loss .................... 24 Diabetes mellitus ................................ ................................ .............................. 24 Type 1 diabetes ................................ ................................ ................................ 24 Type 2 diabetes ................................ ................................ ................................ 26 Diabetes mellitus and epidemiological link to inflammatory bone loss ............. 28 Contributions of Intrinsic Defects ................................ ............................... 30 Presence of chronic inflammation ................................ .............................. 34 Contributions of hyperglycemia ................................ ................................ .. 36 Diabetes and Inflammatory Bone Pathologies ................................ ........................ 40 Periodontal Disease ................................ ................................ ......................... 40 Periodontitis and diabetes co morbidity ................................ ..................... 40 Pathology: bacterial colonization, inflammation, and alveolar bone loss .... 41 Inflammatory Arthritis ................................ ................................ ........................ 44 Inflam matory arthritis and diabetes co morbidity ................................ ........ 45 Pathology: inflammation and bone destruction ................................ .......... 46 Hypotheses ................................ ................................ ................................ ............. 48 2 AUGMENTED LPS RESPONSIVENESS IN TYPE 1 DIABETES DERIVED OSTEOCLASTS ................................ ................................ ................................ ..... 50 Introduction ................................ ................................ ................................ ............. 50 Materials and Methods ................................ ................................ ............................ 52 Mouse Models ................................ ................................ ................................ .. 52 Osteoclast Differentiation ................................ ................................ ................. 53 TR AP Staining ................................ ................................ ................................ .. 53 Osteoclast Stimulation ................................ ................................ ...................... 54 Flow Cytometry for Osteoclast Culture Purity ................................ ................... 55 Cell Viability Assays ................................ ................................ ......................... 55

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7 Scanni ng Electron Microscopy ................................ ................................ ......... 56 Collagen Telopeptide ELISA ................................ ................................ ............ 56 Cathepsin K ELISA ................................ ................................ ........................... 57 MMP 9 ELISA ................................ ................................ ................................ ... 57 Soluble Mediator Analysis ................................ ................................ ................ 57 Hi stological Analysis of Pancreas ................................ ................................ ..... 58 Statistical Analysis ................................ ................................ ............................ 59 Results ................................ ................................ ................................ .................... 59 NOD derived Osteoclasts Display Altered Differentiation ................................ 59 NOD derived Osteoclasts Have Increased Bone Resorption Capabilities in Response to RANK L Stimulation ................................ ................................ 60 NOD derived Osteoclasts Degrade More Type 1 Collagen than Controls via Enhanced Cathepsin K and MMP 9 Secretion ................................ .............. 61 NOD derive d Osteoclasts Respond Aberrantly to LPS ................................ ..... 62 NOD derived Osteoclasts Secrete Increased Soluble Osteoclastogenic Mediators in Response to LPS ................................ ................................ ...... 62 NOD derived Osteoclasts Respond Aberrantly to Inflammatory Mediators ...... 63 NOD derived BM OC Conditioned Media Leads to Increased Bone Resorption in Control Cultures ................................ ................................ ...... 64 NOD derived Osteoclasts are from Pre diabetic/euglycemic Mice ................... 65 Discussion ................................ ................................ ................................ .............. 65 3 EXACERBATED RESPONSE TO MBSA INDUCED INFLAMMATORY ARTHRITIS IN NOD MICE ................................ ................................ ..................... 81 Introduction ................................ ................................ ................................ ............. 81 Materials and Methods ................................ ................................ ............................ 83 Mouse Models ................................ ................................ ................................ .. 83 Arthritis Induction ................................ ................................ .............................. 84 Collagen Telopeptide ELISA ................................ ................................ ............ 84 Histological Analysis of Joints ................................ ................................ .......... 84 Results ................................ ................................ ................................ .................... 85 Intra articular Injection of mBSA Causes an Acute Inflammatory Arthritis in NOD, NOR and C57BL/6 Mice ................................ ................................ ...... 85 NOD Mice do not have Increased Joint Swelling After Induction of Arthritis ..... 85 NOD Mice Display Increased Bone Destruction via Collagen Degradation in Arthritic Joints ................................ ................................ ................................ 86 Discussion ................................ ................................ ................................ .............. 86 4 HYPERGLYCEMIA INDUCED AND INTRINSIC ALTERATIONS IN TYPE 2 DIABETES DERIVED OSTEOCLAST FUNCTION ................................ ................. 92 Introduction ................................ ................................ ................................ ............. 92 Materials and Methods ................................ ................................ ............................ 95 Participant Population ................................ ................................ ....................... 95 Peripheral Mononuclear Cell [PBMC] Isolation, Osteoclast [OC] Differentiation and Activation ................................ ................................ ......... 9 5

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8 Mouse Models ................................ ................................ ................................ .. 96 Bone Marrow Oste oclast [BMOC] Differentiation and Activation ...................... 97 TRAP Staining ................................ ................................ ................................ .. 98 Collagen Telopeptide ELISA ................................ ................................ ............ 98 Cathepsin K ELISA ................................ ................................ ........................... 99 Soluble Me diator Analysis ................................ ................................ ................ 99 Statistical Analysis ................................ ................................ .......................... 100 Results ................................ ................................ ................................ .................. 100 Hyperglycemia Enhances Differentiation of T2D derived OCs ....................... 100 Enhanced Differentiation Results in Exacerbated Bone Resorption ............... 101 T2D derived Osteoclasts Respond Aberrantly to LPS ................................ .... 102 Elevated Pro Osteoclastic Milieu in T2D derived OC Cultures is Augmented by LPS ................................ ................................ ................................ ......... 102 Discussion ................................ ................................ ................................ ............ 103 5 DISCUSSION ................................ ................................ ................................ ....... 113 Differentiatio n ................................ ................................ ................................ ........ 113 Type 1 Diabetes Derived Osteoclasts ................................ ............................ 113 Type 2 Diabetes Derived Osteoclasts ................................ ............................ 114 RANK L Activation ................................ ................................ ................................ 114 Normal Osteoclasts ................................ ................................ ........................ 114 Diabetic Osteoclasts ................................ ................................ ....................... 115 LPS induced Deactivation ................................ ................................ ..................... 116 Normal Osteoclasts ................................ ................................ ........................ 116 Diabetic Osteoclasts ................................ ................................ ....................... 116 Osteoc last Regulation by Inflammatory Mediators ................................ ................ 118 Precursor Mobilization ................................ ................................ .................... 118 Differentiation ................................ ................................ ................................ 118 Activation ................................ ................................ ................................ ........ 119 Hyperglycemic Effects on Osteoclasts ................................ ................................ .. 121 Differentiation ................................ ................................ ................................ 121 Activation ................................ ................................ ................................ ........ 122 Possible Adjunct Therapies ................................ ................................ .................. 124 Addition of anti osteoclastic drugs: Anti RANK L ................................ ........... 124 Addition of anti osteoclastic drugs: Bisphosphonates ................................ .... 125 Future Directions ................................ ................................ ................................ .. 126 Mechanism of inhibition of LPS induced deactivation ................................ .... 126 Mechanism of decreased fusion in T1D osteoclasts ................................ ...... 127 Regulating receptor expression: Activating (RA NK, TNF R, and IL 1R) ......... 127 Regulating receptor expression: Deactivating (Calcitonin Receptor) .............. 128 LIST OF REFERENCES ................................ ................................ ............................. 129 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 163

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9 LIST OF FIGURES Figure page 2 1 Flow cytometric analysis of osteoclast culture purity ................................ .......... 71 2 2 NOD derived osteoclasts display altered differentiation. ................................ .... 72 2 3 NOD derived osteoclasts have increased bone resorption capabilities in response to RANK L stimulation ................................ ................................ ......... 72 2 4 NOD derived osteoclasts degrade more type 1 collagen than controls via enhanced cathepsin K and MMP 9 secretion. ................................ .................... 75 2 5 NOD derived osteoclasts respond aberrantly to LPS. ................................ ........ 76 2 6 NOD derived osteoclasts secrete increased soluble osteoclastogenic mediators in response to LPS. ................................ ................................ ............ 77 2 7 Pro inflammatory cytokines do not in hibit LPS deactivation of OCs .................. 78 2 8 NOD derived soluble mediators do not inhib it LPS deactivation of OCs ........... 79 2 9 NOD derived BM OCs are from pre diabetic/euglycemic mice ( A) Blood glucose was measured at time of sacrifice ................................ ........................ 80 3 1 Intra articular injection of mBSA causes an acute inflammatory arthriti s in NOD, NOR, and C57BL/6 mice ................................ ................................ .......... 89 3 2 NOD mice do not have increased joint swelling after induction of arthritis ......... 90 3 3 NOD mice display increased bone destruction via collagen degradation in arthritic joints ................................ ................................ ................................ ..... 91 4 1 Glycemic indices of murine and human cohorts. ................................ .............. 107 4 2 Db/db bone marrow derives increased numbers of larger osteoclasts. ........... 108 4 3 Type 2 diabetes derived osteoclasts are less responsive to LPS induced deactivation. ................................ ................................ ................................ ..... 109 4 4 LPS induced elevation of pro inflammatory and pro osteoclast ic soluble mediators in db/db osteoclast cultures. ................................ ............................ 111 4 5 LPS induced elevation of pro inflammatory and pro osteoclastic soluble mediators in human type 2 diabetes osteoclast cultures. ................................ 112

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10 LIST OF ABBREVIATION S ACPA Antibodies to citrullinated protein antigens ADAM A disintegrin and metalloproteinase domain containing protein AGE Advanced glycated end product ALP Alkaline phosphatase MEM Minimum essential medium, alpha modification AP 1 Activator protein 1 APC Antigen presenting cell ATP Adenosine triphosphate Alpha v beta 3 integrin cells Beta cells BM OC Bone marrow derived osteoclast BMP Bone morphogenic protein Breg Regulatory B cell C57BL/6 C57 black 6 mouse strain CA Carbonic anhydrase CaCl 2 Calcium chloride Cbfa1 Core binding factor alpha 1 CD25 Interleukin 2 recepto r c fms Receptor for macrophage colony stimulating factor CFU GM Colony forming unit granulocyte macrophage ClC 7 Chloride channel 7 CRP C reactive protein CTLA 4 Cytotoxic T lymphocyte antigen 4 CTR Calcitonin receptor

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11 CTx Carboxy terminal collagen cross link db/db Diabetic mutation DC Dendritic cell DC STAMP Dendritic cell specific transmembrane protein DPD Deoxypyridinoline crosslink ELISA Enzyme linked immunosorbent assay EtOH Ethanol FACS Fluorescence activated cell sorting Flt3 Fms like tyrosine kinase receptor 3 GAD65 65kDa isoform of glutamic acid decarboxylase G CSF Granulocyte colony stimulating factor GLUT2 Glucose transporter 2 GM CSF Granulocyte macrophage colony stimulating factor HbA1c Glycated hemoglobin A1c HGF Hepatocyte growth factor HLA Human leukocyte antigen hOC Human osteoclast HRP Horse radish peroxidase IA 2A Insulinoma associated protein 2 antibody ICA Islet cell antibody IDDM Insulin dependent diabetes susceptibility loci IFN Interferon gamma IGF 1/2 Insulin like growth facto rs 1 and 2 IgM Immunoglobulin M Inhibitor of kappa B

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12 IL 2 Interleukin 2 IP 10 Interferon gamma induced protein 10 IRB Institutional review board KCl Potassium chloride Lepr Leptin receptor LPS Lipopolysaccharide MAP Mitogen activated protein mBSA Methylated bovine serum albumin MCP 1 Monocyte chemoattracant protein 1 M CSF Macrophage colony stimulating factor MHC Major histocompatibility complex MIP Macrophage inflammatory protein 1 alpha Mitf Microphthalmia associated transcription factor MMP Matrix metalloproteinase mOC Mouse osteoclast MSC Mesenchymal stem cell MTT 3 (4,5 Di methyl thiazol 2 yl) 2,5 di phenyl tetrazolium bromide NaCl Sodium chloride NFATc1 Nuclear factor of activated T cells, cytoplasmic 1 N F Nuclear factor kappa B NK Natural killer cell NO Nitric oxide NOD Non obese diabetic mouse strain NOR Non obese resistant mouse strain NSAID Non steroidal anti inflammatory drug

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13 NTx Amino terminal collagen crosslink OB Osteoblast OC Osteoclast OC STAMP Osteoclast specific transmembrane protein OPG Osteoprotegerin op/op Osteopetrotic mutation OSCAR Osteoclast associated receptor PBMC Peripheral blood mononuclear cell PBS Phosphate buffered saline PE Phycoerythrin P.g. Porphyromonas gingivalis PGE 2 Prostaglandin E2 PlGF Placental growth factor Peroxisome proliferator activated receptor gamma PTH Parathryoid hormone PTPN22 P rotein tyrosine phosphatase non receptor type 22 RA Rheumatoid arthritis RAGE Receptor for advanced glycated end products RANK Receptor activator of nuclear factor kappa B RANK L Receptor activator of nuclear factor kappa B ligand RANTES Regulated upon activation, normal T cell expressed, and secreted RF Rheumatoid factor RGD Arginine glycine asparagine motif ROS Reactive oxygen species rpm Revolutions per minute

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14 RUNX2 Runt related transcription factor 2 SAV Streptavidin SDF 1 Stromal cell derived factor 1 Signal regulatory protein alpha SLE Systemic lupus erythematosus SNP Single nucleotide polymorphism SPF Specific pathogen free STZ Streptozotocin T1D Type 1 diabetes T2D Type 2 diabetes TGF Transforming growth factor beta T H 17 T helper 17 cell T H 2 T helper 2 cell TLR Toll like receptor TMB 3,3',5,5' Tetramethylbenzidine TNF Tumor necrosis factor alpha TRAP Tartra te resistant acid phosphatase Treg Regulatory T cell TSH Thyroid stimulating hormone UV Ultraviolet V ATPase Vacuolar proton ATPase VEGF Vascular endothelial growth factor VNTR Variable number tandem repeats

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ALTERED BONE RESORPTION IN TYPE 1 AND TYPE 2 DIABETES By Dana Lynn Catalfamo December 2012 Chair: Shannon M. Wallet Major: Medical Sciences Immunology & Microbiology Inflammation perturbs bone homeostasis by inducing bone loss. Thus, inflammatory diseases such as diabetes mellitus are epidemiologically linked to bone pathologies. However, the mechanisms of b one loss in these inflammatory diseases are complex where disturbances in the control of bone remodeling are involved. T wo major cell types are involved in the regulation of bone remodeling: osteoclasts are responsible for removal of bone and osteoblasts are respon sible for formation of new bone While decreased function by osteoblasts from hosts with diabetes have been implicated in mechanisms of altered bone homeostasis, osteoclasts and their specific contributions were previously unclear B one marrow derived osteoclasts were isolated from pre diabetic T1D prone non obese diabetic [ NOD ] mice and hyperglycemic T2D prone C57BL/6 Lepr db3J [db/db], along with peripheral blood mononuclear cell derived osteoclasts from participants with well controlled type 2 diabetes [ T2D ] Osteoclasts derived from NOD mice had decreased fusion during differentiation, along with elevated RANK L induced enzyme production resulting in enhanced bone resorption Conversely, osteoclasts derived from hyperglycemic db/db mice dis played enhanced fusion along with similar RANK L

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16 induced bone resorption compared to controls Osteoclasts prepared from well controlled T2D participants displayed no difference in fusion along with similar RANK L induced bone resorption compared to contr ols. All diabetes derived o steoclasts overcame LPS induced inhibition where by diabetes derived cultures continued to resorb bone in the presence of LPS while control derived cultures resorbed little to no bone in the presence of LPS. In addition, diabetes derived cultures also contained elevated levels of pro osteoclastogenic mediators compared to controls. Importantly, the altered LPS responsiveness observed was not solely due to soluble mediators produced by the cultures as a pro inflammatory cocktail nor conditioned supernatants from diabetes derived cultures could overcome LPS induced inactivation in c ontrol cultures. Together these data indicate that diabetes derived osteoclasts are more resorptive in nature through different mechanisms depending on type and state of disease. In addition, all diabetes derived osteoclasts are refractory to LPS induced deactivation whereby enhanced pro osteoclastic mediator expression is also evident.

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17 CHAPTER 1 LITERATURE REVIEW Diabetes and Inflammatory Bone Loss Inflammation perturbs normal bone homeostasis and is known to induce bone loss (1 3) Thus, not surprisingly, inflammatory diseases such as diabetes mellitus are epidemiologically linked to local and general bone pathologies (4 10) However, the mechanisms that target bone loss in these inflammatory diseases are complex and diverse ranging from attack on bone and cartilage by imm une cells to disturbances of the systemic control of bone remodeling (6, 11 22) Diabetes mellitus afflicts over 21 million Americans, including >9% of the adult population (23) Bone and joint abnormalities are frequent co morbidities of both type 1 [T1D] and type 2 [T2D] diabetes including destruction due to inflammatory diseases such as rheumatoid arthritis [RA] (17, 24, 25) and periodontal disease [PD] (26 29) Both T1D and T2D originate from complex etiology with intrinsic genetic risk factors and extrinsic environmental factors (30 45) Diabetes associated bone and joint pathologies likewise may originate from shared intrinsic genetic factors or from extrinsic sources of inflammation such as infection (occult or symptomatic) (17, 19, 22, 46 60) Furthermore, bone and joint pathology may develop secondary to autoimmune inflammation [T1D], insulin resistance [T2D], or as a consequence of hyperglycemia [T1D and T2D] (17, 19, 25, 27, 50, 61 65) Bone Remodeling Normal process versus inflammatory bone los s: Developmental bone growth as well as post developmental maintenance and repair of bone are dependent on a dynamic process called bone remodeling. Remodeling of bone is a continuous process

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18 necessary to maintain the integrity of m ineralized tissue (66) When bone remodeling is not coupled correctly, either too much bone can be formed or too much can be lost (67) Bone remodeling consists of 5 distinct phases: activation, resorption, reversal, formation, and termination, where two major cell types are involved in the regulation of these phases. Specifically, osteoclasts are responsible for removal of old or damaged bone while osteoblasts are respon sible for formation of new bone (68) A proper coupling between bone formation and bone destruction is essential to maintain bone integrity which is regulated at three different levels: 1) lo cally by a direct interaction between osteoblasts and osteoclasts, 2) by the local interaction between cells of the immune system and bone cells as well as the 3) control by the neuroendocrine systemic on bone metabolism (66, 68 82) Dysregulation of one or more of these levels of regulation are thought to determine the various skeletal manifestations associated with inflammatory diseases such as diabetes mellitus (6 9, 11, 13, 14, 83, 84) Bone remodeling during inflammation is altered where bone formation ceases and bone resorption takes precedenc e. Inflammatory bone loss is initiated after an inflammatory stimulus, such as a bacterial product, is sensed by the innate immune cells residing near the bone (70) Pro inflammatory cytokines such as tumor necrosis factor alpha [TNF beta [IL 6] are produced by these cells in response to the stimulus and can act as direct activators of osteoclast differentiation (osteoclastogenesis) as well as initiate the resorptive process (2, 85 91) CD4+ T cells activated by the stimulus and cytokines also produce pro osteoclastic mediators to initiate resorption (92, 93) Th ese cytokines also induce osteoblasts to produce pro osteoclastic mediators and suppress

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19 bone formation (13, 94 96) Thus, inflammation induces bone loss and suppresses bone healing. Osteoclasts Differentiation, activation, and function : While osteoblasts are derived from mesenchymal stem cells of the bone marrow, o steoclast s are derived from the hematopoietic stem cell lineage. Osteoclasts differentiate from monocytic progenitors at the expense of other monocyt ic lineages such as dendritic cells, granulocytes, macrophages and microglia (97) Osteoclast differentiation is regulated by two essential cytokines: macrophage colony stimulating factor (M C SF) and the receptor activator of nuclear factor kappa B ligand (RANK L). M CSF promotes proliferation and survival of osteoclast precursors while RANK L induces the commitment and differentiation of the precursors (98 1 00) Osteoclast differentiation is strictly dependent on the presence of supportive cells that express on their surface or secrete these two essential pro osteoclastogenic cytokines (101) The earliest osteoclast progenitor known is the colony forming unit g ranulocyte macrophage [CFU GM] (97) These precursors account for 1 4% of the pool of circulating monocytes and express monocyte/macrophage markers yet do not express many of the osteoclast specific markers such as tartrate resistant ac id phosphatase [TRAP], the adhesion molecule beta 3 integrin [ 3 integrin] or the calcitonin receptor [CTR] (101 103) Loss of some monocyte/macrophage markers and upregulation of osteoclast markers are the earliest signs that a mononuclear precursor is committed to the osteoclast lineage (104) On the other hand, other macrophage markers such as Mac 1 (CD11b/CD18) are retained on these mononuclear precursors but are lost once multi nucleation begins In addition, at this stage osteoclast precursors are post mitotic

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20 and thus do not replicate once they are become committed to the osteoclast lineage (105) Osteoclast differentiation is controlled by a highly ordered cascade of osteoclast specific gene expression. The transcription factor PU.1 expressed i n CFU GM induces the expression of c fms the receptor for M CSF (106) This receptor is of the tyrosine kinase super fami ly and upon ligation, dimerizes and auto phosphorylates. M CSF induced signaling results in the up regulation of RANK whose ligation by RANK L is required for the completion of osteoclastogenesis and induction of osteoclast activation (79) Specifically, M CSF ligation to c fms induces the transcription factor c Fos, whereby if M CSF ligation is accompanied by RANK ligation, these precursors commit to an o steoclast lineage (107, 108) Microphthalmia associated transcription factor [ Mitf ] is then activated resulting in the induction of carbonic anhydrase 2 [CA II], TRAP, and add itional c fms (106, 109, 110) Simultaneously, RANK ligation leads to induction of NF (111) inducing the expression of 3 integrin, CTR, cathepsin K, and matrix metalloproteinases [ MMP s] (112, 113) completing the commitment to the osteoc last lineage where n o further proliferation of these cells occurs (99) In addition to its roles in differentiation, M CSF induced signaling enhances pre osteoclast survival and proliferation while preventing apoptosis and enhancing motility in mature osteoclasts (98, 99, 112) M CSF is a homodimeric glycoprotein that can be produced in a soluble or membrane found form by osteoblasts, fibroblasts, epithelial cells, and activated macrophages (101) Soluble M CSF is more involved in early osteoclast differentiation while the membrane bo und form is essential for proper cell fusion during subsequent differentiation stages (114) M CSF also act s as a negative

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21 regulator of its receptor, c fms by reducing i ts transcription which helps to control the resorptive response once it has begun (98, 101, 115) RANK L the l igand for RANK, is a member of the TNF receptor ligand super family and is expressed by stromal cells and osteoblasts in bone as well as activated T cells (93, 101) whereby it can be induced by active vitamin D3, parathyroid hormone [ PTH ] and TNF (101) This molecule is expressed in a membrane bou nd form which is later cleaved into a soluble form by MMPs (116) Through the signaling pathways described above, RANK L along with M CSF, promotes the fusion of mononuclear osteoclast precursors and acts to activate the mature osteoclasts to resorb bone. Only recen tly have the mechanisms associated with the f usion of mononuclear osteoclast precursors begun to be deciphered where the literature describes several candidates involved in cell cell recognition and attachment : 1) the Ig superfamily member CD200, 2) signal regulatory protein and 3) d endritic cell specific transmembrane protein [DC STAMP] (117) Specifically, d uring osteoclast differentiation, CD200 and its receptor CD200R are up regulated just before fusion where their interactions act to enhance fusion promoting RANK sign aling (118 ) Upon a nother member of the CD200 family, not only induces osteoclast differentiation (117) but also promote s fusion initiation although it is dispensable once the process has begun (119) On the other hand, DC STAMP has been called a where it is highly expressed in multinucleated osteocl asts Interestingly, DC STAMP only nee ds to be expressed on one cell and preferably in low levels in order to allow for fusion to occur (120) Similarly, OC

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22 STAMP, which is similar to DC STAMP but only expressed on osteoclasts, is also required for fusion where its up regulation occurs early in the fusion process (121) Osteoclast me diated r esorption requires the following cellular activities: migration of the osteoclast to the resorption site, its attachment to bone, polarization and formation of new membrane domains, dissolution of hydroxyapatite, degradation of organic matrix, and removal of degradation products from the resorption lacuna (97) T he reorganization of the actin cytoskeleton and polarization of the plasma membrane results in the development of f our comp artm ents in the plasma membrane: 1) sealing zone, 2) ruffled border, 3) funct ional secretory domain, and 4) basolateral membrane (97) Unpolarized inactive osteoclasts have dispersed podosomes. During osteoclast activation, these podosomes coalesce into a peripheral belt and subsequently into a the bone surface (122) The podosomes that attach to the bone utilize the alpha v beta 3 integrin which binds to RGD motifs in the bone matrix (123) The resulting actin ring in the sealing zone completely restricts osteoclastic enzyme activity to insid e the resorption lacuna. After the formation of the actin ring, trafficking of late endosomes/lysosomes toward the bone surface allow for formation of the ruffled border, the resorptive portion of the osteoclast. Finally, the functional secretory domain forms at the top of the cell, to which transcytotic vesicles formed at the ruffled border are targeted. T he functional secretory domain allows for the transcytosis of digested bone matrix from the lacuna to the extracellular milieu (124) The main physiological function of osteoclasts is to degrade mineralized bone matrix that requires the dissolution of hydroxyapatite and proteolytic cleavage of the

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23 organic matrix that is rich in collagen. Before proteolytic enzymes can reach and degrade collageneous bone matrix, tightly packed hydroxyapat ite crystals must be dissolved which occurs by targeted secretion of HCl through the ruffled border into the resorption lacuna (97) Vesicles containing vacuolar proton ATPases [V ATPases] are transported to the ruffled border along actin filaments and utilize adenosine triphosphate [ATP] to pump hydrogen ions against a concentration gradient into the resorption site (125, 126) Here, the a3 subunit of the V ATPase aid s in targeting of this normally lysosomal enzyme to the ruffled border (127, 128) Protons are supplied by CA II which acts to hydrate carbon dioxi de and produce bicarbonate that later dissociates into hydrogen ions and HCO 3 (129) Chloride channel 7 [ClC 7] aids in pumping chlorine ions, which along with the hydrogen i ons, creates hydrochloric acid. This creates an environment with a pH of 3 to 4 that is effective at dissolving hydroxyapatite mineral from the organic bone matrix (130) After solubilization of the mineral phase, several proteolytic enzymes degrade the organic bone matrix where t h e acidic environment described above also serves to activate these enzymes (97) Specifically, cathepsin K and MMP 9 are responsible for the degradation of organic bone matrix where both degra de type I collagen into small telopeptides (131, 132) Osteopontin a non collagenous bone protein is also degraded by cathepsin K (97) In addition, c athepsin K acts to activate TRAP, a hallmark enzyme produced by osteoclasts and their precursor s (133, 134) TRAP is a phosphatase that is thought to 1) generate ROS which help degrade collagen 2) aid in degradation of phosphoproteins in the bone matrix such as osteopontin and 3) aid in the release of osteoclasts from the site of resorption so that the cell can migrate (133 136)

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24 As mentioned above, all of these d egradation products are transcytosed through the osteoclast and released out into the extracellular milieu via intr acellular vesicle trafficking where t hese vesicles travel to the functional secretory domain fuse, and empty their contents i nto the extracellular milieu (124) Ef fects of Diabetes on Osteoclast Mediated Inflammatory Bone Loss Diabetes mellitus Although once controversial, the evidence that bone health is compromised in diabetes mellitus is now strong. Bone mineral density is lower, the risk of fractures is increa sed, and there are strong epidemiological links between inflammatory bone diseases and diabetes mellitus all suggesting significant alterations in bone health (5, 7, 8, 10, 11, 14, 24, 26, 27, 84, 137 139) Several mechanisms have been proposed for diabetes related alterations in bone health a nd include the co morbidities of diabetes and more direct pathophysiological effects of the diseases themselves (5, 7, 8, 11, 14, 15, 72, 140) Diabetes mellitus is a heterogeneous group of metabolic disorders characterized by chronic hyperglycemia with disturbances of carbohydrate, fat, and protein metabolism resulting from defects in insulin secretion, insulin action, or both (141) The two major classifications will be discussed in brief below. Type 1 d iabetes Type 1 Diabetes [T1D] or insulin dependent diabetes mellitus [IDDM] is an endocrine disease in humans that, in most cases, results from the autoimmune destruction of the insulin cells] located in the islets of Langerhans of the pancreas that secrete insulin in response to postprandia l elevations in blood glucose (142) Clinical presentation of the symptoms o f T1D includes fasting

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25 hyperglycemia, glycosuria and ketoacidosis. These symptoms are usually abrupt in onset and are indicative of destruction of cell mass. Thus, o ver time, individuals with T1D require exogenous insulin to maintain blood glucose home ostasis since insulin production by the pancreas declines (143) cell destruction is believed to be chronic in nature and can be detected earlier than the onset of clinical symptoms by the presence of certain autoanti bodies including those specific for glutamic acid decarboxylase [GAD], islet cell antibody [ICA], and insulinoma associated protein 2 antibody [IA 2A] as well as insulin (144) Individuals who have one or more of these antibodies can be sub classified as having type 1A, or immune mediated type 1 diabetes (145) Interestingly, the time between the appearance of autoantibodies and full blown insulitis (infiltration of the pancreas by immune cells) is highly variable and can range from a few months to even a decade or more (30, 146) It is important to note that T1D can occur in the absence of autoimmune antibodies and without evidence of any autoimmunity. Such individuals are classified as having type 1B, or idiopathic diabetes, where progressive disease marked by hyperglycemia results in the requirement of insulin for the prevention of ketosis and survival much like type 1A (147) In that type 1A diabetes is of immune etiology, it represe nts an inherited failure in the maintenance of self tolerance and thus it is not surprising that individuals with diabetes are at increased risk for a series of autoimmune and inflammatory disorders including those associated with bone loss/destruction (17) Lessons learned from murine models, but not necessarily confirmed in human T1D, reflect a significant lymphoaccumulatio n that is not restricted to the pancreas suggesting a heightened level of local and systemic inflammation, which could contribute to the manifestation of many

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26 of the inflammatory co morbidities associated with T1D (148) Similar inflammatory complications also arise from the consequences of hyperglycemia that is characteristic of all classifications of diabetes and thus al so could contribute to the manifestation of many of the inflammatory co morbidities associated with T1D (141, 149 152) including bone loss/destruction (5, 27, 153) Such contributions are discussed in detail below. B ecause of the multifaceted nature of diabetes, etiological dissection of diabetes and its co morbidities in humans has been difficult. Therefore, much of the current understanding of diabetes pathogenesis and its co morbidities have been learned from spon taneous animal models. Thus, one of the most extensively studied rodent models of T1D, the NOD mouse, will be utilized in the studies presented here. A detailed description of this model can be found in later Chapters. Type 2 d iabetes Type 2 diabetes [T 2D] or non insulin dependent diabetes is the most common form of diabetes and is characterized by disorders of insulin action and/or insulin secretion where either of which can be the predominant feature but both are usually present at the time of clinical manifestation (154) In many cases, onset of T2D occurs when compensatory mechanisms for insulin resistance drive cell failure (155) The risk of developing T2D increases with age, obesity and physical inactivity although T2D does show strong familial aggregation (42, 43) Environmental f actors implicated in the triggering of T2D in susceptible individuals include smoking, psychological stress, endocrine modulating chemicals, aging, and infections (44, 156) Thus, similar to T1D T2D is a multi factorial disease where genetic and environmental factors influence its development (156, 157)

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27 T2D patient s do not usually require insulin due to their insulin resistance rather than absolute insulin deficiency, although progressive cell loss may require insul in therapy for glycemic control. Glucose tolerance first becomes impaired due to the resistance of the insulin receptors to its ligand thereby leading to both higher fasti ng glucose and hyperinsulinemia (154) Insulin production eventually decreases over time due to the highly toxic, chronic hyperglycemic environment resulting in loss of cell function and/or mass (155) Importantly, there is a relationship between insulin resistance and systemic inflammation in T2D whereby adipose tissue produces many pro inflammatory molecules including TNF IL 6, transforming growth factor [TGF ], and monocyte chemoattractant protein 1 [MCP 1]. These pro inflammatory molecules not only induce systemic insulin resistance, but also contribute to the pathogenesis of many inflammatory complications of T2D including inflammatory bone loss (87, 95, 149, 156, 158 162) As described with T1D, the etiological dissection of T2D and its co morbidities in humans is difficult. T herefore, much of the current understanding of pathogenesis and its co morbidities have been learned from animal models. A large array of rodent models exists for the study of T2D where all models manifest hyperglycemia. Importantly, the stresses needed for the onset of diabetes as well as the associated phenotypes are drastically different. In the studies presented here, the B6.BKS(D) Lepr db /J mouse model was employed where hyperinsulinemia, hyperglycemia, and obesity are all evident (163, 164) A detailed description of this model and its rationale can be found in later Chapters.

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28 Diabetes mellitus and epidemiological link to inflammatory bone loss Although many factors influence bone health, th e most significant factor is bone mineral density or bone strength. Bone densitometry techniques have become much more sophisticated in the past two decades allowing for the detection of more sensitive differences in both T1D and T2D. Low bone density or osteopenia was initially described in adolescents with diabetes, where 25 50% were found to have decreased cortical and trabecular bone mineral density (165, 166) Most studies in adults confirm that bone mineral density is lower in patients with T1D than in subjects without diabetes. In contrast, studies in participants with T2D show bone mineral density that is either the same or greater than diabetes free subjects (138) If the relationship between bone loss and diabetes were related only to hyperglycemia, one would expect a similar incidence of poor bone density in patients with type 1 and type 2 diabetes. Thus, this data suggests that differences between types of diabetes other than glucose control impact bone loss. With that said, hypercalciuria, a marker for bone loss, has been noted in patients with poorly controlled diabetes (type 1 and typ e 2), which can be reversed upon improved glycated hemoglobin [HbA1c] (167) Thus, metabolic control appears to be at least one major factor in the increased incidence of bone loss in patie nts with diabetes. Importantly, e xcessive osteoclast activity as measured by urine concentrations of bone degradation products have been found to be elevated in both T1D and T2D pa tients. On the other hand, osteoprotegerin [ OPG ], the negative regulator of osteoclasts produced by osteoblasts has been found to be reduced in T1D pa tient s yet increased in individuals with early T2D (7, 11) This may be attributed to the hyperinsulinemia associated with early T2D but not late T2D or T1D discussed above.

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29 Insulin is an anabolic hormone whereby it activates osteoblasts to synthesize proteins necessary for bone formation (168) Similarly, insul in like growth factors induce the synthesis of bone collagen and deposition of calcium (169) These findings suggest that hyperinsulinemia can stimulate boney overgrowth (170) Similarly, inflammatory bone pathologies such as rheumatoid arthritis [RA] and periodontitis associated alveolar bone loss are also more prevalent, more severe, and carry with them highe r morbidity in patients with diabetes than in those without diabetes (17, 24 29, 171) Rheumatic diseases such as osteoarthritis and RA have a higher prevalence in patients with both T1D and T2D where damage begins earlier and is more severe than in diabetes free patients (17, 172) While diabetes is not currently conside red a risk factor for developing RA, it is widely known that both conditions do occur simultaneously (24) RA is a chronic inflammatory arthritis which is also classified as an autoimmune disease (46) Thus, similar mechanisms of pathology associated with inflammation in T1D and T2D, as well as those associated with predisposition for T1D autoimmunity, contribute to these epidemiological links (46, 50 52, 55) As with T2D, o besity is a common risk f actor for RA and although the mechanism is unknown one could speculate that the effect of obesity on systemic inflammation discussed above could play a role (55, 61) Rheumatoid arthritis and its co morbidity with diabetes will be discussed further in the sections below. Periodontal diseases are inflammatory processes that occur in the tissues surrounding the teeth in resp onse to bacterial accumulations. This chronic and progressive inflammatory response results in the loss of soft tissue stability and alveolar bone destruction (173) Numerous studies have found that p atients with both T1D and

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30 T2D have a greater susceptibility to and more severe periodontal disease than diabetes free patients as measured by increases in clinical attachment loss and alveolar bone resorption (26, 29) Here, the most common view is that periodontal disease development results from complications of diabetes although recent studies present evidence of a bidirectional adverse interrelationship between both T1D and T2D with perio dontal disease (20, 22, 26, 28, 60, 64, 171, 174, 175) Specifically, epid emiological studies suggest that the level of glycemic control seems to play a key role whereby poor glycemic control positively correlates with high levels of soft tissue damage and alveolar bone loss (174, 176) In addition, as discussed with RA, similar mechanisms of pathology associated with inflammation in T1D and T2D may also contribute to these epidemiological links (18 20, 22, 60, 177, 178) Mechanisms of association between diabetes and periodontal disease will be discussed in detail below. Contributions of Intrinsic Defects While chronic inflammation and hy perglycemia do affect bone metabolism and osteoclast function, as will be discussed later, intrinsic factors such as defects in immune tolerance leading to pro inflammatory signaling and hyper responsiveness of osteoclasts to RANK L stimulation in patients with diabetes are also contributing elements to increased propensity for bone pathology in these individuals (8, 179 181) Lessons from related cell types: macrophages and dendritic cells : Hematopoietic stem cells give rise to the earliest precursor of the osteoclast lineage, the CFU GM. Depending on the growth fact ors and cytokines given, this cell type can give rise to osteoclasts, dendritic cells, monocytes, and macrophages (97) Dendritic cells [DCs], major antigen presenting cells have an alter ed phenotype and function in patient s with T1D where fewer numbers of DCs with decreased

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31 expression of the co stimulatory molecules B7.1/2 that are necessary for the activation of T cells after antigen presentation lead to an impaired ability to activate na ve T cells (182, 183) Similarly, the NOD mouse model also has defects in DC differentiation resulting in lower expression of MHC class II and co stimulatory molecules (184, 185) Interestingly, even with this less mature phenotype, NOD derived DCs display increased NF translocation to the nucleus leading to increased production of pro inflammatory cy tokines, along with more efficient activation of nave antigen specific CD8+ T cells (186 188) Similarly, monocytes and macrophages in p atient s with T1D and T2D have been found to be not only constitutively activated due to constantly circulating t oll like receptor [TLR] ligands but hyper responsive to these ligands (20, 41, 179, 181, 189) This again results in increased levels of multiple pro inflammatory cytokines such as IL (45, 179) Interestingly, studies of NOD derived macrophages again demonstrate a less mature phenotype along with this hyper reactive trait, whereby alterations in NF DCs above (190 192) Thus, one co uld hypothesize that since these related cell types have altered and increased activation, osteoclasts derived from the same hematopoietic lineage may also display aberrant function thereby leading to exacerbated bone destruction. Indeed, osteoclasts util ize the NF regulation of critical genes during differentiation and resorption and thus, heightened translocation as described would

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32 therefore lead to increased resorption under both homeostatic and inflammatory conditions (1, 193) Lack of LPS tolerance : Endotoxin, or LPS, tolerance is defined as a reduced capacity of the host to respond to LPS activation following a first exposure to this stimulus (194) LPS tolerance has also been termed hypo responsiveness, refractoriness, adaptation, deactivation and desensitization (195) However, LPS tolerance is not a global down regulation of signaling protein and mediator production, but rather a change in the expression of specific genes and proteins upon second challenge. The purpose of LP S tolerance has been thought to be a mechanism for controlling innate immune responses (inflammation) important for fighting Gram negative bacteria to prevent host tissue damage and/or to prevent reactivity to commensal flora (196) Lack of tolerance can therefore lead to a constant state of inflammation w hich could directly exacerbate inflammation induced bone pathologies (41) Macrophages derived from diabetes free mouse models such as C57Bl/6, down regulate the receptor for LPS, TLR 4, in response to LPS treatment resulting in a tempered response to subsequent challenge (197) On the other hand, T1D prone NOD mouse derived mac rophages do not down regulate TLR 4 after LPS treatment suggesting defective tolerance to LPS due to aberrant, constitutive TLR 4 expression (180) Interestingly, tolerance al so seems to be defective in monocytes derived from p atients with T2D, whereby hyper secretion of pro inflammatory mediators is observed when using traditional tolerance inducing assays (45, 179) As mentioned above, monocytes and macrophages from T2D patients appear to be constitutively activated

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33 apparently due to constantly circulating T LR ligands derived from endogenous and/or pathogen ic s ources (179) While TLR responses indirectly modulate osteoclast function th rough the interplay between the immune system and bone metabolism, TLRs, in particular TLR 4, are capable of directly modulating bone cell metabolism (198 201) Specifically, LPS can act directly on osteoclasts by inhibiting the differentiation of monocytic precursors into bone resorbing o steoclasts and bone resorption by mature osteoclasts (200, 202) Thus, one could hypothesize that similar to the diabetes specific alterations in LPS responsiveness des cribed above, diabetes derived osteoclasts could also have alterations in their LPS responsiveness. RANK L hyper responsiveness : As previously mentioned, differentiation and activation of osteoclasts require RANK L/RANK interactions whereby RANK L is expressed on a variety of cell types including osteoblasts, T cells, dendritic cells, endothelial cells, and fibroblasts (93, 101) RANK L/RANK interactions are counterbalanced by osteoprotegerin [OPG], which acts as a soluble decoy receptor for RANK L, thus regulating the extent of osteoclast formation and resorption (203, 204) Interestingly, RANK and TLR signaling share common signaling molecules (111, 205) where these molec ules play a role in the augmented TLR responses in T1D and T2D discussed above (179 181, 187, 190) suggesting the potential for RANK L hyper responsiveness as well. Indeed, Mabilleau et al. have demons trated that o steoclasts derived from the peripheral blood of patients with diabetes suffering from arthropathy are more sensitive to RANK L as indicated by increased differentiation capability and resorption. In addition, these diabetes derived osteoclasts were less able

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34 to be deactivated by OPG compared to diabetes free controls suggesting a RANK L dependent and RANK L independent mechanism for augmented osteoclast function (8) Therefore, patien ts with diabetes that also suffer from bone pathologies may have intrinsically hyper responsive osteoclasts to RANK L stimulation thereby leading to exacerbated bone destruction even under homeostatic conditions which may be further augmented with inflamma tion. Presence of chronic inflammation As highlighted above both T1D and T2D are accompanied by a chronic state of inflammation (45, 179, 181, 189, 206 209) Recent reviews have highlighted the interactions between bone and immune cells as well as their overlapping mechanisms (3, 69, 70, 210 212) Furthermore many of the soluble mediators of immune cells including cytokines, chemokines and growth factors regulate the activities of osteoblast s and osteoclasts. Thus mechan isms governing the interaction between skeletal and immune systems most likely play a significant role in elements leading to increased bone pathology in individuals with diabetes mellitus. Importantly, the examination of the interface between these two s ystems is by no means complete, thus further examination should contribute to novel therapeutic strategies to treat disease states mediated by both systems as in the case of diabetes related bone pathologies. Pro osteoclastic milieu : As previously mentioned, both M CSF and RANK L are indispensable mediators for osteoclast differentiation and activation, yet many other cytokines and local immune cell factors also regulate these processes. TNF can stimulate osteoclast formation and bone resorption w here it was demonstrated to be IL 1 dependent (213) In addition, TNF inhibits osteoblast differentiation and collagen synthesis as well as induces their apoptosis (214 216) On

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35 the other hand, TNF can induce osteolysis in an M CSF dependent manner (217) Thus, the osteoclastic milieu of mediators is key in determining osteoclast fate and function as well as the balance of bone remodeling. IL 1 and IL 6 are bot h potent stimulators of bone resorption where they act indirectly to augment osteoclast function. Specifically, they both enhance RANK L production and activity as well as the induction of prostaglandin synthesis in bone cells (74, 96, 218, 219) Interestingly, while TNF enhanced osteoclast activity is dependent on IL 1, IL 1 induced activity is independent of TNF (213, 220) Importantly, IL 1 mediated RANK L production depends on a key signaling molecule also involved in TLR signal ing known as MyD88 but is independent of the additional TLR adaptor molecule (TRIF) (221) This may be important in the context of diabetes mellitus where augmented MyD88 dependent TLR signaling is present (20, 45, 179, 181, 208) Excessive IL 6 has been linked to th e mediation of multiple diseases associated with increased bone resorption (19, 222 225) Interestingly, members of the IL 6 family of cytokines including oncostain M and LIF may play inhibitory roles in osteoclast activation although the data is conflicting (226 229) Other inhibitors of osteoclas togenesis include IL 10 and IFN IL 10 specifically inhibits RANK L mediated NFATc1 expression and nuclear translocation (230) In addition, IL 10 inhibits RANK L expression while inducing OPG expression in supporting ce lls of the bone microenvironment (231, 232) IFN signaling in a TRAF6 dependent manner (233) TRAF6 is an additional signaling molecule also involved in TLR signaling, thus a lterations in the expression/function of these intermediates in diabetes has the potential to alter the effect of IFN

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36 osteoclastogenesis. In addition to its direct inhibitory effects on osteoclasts, IFN indirect affects on osteoclastogenesis by increasing RANK L expression in T lymphocytes and TNF (234, 235) highlighting the fact that not only is the osteoclastic milieu of mediators key in determining osteoclast fate and function, but the cellular inflammatory milieu is as well. The immunological conditions associated with T1D and T 2D exhibit a chronic pro inflammatory skewing of the immune system where pro osteoclastic TNF 1, and IL 6 are found in abundance over anti osteoclast soluble mediators such as IL 10 (30, 144, 236) On the other hand, the anti osteoclast mediator IFN elevated levels under the conditions of diabetes (237, 238) But as eluded to above, in the presence of heavy T cell infiltrate, also present in diabetes, this cytokine induces a more pro osteoclastic environment. Importantly, as discussed above, secondary to the chronic inflammatory conditions, diabetes derived immune cells respond to insulin in an exacerbated fashion, whereby more severe bone loss would be expected given the effects of these soluble mediators on osteoclast activation and function In addition, the aberrant TLR and cytokine ind uced res ponsiveness of these cell types and the phylogenetic relationship of osteoclasts to these hyper active immune cells suggest that diabetes derived o steoclasts may also have heightened sensitivity to stimulation further augmenting bone resorption Contributions of h yperglycemia A major clinical symptom of type 1 and type 2 diabetes is hyperglycemia, or high blood sugar. While the mechanisms associated with the development of hyperglycemia differ in the two diseases, the outcome of increased glucose in the extracellular milieu leads to similar bone and joint pathologies (141)

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37 Effects of excess glucose on osteoclast differentiation and function : While many defects in osteoblast differentiation and function have been documented and confirmed in hyperglycemic conditions findings of aberrant differentiation and/or function in osteoclasts have been controversial (4, 153, 239 245) Hyperglycemic conditions have been shown to increase (246 248) and decrease (240, 249) osteoclast differ entiation and activity depending on the model from which the cells were derived, the method of osteoclast derivation, and the experimental conditions utilized (241, 242, 250 252) This is also the case with studies using rodent models of diabetes mellitus. For instance, studies using the obese Zucker fatty rat T2D prone model demonstrated increased osteoclast activity (253) while decreases in osteoclast function were observed in the non obese T2D Torii models (254) As mentioned above, it is important to note that while both of these models manifest hyperglycemia, the stresses needed for the onset of diabetes as well as the associated phenotypes are drastically different. Here, Zucker fatty rat models display obesi ty and insulin resistance, while the Torii model displays only insulin resistance, both of which lead to hyperglycemia (255, 256) Thu s, the differences in the effect on osteoclast function are most likely attributed to the effect of obesity on chronic inflammation which directly and indirectly affects osteoclastogenesis as described above. Drug induced hyperglycemic models such as the streptozotocin [STZ] treated (257) In these models, decreased osteoclastogenesis and resorp tion concomitant with lowered urine bone metabolism markers have been observed (258, 259) In addition, defects in the fusion

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38 of osteoclast precursors as a result of decreased DC STAMP expression was also found in th e STZ treated mouse model, although more osteoclasts were observed (259) However, o ther markers for osteoclast activation such as RANK L, M CSF, and TNF found to be up regulated in the STZ treated mouse model suggesting increased osteoclast function in this hyperglycemic environment (260, 261) Therefore, the effects of hyperglycemia in this mouse model on osteoclast differentiation and function are controversial and warrant further study. Hyperglycemia can directly initiate pr o inflammatory cytokine production by immune cells such as monocytes via NF activation, specifically TNF both potent inducers of resorption (74, 86, 87, 152, 262) Therefore, hyperglycemia is in itself an inflammatory stimulus and can lead to a chronic pro inflamma tory state which is pro osteoclastic (263) Long term hyperglycemia lead s to increased formation of advanced glycation end products [AGEs] (264) AGEs are formed by th e reaction between free amino groups of proteins lipids, and nucleic acids with oxo groups of sugars. These converted AGEs have altered confirma tion, turnover, and/or function. In addition, AGEs can b ind to an additional receptor aptly called the recep tor for advanced glycated end products [ RAGE ] The interaction of AGE s and RAGE on innate immune cells results in the initiation of NF athway that leads to the productio n of pro inflammatory cytokines thus promoting a pro osteoclastic environment (264) In addition, AGEs inhibit osteoblast proliferatio n and activity while promoting osteoblast apoptosis resulting in decreased bone formation (239, 265)

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39 In addition to being the receptor for AGEs, RAGE is actually a pattern recognition receptor which binds a host of endogenous and exogenous proteins (266) R AGE is expressed on osteoclasts where its ligation is required for bone resorption although the ligand is only known to be a serum component (267 271) Thus, hyperglycemia induced over activation of RAGE would activate a pro oste oclastic response as well as perpetuate an already heightened inflammatory environment providing a cyclical enhancement of bone resorption. Disruption of insulin signaling on bone metabolism : While hyperglycemia is a consequence of both T1D and T2D, T1D r esults in hypoinsulinemia while early T2D is associated with hyperinsulinemia which can progress to hypoinsulinemia later in disease progression. Insulin has direct and indirect effects on osteoclastogenesis whereby i nsulin is considered an anabol ic bone agent. Specifically, insulin signal s osteoblasts to proliferate and function (72, 168) while inhibiting osteoclast pit formation (272) Therefore, i ncreased insulin production in early T2D may lead to increased bone mass partially explaining the high bone mineral density in T2D subjects in the first stages of the disease (138) With declining insulin production durin in later stages of T2D, bone cell insulin signaling would decrease resulting in decreased b one formation as well as increased osteoclast function thereby setting the stage for increased bone destruction and reduced bone repair In sup artilage loss is accelerated during fracture repair in hyperglycemic STZ t reated mice leading to a smaller callus, yet insulin treatment reverses this anomaly by decreasing osteoclast function and incr easing bone formation (260, 261, 272) Further, m ouse models with

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40 hypoinsulinemia such as the NOD mouse and humans with T1D also display low bone mass with osteopenia and osteoporotic phenotypes (10, 14, 84, 273, 274) Therefore, without proper insulin action as seen in diabetes, bone f ormation would decrease and destruction would be left unchecked. Diabetes and Inflammatory Bone Pathologies Periodont al Disease Periodontal disease is characterized by a progressive, destructive host response to bacterial accumulations surrounding the teeth. Over time, soft and hard tissue such as the gingiva and alveolar bone, respectively, are destroyed by the chronic inflammati on eventually leading to loss of the tooth (173) Periodontal disease is now considered to be the sixth complication of diabetes and is more prevalent and severe in individuals with both T1D and T2D (26, 29, 275) Alveolar bone resorption in periodontal disease, in particular, is caused by over activation of osteoclasts, yet the exact mechanism for why this occurs to such a great degree in patients with diabetes is unclear (174, 176, 276) Periodon titis and diabetes co morbidity Patients with both type 1 and type 2 diabetes have a greate r susceptibility to and more s evere periodontal disease than individuals without diabetes as measured by clinical attachment loss and tooth loss (26, 29) Gingivitis, periodontal abscesses, and granulation tissue are also more prevalent in participants with diabetes suggesting an alteration of the host response to the bacterial plaque (277) While this plaque is the necessary initiating factor in periodontal disease, quantity of plaque does not determine progression to periodontitis. In fact, levels of plaque were found to be the same in pa tient s with and without diabetes with periodontal disease, however the severity of

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41 disease was increased in those individuals with diabetes (277) Types of bacterial flora d iffer between T1D and T2D where T1D individuals carry flora consisting mostly of anaerobic vibrios while T2D flora is comprised more often of gram negative anaerobes such as Porphyromonas gingivalis [ P.g ], although the reason for this skewing is unclear (57, 58) Pe riodontal disease is mainly seen as a complication of diabetes, yet there is also evidence that im proving periodontal status may also improve glycemic control (20, 22, 26, 28, 60, 64, 171, 174, 175) Here, poor glycemic control is associated with poor periodontal prognosis and thus an individual with well controlled diabetes displays less severe periodontal disease than their poorly controlled counterpart (174, 176) Mortality due to other diabetic complications such as nephropathy and ischemic heart disease is also increased in individuals with diabetes who also suffer from periodontal disease (171) While there is a clear association between diabetes and the severity of periodontal disease and ass ociated alveolar bone loss, the role of osteoclasts derived from hosts with diabetes and their functional status has not been fully elucidated. Pathology: bacterial colonization, inflammation, and alveolar bone loss The periodontal tissues, or periodontium are those tissues that support the teeth including the root cementum, periodontal ligament, alveolar bone, and gingiva surrounding each tooth. Alveolar bone lines and forms the sockets of the teeth. This type of bone has a high turnover and is constantly remodeled by osteoclasts and osteoblasts. Once a tooth is removed, this alveolar bone is resorbed and will not reform. Alveolar bone is uniqu e in that during tooth movement, bone is lost from one side of the tooth and formed on the opposite side of the tooth leading to asynchronous resorption (173)

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42 While the periodontium does provide an effective barr ier against environmental insults such as bacterial colonization, the state of the host immune system and types of bacteria present can lead to breakdown of tissue integrity and inflammation induced activation of bone resorption Innate immune cells in th e periodontium such as epithelial cells, macrophages, neutrophils, and DCs are the first to respond to pathogenic bacteria and normally keep the infection in check without inducing tissue destruction However, a shift occurs when the host responds too rob ustly or is unable to resolve inflammation as seen in diabetes mellitus, whereby the immune response results in host tissue damage. Specifically, pro inflammatory cy tokines, chemokines, and MMPs serve to activate the innate immune response, mark the site of infection and recruit adaptive immune cells such as CD4+ T H 1 and T H 2 T cells a s well as B cells (plasma cells) However, MMPs and pro inflammatory cytokines such as TNF also have the ability to destroy host tissues (278, 279) These inflammatory processes lead to connective tissue destruction resulting in the migration of the junctional epithelium down along the root surface leading to pocket formation. This free epithelial surface is now extended and therefore expose d to more bacterial biofilm The connective tissue also migrates deeper towards the root and thus the attachment of the tooth to the alveolar bone is compromised. In addition, the neutrophils once found in the sulcus bec ome displaced near the root surface (173) The inflammatory response to periodontal pathogens eventually leads to alveolar bone resorption by osteoclasts whereby IL 1 IL 6, and TNF inflammatory cytokines found in the inflamed periodontium and pro osteoclastic mediators described above can lead to the differentiation and activation of osteoclasts

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43 to resorb bone (90, 280) Osteoblasts in the periodontium can also be activated by LPS from invading bacteria and p roduce RANK L, IL 1 IL 6, and TNF (95, 201, 221) Simi larly, RANK L produced by activated CD4+ cells is also in high abundance in the inflamed periodontium which directly differentiates osteoclast precursors and activates mature osteoclasts to resorb bone (92, 281) Contributions of diabetes to exacerbation of periodontal disease: The status of the host immune system is a major factor in determining the development and subsequent severity of periodontal disease (278, 281) Patients with diabetes display many innate immune system abnormalities lead ing to defective recruitment and/or action of leukocytes that result in decreased ability to fight infection, defective wound healing and exacerba ted inflammation (282) N eutrophil chemotaxis to the site of infection and phagocytosis of extracellula r pathogens are impaired in a hyperglycemic environment th ereby allowing microbes to accumulate that would normally be destroyed, leading to uncontrolled infections (22, 157, 283) Responses to bacterial components are also altered where pro inflammatory cytokine secretion is augmented, as described in detail earlier. In particular importance to the gingiva, h yper secretion of pro inflammatory cytokines such as TNF patient s in response to P.g. compared to diabetes free participants and may explain why the tissue destruction is so severe in these individuals (20) The accumulating b acterial components such as LPS in patients with diabetes can act on activated T cells and osteoblasts to produce RANK L and indirectly activat e osteoclasts (92) LPS can also induce the expression of pro inflammatory cytokines such as IL 1, IL 6, and TNF osteoblasts, monocytes, and macrophages (the latter which are hyperactive in both T1D

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44 and T2D) that can act on osteoclasts directly or indirectly via RANK L up regulation (70) Collagen breakdown is also increased in participants with diabetes via augmented MMP production, which leads to impaired wound healing (282, 284, 285) AGEs which are more prevalent in periodontal tissues from rodent models of diabetes lead to increased oxidative stress and tissue destruction wh ile promoting a pro inflammatory environment that in itself is destructive to host tissues (178, 268) Supporting the rodent model findings, g ingival crevicular fluid has been found to contain high levels of glucose in patients with diabetes compared to diabetes free individuals suggesting diffusion of glucose into the periodontal pocket and possible metabolic changes in the surroundi ng tissues such as the formation of AGEs that can increase host tissue destruction (286, 287) While osteoclasts are the cell type responsible for the loss of alveolar bone, the contributions of diabetes and its possible alterations on osteoclast differentiation and function from hosts with diabetes has not been elucidated. Yet with increased bacterial burden and augmented pro inflammatory cytokine production in patient s with diabetes, one can imagine an environment conducive to exacerbated host tissue destruction, especially in the alveolar bone. Inflammatory Arthritis Inflammatory arthritis is a general term for a group of bone pathologies i n the joint caused by an initial inflammatory stimulus and includes rheumatoid arthritis psoriatic arthritis, and reactive arthritis. RA, a chronic autoimmune disease, is considered the prototypical inflammatory bone pathology (262) This disease affects an estimated 1.5

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45 mill ion Americans with women being predominantly affected (263) and has been found to be associated with other autoimmune disorders such as T1D (17) Inflammatory arthritis and diabet es co morbidity Diabetes is not considered a risk factor for RA, although RA is positively correlated with T1D and T2D (24, 25) RA is more severe and occurs earlier in patients with T1D than in those without diabetes (17, 172) In addition, obesity as seen in individuals with T2D, can lead to the development of RA (55, 61) Individuals who are morbidly obese (body mass index of 30+) have the highest risk of developing RA compared to normal body weight participants (46, 55) Both RA and diabetes are considered disorders caused by chronic activation of the immune system. Pro inflammatory cytokines such as IL 6 and TNF regulat ed in both diabetes and RA (264 266) Therefore, the chronic inf lammation seen in RA can lead to insulin resistance and thus may predispose one to T2D (25, 55) Insulin resistance in RA may also be caused by the reduction in lean muscle mass and increase in adipose tissue from the sedentary l ifestyle of individuals with the disease due to its debilitating nature, which in turn leads to chronic inflammation via adipocyte hyper secretion of pro inflammatory soluble mediators (2 67, 268) Interestingly, medications used to treat RA such as corticosteroids, non steroidal anti inflammatory drugs [NSAIDs], TNF improve glycemic control by promoting insulin secretion and insulin sensitivity (25, 63) Thus, improving the chronic inflammation in RA could theoretically improve glycemic control in patients with T2D. As mentioned previously, osteoclasts can become activated to resorb bone by pro inflammatory soluble mediators, which are in high abundance in RA. When inflammation runs rampant as in the case of RA, excessive secretion of these mediators

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46 can lead to inappropriate osteoclast activation and exacerbated bone loss. The contributions of diabetes derived osteoclasts to RA pathogenesis, however, h ave not been investigated. Pathology: inflammation and bone destruction The joint in health consists of articulating bones covered with cartilage to protect against erosion during movement and ligaments and tendons which surround the joint for stabilizatio n contained in a joint capsule. The intra articular side of the joint capsule is lined with a synovial membrane and is filled with s ynovial fluid which consists primarily of hyaluronic acid (294) This synovium also consists of extracellular matrix consisting of collagen fibrils, adipose tissue, elastin, and other proteoglycans. The synovial membrane provides nutrients and also lubricates the joint and consists of two ty pes of synoviocytes. Type A synoviocytes are macrophages derived from bone marrow and type B cells are fibroblast like (295) Synovial fluid and matrix components are produced by type B cells and cleared by type A cells to maintain joint nutrient and fluid homeostasis. Type B synoviocytes also produce MMPs, cathepsins, and serine proteases, all which aid in joint matrix remodeling (296) Few immune cells such as macrophages and mast cells along with fibroblasts and adipocytes also reside in the synovial membrane in the side proximal to the bone (297) RA is a chronic inflammatory arthritis with features of autoimmunity including autoan tibodies to citrullinated protein antigens [ACPA] and immunoglobulin M [IgM] rheumatoid factor [RF]. The joint synovium is the primary site of inflammation where this synovitis leads to joint damage and eventual bone destruction and subsequent deformity (298) Fibroblast like synoviocytes also produce chemokines along with resident macrophages to recruit immune cells to the inflamed synovium (299, 300)

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47 Leukocytes such as T and B cells, DCs, and macrophages infiltrate the synovium and produce many pro inflammatory cytokines and chemokines (301) The synovium then thickens and becomes highly vascularized and invade the cartilage covering the bone. The destruction is mediated by MMPs and aggrecanases, destructive enzymes that target collagen and aggregan (another proteoglycan found in cartilage), respectively (298) B cells are important in the progression of RA and serve to produce ACPAs and RF as well as present antigen to T cells in the lesion (298, 302) Interestingly, presence of ACPA and RF can be detected up to 10 years before onset of clinical disease (303, 304) Erosion of the bone underlying cartilage (subchondral bone) is mediated by osteoclast s (288) T hese cells are found both at the front of the pannus as well as within the subchondral bone and are found early within the first few days, in the arthritic process (305, 306) However, bone destruction is not detected on conventional x rays until at least two months after onset of clinical symptoms (307) In c fos deficient mice that do not develop osteoclasts, bone destruction does not occur, yet synov itis and arthritis are still evident (89) The inflammatory environment of the arthritic joint serves to attract osteoclast precursors and allow them to differentiate into fully resorptive mature osteoclasts. TNF inducer of osteoclastogenesis and resorption. Other important cytokines that activate osteoclast mediated reso rption that are found in RA joints include IL 1, IL 17, and RANK L, all produced by CD4+ T cells (T H 1 and T H 17) (288) Contributions of diabetes to exacerbation of periodontal disease: While the exact etiology is unknown, genetics play a significant role in the dev elopment of RA yet

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48 environmental stimuli are necessary for triggering the inflammatory process similar to T1D and T2D (48, 298) Interestingly, some genes associated with RA including PTPN22 and CTLA4 which negatively regulate T cell activation, are also associated with the development of T1D thereby allowing for multiple autoimmunities (50, 51, 308) A loss of function variant of the IL 4 receptor was also fo und to be associated with RA which can lead to skewed Th responses away from the T H 2 lineage (309) as is commonly seen in T1D individuals that exhibit extreme T H 1 polarization (209) Therefore, in a T1D environment where there is chronic T H 1 activation and lack of regulation of T responses, one can imagine an environment suita ble for the development of autoimmune RA. Interestingly, periodontal disease which is a complication of diabetes, is found to be more prevalent in participants with RA with P.g. antibodies correlated with ACPA positivity (53, 54) Thus, individuals with diabetes who suffer from periodontal disease may also develop RA, although the exact relationship between the latter two is unclear. Hyper responsive monocytes and macrophages, as commonly seen in patients with diabetes (179, 181, 189) may also exacerbate RA. Pro inflammatory cytokines such as TNF inflammatory response in the joint and thus induce more bone destruction (86, 310) Therefore, genetic predisposition similarities between the two diseases and intrinsic hyper reactive defects in osteoclast modulating cell types could set the stage for an even more severe inflammatory arthritis in pati ents with diabetes. Hypotheses Due to the fact that innate immune cells such as macrophages, monocytes, and DCs have bee n found to be hyperactive in diabetes mellitus to inflammatory stimuli and

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49 that these cells share the same lineage with oste oclasts, it is possible that diabetes derived osteoclasts are intrinsically more easily activated to resorb bone than those derived from diabetes free sources. In addition, the chronic inflammatory environment and hyperglycemia present in diabetes may further augm ent differentiation and/or resorptive function of osteoclasts derived from these hosts compared to diabetes free sources.

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50 CHAPTER 2 AUGMENTED LPS RESPON SIVENESS IN TYPE 1 D IABETES DERIVED OSTEOCLASTS Introduction B one and joint abnormalities are fre quent co morbidities of type 1 diabetes [T1D] (140, 311, 312) T1D originates from a complex etiology with intrinsic genetic risk factors and extrinsic environmental factors. T1D associated bone and joint pathologies likewise may originate from shared intrinsic genetic fac tors or from extrinsic sources of activation. Furthermore, bone and joint pathology may develop secondary to autoimmune inflammation or as a consequence of hyperglycemia. The complexity of T1D itself and added complexity of bone and joint co morbidities necessitates well controlled and innovative approaches to assess the totality of potential causes. Physiological bone remodeling is a highly coordinated process that orchestrates five sequential phases: activation, resorption, reversal, formation and term ination (68) In addition to traditional bone cells including osteoclasts and osteoblasts which are responsible f or bone resorption and formation, respectively, immune cells have also been implicated in the regulation of this process. Thus, alterations in immune function have been implicated in many bone diseases (68) While decreased skeletal health in T1D involves alterations in osteoblast maturation and function, the role of osteoclasts in inflammation induced bone and join t loss is less understood (140) Osteoclast differentiation is regulated by macrophage colony stimulating factor [M CSF] and the receptor activator of nuclear factor kappa B ligand [RANK L]. Resorption of bone is initiated by binding of the osteoclasts to the mineralized bone surface via sealing zone membrane and a resorption lacuna (313) Vesicles containing osteo clastic enzymes such as tartrate resistant acid

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51 phosphatase [TRAP], the serine protease cathepsin K, and matrix metalloproteinase 9 [MMP 9] induce collagen degradation after bone demineralization by the vacuolar H+ ATPase (133) Osteoclasts are also negatively regulated through soluble me diators including calcitonin and osteoprotegerin [OPG] (69) Activation of osteoclast mediated bone resorption can be augmented by infection and in flammation, as well as hormonal alterations (69) Lipopolysaccharide [LPS], a cell wall component of gram negative bacteria, has been found to be hi ghly immunogenic and induces the production of pro inflammatory cytokines by various immune cells. Osteoclasts and their precursors, which share the same lineage as macrophages and dendritic cells, express many innate immune receptors including toll like receptors [TLRs] and thus can respond to bacterial components (71, 199, 314) In a co culture of osteoclasts and osteoblasts, LPS, the ligand for TLR4, augments bone resorption (201) However, when supporting cells such as osteoblasts or other immune cells are ab sent, LPS inhibits bone resorption in osteoclast pure cultures, although the exact mechanism(s) is not known (200) In addition, pro inflammatory cytokines including TNF 6 stimulate differentiation and activation of osteoclasts with IL and TNF ing resorption (91, 160) In both murine and human T1D, macrophages and dendritic cells have been shown to be hyperactive to TLR stimulation resulting in excessive pro inflammatory cytokine production (186, 190, 315) In addition, individuals with T1D are less able to clear bacterial infections thereby a llowing bacterial components to accumulate further amplifying the inflammatory process (316)

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52 Since individuals with T1D respond to inflammation in such an aberrant fashion, it is entirely possible that these individuals would be more susceptible to severe bone loss than T1D free individuals given the effects of these soluble mediators on osteoclas t activation and function. Similarly, given the phylogenetic relationship of osteoclasts to macrophages and dendritic cells and the aberrant TLR responsiveness of these cell types in T1D, we postulate that TID derived osteoclasts have heightened sensitivi ty to stimulation resulting in augmented differentiation and activation. Non obese diabetic [NOD] mice, a model for spontaneous autoimmune diabetes with pathology similar to individuals with T1D, have an osteoporotic phenotype (274) In addition, NOD mice are more susceptible to spontaneous and in duced arthritis (317) Therefore in the present study, we characterized T1D associated osteoclast specific differentiation, activation and function in the presence and absence of inflammatory stimuli utilizing the NOD mouse model. Materials and Methods Mouse Models NOD/LtJ [NOD], NOR/LtJ [NOR], C57BL/6 J [ C57BL/6 ], and BALB/c J [ BALB/c ] mice were maintained in a specific pathogen free [SPF] environment at th e breeding facilities of the University of Florida. The NOD mouse spontaneously develops an autoimmune cells within the islets of Langerhans of the pancreas producing very similar pathology as seen in individuals with T1D, wh ere by 10 weeks of age severe insulitis is apparent, but mice are normoglycemic (148) The NOR mouse model has a simila r genetic background as the NOD mouse but does not develop insulitis and diabetes due to the dispersal of C57BL/6 genome within diabetes susceptibility loci (318) Thus the C57BL/6 strain serves as an additional genetic

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53 background control as well as an immunological control along with the BALB/c strain C57BL/6 mice are considered T H 1 responders as are NOD mice (148) while BALB/c mic e respond predominately in a T H 2 dir ected manner (319) Blood glucose levels were measured at time of sacrifice with the Ascensia Contour Bloo d Glucose Meter (Bayer). Bone marrow was harvested from female mice of all strains at 10 12 weeks [wks] of age. Pancreata were also harvested from NOD and NOR mice. All experimental procedures were conducted in accordance with the guidelines of the Univ ersity of Florida Institutional Animal Care and Use Committee. Osteoclast Differentiation Femora and tibiae were surgically isolated, excess tissue removed, and marrow MEM complete media (Sigma Aldrich) [10% feta l bovine serum (Mediatech), 1% L glutamine (Thermo Scientific), 1% penicillin/streptomycin/amphotericin B (Fisher)]. Cells were seeded in T75 flasks at a concentration of 1.5x10 6 cells/mL supplemented with 5ng/mL recombinant murine M CSF [rmM CSF] (Peprot ech) and allowed to culture for 24 h at 37 C and 5% CO 2 Non adherent cells were removed and 5.9x10 5 cells/mL of adherent cells were seeded in 24 well plates on either glass coverslips (Fisher) or 1 cm 2 bovine bone slices cut with an Isomet Low Speed Saw (Buehler). All cultures were supplemented with 10ng/mL rmM CSF and 50ng/mL recombinant murine soluble RANK L [rmsRANK L] (Peprotech) and allowed to culture for 6 d with complete media refreshed every 3 d. TRAP Staining After 6 d or 9 d of differentiation cells plated on glass coverslips were fixed with 2% paraformaldehyde/PBS (Fisher). Cells were washed with PBS and permeablized in 0.5% Triton X 100/PBS (Fisher). Cells were washed and probed for leukocyte acid

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54 phosphatase (TRAP) [1:1:1:2:4 Fast Garnet GBC Base Solution:Sodium Nitrite Solution:Napthol AS BI Phosphate Solution:Tartrate Solution:Acetate Solution] (Sigma Aldrich) after which cells were washed and mounted on glass slides with MOWIOL 4 88 solution (Calbiochem). TRAP positive cells [purple in color] were counted according to number of nuclei present: mononuclear cells [1 nucleus], multinucleated osteoclasts [2 10 nuclei], and giant osteoclasts [11+ nuclei] using light microscopy at 40x magnification. Whole coverslips were counted for each cel l type. Percentage of total nuclei was calculated by the number of nuclei in each cell type divided by total number of nuclei counted. Osteoclast Stimulation After 6 MEM complete media supplemented with 10n g/mL rmM CSF and 50ng/mL rmsRANK L. Cells were allowed to resorb bone for 72 h in the presence or absence of the following: 1) 1ug/mL Escherichia coli LPS [LPS] (Sigma), 2) pro inflammatory cytokine cocktail [10ng/mL recombinant human TNF D Systems) + 10ng/mL recombinant murine IL 6 (Peprotech)], 3) NOD conditioned media [from M CSF and RANK L stimulated bone resorption cultures], or 4) C57BL/6 conditioned media [from M CSF and RANK L stimulated bone resorption cultures]. Cultures were permeablized with 1% Triton X 100 and supernatants stored at 80 C until cathepsin K ELISA, collagen type I telopeptide ELISA, MMP 9 ELISA, and Luminex cyto/chemokine analyses were performed. Bone was made devoid of c ells with 10% fixative (Fisher) at 4 C until scanning electron microscopy [SEM] could be performed. All bone resorption outcome measures were normalized to number of TRAP+ cells

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55 [outcome measure x (total number of TRAP+cells/total number of cells plated)], mononuclear cells [outcome measure x (total number of mononuclear cells/total number of TRAP+cells)] and multi nucleated cells [outcome measure x (total number of multi nu clear cells/total number of TRAP+cells)]. Flow Cytometry for Osteoclast Culture Purity After 6 MEM complete media supplemented with 10ng/mL rmM CSF and 50ng/mL rmsRANK L in the presence or absence of 1ug/mL non pure E. coli LPS for 72 h. Cells were allowed to dissociate from the bottom of UpCell (ThermoScientific) coated plates at room temperature. Suspended cells were washed with FACS Buffer [1x PBS + 5% FBS + 0.372g EDTA] and allowed to incubate with the following primary [1:200] and secondary antibodies [1:200]: goat anti mouse calcitonin receptor [CTR] (Santa Cruz) with anti goat Alexa Fluor 647 (Invitrogen) and biotin conjugated rat anti mouse RANK (eBioscience) with PerCP Cy5.5 conjugated streptavidin (eBioscience). Cells were acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FCS Express (De Novo Software). Cell Viability Assays Viability was assessed using a colorimetric MTT Cell Growth Assay (Millipore, Billerica, MA) at 6 d and 9 d post differentiation described above. MTT assay was absorbance was quantified with a spectrophotometer set at a dual wavelength reading of 570nm with a reference of 630nm. Culture media al one was used as a negative co ntrol while cells lysed with 1% T riton X 100 were used as a positive control.

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56 Scanning Electron Microscopy Bone slices were sputter coated with gold and visualized with S 4000 FE SEM scanning electron microscope (Hitachi). Thr ee r andom scanning electron micrographs [8 bit grayscale] of bone slices were acquired at 40x magnification with a 2048x1594 Identical procedures were applied to ever y image from all experimental groups utilizing NIH ImageJ software to quantify the surface area resorbed. Prominent repeating elements in the frequency domain were identified and removed after which the inverse fast Fourier transformation was applied yiel ding the original image with reduced saw marks. The CLAHE algorithm (320) was used to increase contrast [block size: 256, histogram bins: 256, maximum slope: 8], and a Gaussian blur [ : 2 pixels] was applied t o the result. The rolling ball algorithm (321) was applied [radius: 100 pixels] to achieve background intensity equalization. A threshold value (85) was used to convert the result to a 1 bit image [0: normal bone, 1: region of resorption] used for quantitative analysis. Images which contained prominent artifacts spanning 5% or more of the total area were not included for analysis. Percentage of area resorbed was calculated by dividing square microns resorbed by total square microns. Collagen Telopeptide ELISA Collagen carboxy t erminal telopeptides were detected using an ELISA according to manufacturer instructions (Immunodiagnostic Systems). Supernatants were pre incubated with biotin conjugated anti telopeptide and horseradish peroxidase [HRP] conjugated anti telopeptide and a dded to an ELISA plate coated with streptavidin [SAV]. Following five washes, tetramethylbenzidine [TMB] substrate was used to develop reactivity followed by quenching with H 2 SO 4 Colorimetric reactions were detected

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57 using an Epoch microplate spectrophot ometer (Biotek) set at a dual wavelength reading of 450nm with a reference of 655nm. Gen5 Software (Biotek) and a standard curve were used to determine nM concentrations. Cathepsin K ELISA Active cathepsin K was detected using an ELISA according to manufac turer instructions (Alpco). Supernatants pre incubated with HRP conjugated anti cathepsin K were added to an ELISA plate pre coated with polyclonal sheep anti cathepsin K. Following five washes, TMB substrate was used to develop reactivity followed by qu enching with STOP solution. Colorimetric reactions were detected using an Epoch microplate spectrophotometer (Biotek) set at a dual wavelength reading of 450nm with a reference of 655nm. Gen5 Software (Biotek) and a standard curve were used to determine pM/L concentrations of active cathespin K. MMP 9 ELISA Total MMP 9 was detected using an ELISA according to manufacturer instructions (R&D Systems). Supernatants were added to an ELISA plate pre coated with anti MMP 9. Following four washes, HRP anti MM P 9 was used to detect reactivity. Following five washes, TMB substrate was used to develop reactivity followed by quenching with HCl. Colorimetric reactions were detected using an Epoch microplate spectrophotometer (Biotek) set at a dual wavelength readin g of 450nm with a reference of 595nm. Gen5 Software (Biotek) and a standard curve were used to determine ng/mL concentrations. Soluble Mediator Analysis Cytokines and chemokines from resorption supernatants were detected and quantified using a mouse 22 cyt o/chemokine multiplex (Millipore) according to the

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58 coated beads were allowed to incubate overnight at 4 C in a 96 well primed plate. Following three washes, reactivity was probed with biotinylated det ection antibodies and SAV phycoerythrin [PE]. All incubations occurred while gently shaking in the dark. Following three washes, beads were resuspended in sheath fluid and reactivity acquired using a Luminex 200 IS system with Xponent software (Millipore ). Milliplex analyst software (Viagene), 5 parameter logistics and a standard curve were used to determine pg/ml concentrations. Outcome measures were normalized to number of mononuclear cells [outcome measure x (total number of mononuclear cells/total nu mber of TRAP+cells)] and multi nucleated cells [outcome measure x (total number of m ulti nuclear cells/total number of TRAP+cells)]. Histological Analysis of Pancreas Pancreata were fixed in 10% formalin (Fisher) and embedded in paraffin. 5m sections were mounted on glass slides and deparaffinized in xylenes and rehydrated using 100% ethanol (EtOH), 95% EtOH, 75% EtOH (Fisher), and dH 2 O. Sections were stained with Harris hematoxylin, incubated in a bluing solution [1.5% NH 4 OH (EMD Chemicals) in 70% EtOH] and stained with eosin [1% aqueous Eosin Y, 1% aqueous phloxyine B (Fisher), 100% EtOH and glacial acetic acid (Mallinckrodt Chemicals)]. Sections were dehydrated with 70% EtOH, 95% EtOH, 100% EtOH, and xylene (2 times), mounted with Permount (Fisher) and observed under light microscopy. Islet infiltration was graded on a 0 2 scale, with 0=no insulitis, 1=peri insulitis, and 2=insulitis where average insulitis score per pancreata was determined.

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59 Statistical Analysis One parisons were used to analyze and determine statistical significance (p<0.05) as appropriate. Results NOD derived Osteoclasts Display Altered Differentiation In order to determine if increased bone resorption observed in multiple T1D complications is due to alterations in osteoclast differentiation, the differentiation of bone marrow derived osteoclasts [BM OCs] from C57BL/6 BALB/c NOR and NOD mice was evaluated. BM OCs were allowed to differentiate for 6d and purity of cultures was evaluated using FACS analysis where the expression of RANK and calcitonin receptor [CTR] was used to define the osteoclast populations (Fig. 2 1). In addition, TRAP staining was used to determine the number of mononuclear cells, multinucleated osteoclasts, and giant osteocla sts (Fig. 2 2). While BM OC cultures from all strains generated similar percentages of RANK + CTR + where on average cultures were 76.5% pure, NOD BM OCs consistently had a population of cells expressing lower levels of CTR [RANK+CTR lo ] (Fig. 2 1 arrow). In addition, while no differences in the number of mononuclear cells were observed (Fig. 2 2B), there were significantly fewer multinucleated and giant osteoclasts observed in NOD derived osteoclast cultures when compared to C57BL/6 BALB/c and NOR derived cultures (Fig. 2 2C,D). Because osteoclast formation is a result of cell fusion events, we also evaluated the percentage of each osteoclast phenotype type compared to the total nuclei present (Fig 2 2E G). Here again, NOD derived osteoclast cultures had a significantly lower percentage of cells which were multi nucleated compared to those derived from all other strains (Fig. 2 2F,G). In order to determine if

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60 NOD BM OC cultures simply had a delay in fusion events, differentiation of BM OCs was also evalu ated at 9d of culture, where no significant difference in the number of multi nucleated or giant cells was observed (Fig. 2 2H). Similarly, to determine if there was a difference in osteoclast survival an MTT assay was performed at 6d and 9d of BM OC cultu re. Again, no significant difference in cell survival was observed in NOD derived BM OCs (Fig. 2 2I). Taken together, these data indicate a possible defect in the differentiation of NOD derived osteoclasts, most likely involving the fusion of mononuclear cells into multinucleated osteoclasts. NOD derived Osteoclasts Have Increased Bone Resorption Capabilities in Response to RANK L Stimulation Although our differentiation data suggests overall smaller osteoclast size, this data does not address the bone res orbing function of these cells. In order to determine the bone resorbing capabilities of NOD derived BM OCs, these cells were seeded onto bovine bone slices and stimulated to resorb bone with RANK L. To quantify the amount of bone resorbed, SEM and Image J analysis were performed where the total area resorbed and number of resorption pits by TRAP+ cells was determined (Fig. 2 3). NOD derived BM OCs were found to resorb more surface area in response to RANK L stimulation compared to C57BL/6 BALB/c and NOR derived cultures (Fig. 2 3A, B). In addition, the number of resorption pits was significantly higher in NOD derived BM OCs cultures compared to those from all other strains (Fig. 2 3A, E). In order to elucidate activity on a per cell basis, the total area resorbed and number of resorption pit data was stratified based on number of TRAP+ mononuclear cells (Fig. 2 3 C, F) and TRAP+ multinucleated cells (Fig. 2 3 D, G). Here again the total area resorbed and the number of resorption pits was significantly higher in NOD derived TRAP+ mononuclear

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61 cells. On the other hand only the total area resorbed was significantly higher in NOD derived TRAP+ multinuclear cells. Together these data suggest that irrespectively of the smaller size, more cells in NOD derived osteoclast cultures resorbed a greater bone surface area. NOD derived Osteoclasts Degrade More Type 1 Collagen than Controls via Enhanced Cathepsin K and MMP 9 Secretion During bone resorption, the organic portion of bone containing type I collagen is degr aded in the resorption lacunae where vesicles containing collagen telopeptides are transcytosed out of the osteoclast and released into the extracellular milieu (322) Therefore, in order to further quantify bone resorption, levels of intra and extra cellular collagen telopeptides were evaluated. Similar to our SEM data, RANK L stimulated NOD derived BM osteo c l ast cultures had signifi cantly higher levels of collagen telopeptides than C57BL/6 BALB/c and NOR derived cultures (Fig. 2 4A, D, G) confirming higher bone resorption activity. Lysosomal cathepsins, such as cathepsin K, and matrix metalloproteinases such as MMP 9 can degrade type I collagen at an acidic pH created in the resorption lacunae (97) Therefore, to investigate mechanisms associated with the observed increased collagen degradation, the amount of cathepsi n K (Fig. 2 4B, E, H) and MMP 9 (Fig. 2 4C, F, I) in the same BM OC cultures was quantified. Increased levels of cathepsin K and MMP 9 were detected in RANK L stimulated NOD derived BM OC cultures when compared to C57BL/6 BALB/c and NOR derived cultur es (Fig. 2 4B, C). Again, activity was evaluated based on nucleation, where collagen, cathepsin K and MMP 9 was significantly higher in NOD derived TRAP+ mononuclear cells (Fig. 2 4D F) with collagen, and MMP 9 but not cathepsin K being significantly higher in NOD derived TRAP+ multinuclear cells (Fig. 2

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62 4G I). These data indicate that RANK L stimulation of NOD derived BM OC results in increased cathepsin K and MMP 9 release leading to increased collagen degradation, with the largest source of bone d egradation being TRAP+ mononuclear osteoclasts. NOD derived Osteoclasts Respond Aberrantly to LPS Inflammatory bone pathologies are often seen in individuals with T1D, including inflammatory arthritis and periodontitis associated alveolar bone loss, where inflammatory mediators including bacterial components are abundant in these milieus (17, 26, 277, 278, 282, 316) Thus, the effect of LPS on T1D derived osteoclast function was evaluated. Osteoclasts were seeded onto bovine bone slices and stimulated to resorb bone with RANK L in the presence of LPS. LPS induced deactivation of osteoclast function was observed in C57BL/6 BALB/c and NOR derived cultures indicated by a decrease in the ar ea resorbed and collagen release as well as decreased cathepsin K and MMP 9 secretion (Fig. 2 5). However, this LPS induced deactivation did not occur in the NOD derived osteoclast cultures as shown by a lack of decrease in the area resorbed and collagen degradation (Fig. 2 5A, B). Interestingly, NOD derived BM osteoclasts also displayed an increase in LPS responsiveness as measured by increases in cathepsin K and MMP 9 secretion compared to RANK L stimulation alone (Fig. 2 5C, D). These data suggest tha t NOD derived BM OCs are not only unable to be deactivated by LPS but more importantly secrete elevated levels of bone resorbing mediators in the presence of LPS. NOD derived Osteoclasts Secrete Increased Soluble Osteoclastogenic Mediators in Response to LPS Because osteoclast function is regulated by many soluble immune mediators, the cytokine and chemokine profile within BM OC cultures from all strains was evaluated.

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63 Increased amounts of the hematopoietic growth factor GM CSF and pro inflammatory cytoki nes IL 10 were elevated in BM OCs cultures from all strains following LPS stimulation (Fig. 2 6). Similarly, the anti osteoclastogenic mediator IL 10 was also elevated in BM OCs followi ng LPS stimulation (Fig. 2 6F, L). Interestingly, NOD derived BM OCs cultures presented with significantly higher levels of all mediators, with the exception of IL 10, compared to those found in C57BL/6 BALB/c and NOR derived cultures when normalized to TRAP+ mononuclear cells (Fig. 2 6A F). While a similar trend was observed when data was normalized for multinucleation (Fig. 2 6G L), it was interesting to note that GM CSF expression was no longer significantly higher (Fig. 2 6G) and IL 10 levels were significantly lower (Fig. 2 6L), in NOD derived BM OCs cultures. These data indicate an exacerbated pro osteoclastic response to LPS by TRAP+ mononuclear NOD derived BM OCs. NOD derived Osteoclasts Respond Aberrantly to Inflammatory Mediators While pro inflammatory cytokines such as TNF activated T cells to indirectly stimulate osteoclast differentiation, they also directly activate osteoclasts to resorb bone (2, 86, 159) Thus in order to determine if the higher levels of these mediators in NOD derived BM OC cultures could be responsible for the abrogation of LPS induced deactivation of osteoclast function, BM OC cultures from all s trains were subjected to a cocktail of pro inflammatory cytokines and their resorptive function evaluated. As expected, increases in collagen release, cathepsin K and MMP 9 secretion were observed in BM OC cultures from all strains in both the presence of the pro inflammatory cytokine cocktail and in the absence of LPS (Fig. 2 7) when compared to those observed in the absence of the pro inflammatory cytokine

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64 cocktail (Fig. 2 4). In the presence of a pro inflammatory cytokine cocktail, NOD derived BM OCs d isplay significantly increased resorptive function when compared to C57BL/6 BALB/c and NOR cultures (Fig. 2 7), similar to responsiveness in the absence of the pro inflammatory cytokine cocktail (Fig. 2 4). In addition, the pro inflammatory cytokine cocktail did not affect abrogation of LPS induced deactivation in NOD derived BM OCs. On the other hand, C57BL/6 BALB/c and NOR derived cultures had significantly lower levels of collagen, cathepsin K and MMP 9 in the presence of LPS than in its absenc e even in the presence of a pro inflammatory cytokine cocktail (Fig. 2 7). These data suggest that the pro inflammatory milieu in NOD derived BM OCs is not solely responsible for the LPS induced hyper resorptive response observed. NOD derived BM OC Condit ioned Media Leads to Increased Bone Resorption in Control Cultures In order to determine if a soluble mediator was responsible for the abrogation of LPS induced deactivation of osteoclast function observed in the NOD BM OC cultures, conditioned media from LPS free NOD BM OC cultures was added to C57BL/6 BALB/c and NOR derived cultures. As a control, conditioned media from LPS free C57BL/6 BM OC cultures was added to BALB/c NOR and NOD derived cultures. Again, collagen release, cathepsin K and MMP 9 secretion were used to evaluate osteoclast function. As expected, conditioned media from NOD BM OC cultures caused an increase in resorption and osteoclastic enzyme production in C57BL/6 BALB/c and NOR derived cultures when compared to conditioned med ia from C57BL/6 BM OC cultures (Fig. 2 8). Importantly, conditioned media from NOD BM OC cultures were unable to inhibit LPS induced deactivation in C57BL/6 BALB/c and NOR derived

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65 cultures (Fig. 2 8). Similarly, conditioned media from C57BL/6 BM OC c ultures were unable to inhibit LPS induced deactivation in BALB/c and NOR derived cultures. C57BL/6 BM OC conditioned media did not affect the inhibition of LPS induced deactivation in NOD derived BM OC cultures (Fig. 2 8). Together these data suggest that inhibition of LPS induced deactivation in NOD derived BM OC cultures is most likely due to NOD os teoclast responsiveness rather than excess or absence of an LPS induced soluble mediator. NOD derived Osteoclasts are from Pre diabetic/euglycemic Mice Bone resorption can be influenced by many factors including glucose concentration (247, 249) Thus, in order to determine if hyperglycemia is contributing to the inhibition of LPS induced deactivation of NOD derived BM OCs, the stage of T1D progression and blood glucose was evaluated. All strains had normal blood glucose at time of bone marrow harvest (Fig. 2 9A). Similarl y, the histology of pancreata from NOD and NOR mice revealed little insulitis with approximately 75% of islets free of infiltration in the NOD model versus 90% in the NOR (Fig. 2 9B, C). Therefore, NOD mice were considered euglycemic and pre diabetic at t he time of marrow harvest, indicating that the events observed are not due to hyperglycemia or conditions associated with fulminant disease. Discussion Individuals with T1D have increased incidence of inflammatory arthritis as well as osteoporosis, two dis eases principally mediated by a dysregulation in bone remodeling (17, 72) Bone formation by osteoblasts is decreased in individuals with T1D which tips the balance of bone remodeling towards that of less bone deposition (72) Here we demonstrate an osteoclast specific contribution to altered bone remodeling, where T1D

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66 derived osteoclasts are more osteoclastic in nature than T1D free derived osteoclasts s uggesting an additional tip in the balance to that of increased bone resorption. While NOD BM OC resorptive capability was heightened in response to RANK L stimulation, NOD derived BM OCs were found to be smaller in size. In addition, NOD derived BM OCs cultures consistently contained a population of RANK + CTR lo cells. Together these data indicate an aberrant maturation process. The fusion of mononuclear cells into multinucleated and giant cells was decreased suggesting an alteration in cell fusion mecha nism(s). Many surface receptors including DC STAMP multinucleated cells. In addition, ADAM8 and ADAM12 which are disintegrins/metalloproteinases secreted prior to fusion, may be af fected in this model (117) Calcitonin, the ligand for CTR, is responsible for lowering calcium levels in the blood by inhibiting bone resorption by inducing morphological changes leading to osteoclast retraction (78) In addition, while stil l controversial, some studies have demonstrated an effect of calcitonin in the fusion of osteoclast precursors (78) Thus it is plausible that expression or function of one or many of the se molecules is altered in the NOD model leading to decreased fusion. RANK L stimulation of NOD derived osteoclasts resulted in increased cathepsin K and MMP 9 secretion leading to increased collagen degradation. This suggests a hyper responsiveness to R ANK L by NOD BM OCs during homeostatic conditions, whereby augmented signaling leads to increased resorptive enzyme production (8, 112) In support of this theory, it has been shown by others that peripheral blood derived osteoclasts from diabetic (both type 1 and type 2) individuals were less

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67 sensitive to the soluble decoy r eceptor for RANK L, osteoprotegrin [OPG] than those derived from diabetes free controls resulting in increased bone resorption, again indicating a heightened sensitivity in RANK signaling (8, 323) In addition, stratification of the resorption data indicates that TRAP+ mononuclear cells within the NOD derived OC cultures are responsible for the augment ed resorption. Bacterial components such as LPS act on osteoblasts and activated T cells to produce more RANK L to stimulate osteoclasts to differentiate and resorb bone (105, 278) However, LPS can also act on osteoclasts directly by inhibiting the differentiation of monocytic precursors into bone resorbing osteoclasts and bone resorption by mat ure osteoclasts (200, 202) In addition, it has recently been demonstrated that osteoclasts have the capacity to phagocytose bacteria and act as a supporting immune cel l (324) While T1D free derived osteoclasts resorb less bone in the presence of high amou nts of LPS, they do produce pro inflammatory cytokines and chemokines. This suggests a shunting of osteoclast precursors to that of an immune cell phenotype to help fight infection rather than towards mobilization to resorb bone. Interestingly, in additio n to being more resorptive, NOD derived osteoclasts were also more inflammatory in nature than the NOR, C57BL/6 or BALB/c models as indicative of increased soluble mediator secretion. Again, stratification of the data indicates that it is the TRAP+ mononu clear cells which have retained this inflammatory function. Macrophages and monocytes (relatives of the osteoclast) from individuals with T1D secrete elevated levels of pro inflammatory cytokines in response to LPS (20, 208) Here NOD derived osteoclasts also secreted elevated levels of the pro inflammatory and pro osteoclastic mediators, GM CSF, RANTES, IP 10, TNF

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68 stimulated with LPS compared to those observed in osteoclast control cultures. Where GM CSF can mobilize osteoclast precursor release from the bone marrow, RANTES and IP 10 can act as chemokines to attract these precursors to the area of inflammation. TNF ion and function (85, 101, 211) In addition, our data describe for the first time that NOD derived BM osteoclasts are refractory to LPS induced inhibition of bone resor ption. Thus one can envision that under a T1D environment, augmented secretion of these mediators can lead to a positive feedback loop where more osteoclasts are recruited to the site of inflammation where abrogation of LPS induced inhibition leads to ex acerbated bone resorption. We found that neither addition of a pro inflammatory cytokine cocktail nor addition of conditioned media from NOD BM OC cultures led to inhibition of LPS induced deactivation in our control cultures suggesting that soluble fact ors alone are not responsible for the aberrant LPS responsiveness of NOD BM OCs. Importantly, C57BL/6 BM OC conditioned media did not affect the inhibition of LPS induced deactivation in NOD derived BM OC cultures suggesting that there is not an absence o f a soluble mediator in NOD derived BM OC cultures responsible for LPS induced deactivation of osteoclasts. Therefore, we hypothesize that it is the response of the NOD BM OC to LPS and other mediators that is the cause of aberrant function rather than a n excess or absence of soluble mediator expression. For instance, h yper responsiveness in the TLR4 signaling pathway as indicated by increased translocation of NF mice (186, 187, 190) In addition, the receptors for TNF R] and RANK L

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69 [RANK], which originate from the same super family of receptors, also utilize the NF pathway and thus T1D assoc iated alteration in these signaling pathways could potentially alter the bone resorbing function of osteoclasts (325) These mechanisms are currently under investigation in our laboratory. In addition to the inflammatory environment, bone resorption can be influenced by many factors including glucose concentration. For instance, Graves and colleagues have elegantly demonstrated in ligature, calvarial and bone fracture models (in the presence and absence of infection) that hyperglycemia and TNF osteoblast apoptosis which contributes to in vivo cartilage and bone loss in models of type 2 diabetes and hyperglycemia (4, 240, 243, 260, 261, 265, 326 332) While our data demonstrates an osteoclast specific augmented function in the absen ce of hyperglycemic contributions, it is plausible that hyperglycemia may further contribute to osteoclast hyperactivity in T1D. Indeed, glucose is the primary energy source of the osteoclast and has been shown to augment osteoclast mediated resorption vi a increases in V ATPase expression (248, 333) Furthermore, lack of insulin, now considered to be a bone anabolic agent, leads to decreased bone formation in participants with T1D (72) While many contributing fact ors to the inflammatory bone loss in participants with T1D have been surmised, the totality of osteoclast mediated pathology was previously unknown. Dissecting osteoclast function and its role in T1D associated bone pathologies can lead to adjunct treatme nt for participants with T1D that cannot respond to conventional anti osteoclast therapies. In the present study, we have shown that a T1D genetic background leads to a hyper reactive osteoclast phenotype which

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70 responds to bacterial components and pro inf lammatory cytokines in an aberrant fashion resulting in excessive bone degradation via enhanced cathepsin K and MMP 9 secretion concomitant with an increased expression of pro osteoclastic soluble mediators.

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71 Figure 2 1. Flow cytometric analysis of os teoclast culture purity. BM OCs were stimulated with RANK L for 72hrs on UpCell plates. Cells were probed for RANK (PerCP Cy5.5) and calcitonin receptor [CTR] (AlexaFluor647) and acquired using a FACSCalibur flow cytometer. FCS Express software was used to determine forward and side scatter population of analyzed BM OCs (circled in upper panel) and percentage of pure RANK+CTR+ BM OCs (lower panel). Black arrow indicates novel population of RANK+ cells expressing low levels of CTR [RANK+CTR lo ] NOD BM OCs. Data shown as representative scatter plots of each mouse strain.

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72 Figure 2 2. NOD derived osteoclasts display altered differentiation A) 6d post differentiation, BM OCs were imaged with light microscopy at 40x magnificat ion. Representative images of I) mononuclear cells II) multinucleated OCs and III) giant OCs. B D) Number of TRAP+ BM OCs per coverslip were enumerated using 4 0x magnification. E G) Data shown as percentage of total nuclei TRAP+ cells C57BL/6 (black b ars, n=18), BALB/c (dark grey bars, n=16), NOR (light grey bars, n=16), NOD (white bars, n=16). H) 6d and 9d post differentiation, multinucleated and giant BM OCs NO D mice were quantified. I) 6d and 9d post differentiation cell viability of C57BL/6 and NO D BM OC cultures was assessed by MTT assay. p value < 0.05. One

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73 Figure 2 3. NOD derived osteoclasts have increased bone resorption capabilities in response to RANK L stimulation. BM OCs were stimulated with RANK L for 72hrs on bovine bone slices which were sputter coated w ith gold and imaged with SEM. A) Representative SEM and computer quantified areas of resorption (blue) at 40x magnification. Image J software was used to determine B D) area of reso rption and E G) number of resorption pits (light blue outlines = borders of r esorption areas) normalized to B, E)TRAP+ cells, C, F) TRAP+ mononuclear cells an d D, G)TRAP+ multinucleated cells. C57BL/6 (black bars, n=18), BALB/c (dark grey bars, n=16), NOR (light grey bars, n=16), NOD (white bars, n=16). p value < 0.05. One way ANOVA with

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74 Figure 2 3. Continued

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75 Figure 2 4. NOD derived osteoclasts degrade more type 1 collagen than controls via enhanced cathepsin K and MMP 9 secretion. BM OCs were stimulated with RANK L for 72hrs on bovine bone slices. Supernatants were collected after 1% Triton X 100 solubilization and ELISA used to quantify: A D, G) collagen I telopeptide, B, E, H) cathepsin K and C, F, I) MMP 9 normalized to A C) TRAP+ cells, D F) TRAP+ mononuclear cells and G I) TRAP+ multinucleated cells. C57BL/6 (black bars, n=18), BALB/c (dark grey bars, n=16), NOR (li ght grey bars, n=16), NOD (white bars, n=19). p value < 0.05. One way ANOVA

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76 Figure 2 5. NOD derived osteoclasts respond aberrantly to LPS. BM OCs were stimulated with RANK L in the presence or absence of E. coli LPS for 72hrs on bovine bone slices. Supernatants were collected after 1% Triton X 100 solubilization and bones were sputter coated w ith gold and imaged with SEM. A) Area of resorption, B) collagen I telopeptide, C) cathepsin K and D) MMP 9 were evaluat ed using A) SEM and B D) ELISA. Data are expressed as percent expression during RANK L stimulation calculated by [(value in the presence of LPS/value in the absence of LPS) x 100] C57BL/6 (black circles, n=18), BALB/c (dark grey circles, n=16), NOR (light grey circles, n=16), NOD (white circles, n=19). Dashed line = expression levels in the absence of LPS. p value < 0.05. NOD vs. all strains. One correction.

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77 Figure 2 6. NOD derived osteoclasts secrete increased soluble o steoclastogenic mediators in response to LPS. BM OCs were stimulated with RANK L in the presence (+) or absence ( ) of E. coli LPS for 72hrs on bovine bone slices. A, G) GM CSF, B, H) IL 1 C, I) TNF D J) RANTES, E, K) IP 10 and F, L) IL 10 levels w ere evaluated in the permeabilized supernatant s using Milliplex technology. A F) Data shown as pg/mL normaliz ed to TRAP+ mononuclear cells. G L) Data shown as pg/mL normalized to TRAP+ multinucleated cells. C57BL/6 (black bars, n=13), BALB/c (dark grey b ars, n=15), NOR (light grey bars, n=14), NOD (white bars, n=17). Open bars = absence of LPS, hatched bars = presence of LPS. p value < 0.05. One multiple correction.

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78 Figure 2 7. Pro inflammatory cytokines do not inhibit LPS deact ivation of OCs. BM OCs were stimulated with RANK L in the presence or absence of E. coli LPS and a pro inflammatory cytokine cocktail [TNF 6] for 72hrs on bovine bone slices. Supernatants were collected after 1% Triton X 100 solubilizati on and ELISA used to quantify: A) collagen I telopeptide B) cathepsin K and C) MMP 9. C57BL/6 (black bars, n=5), BALB/c (dark grey bars, n=5), NOR (light grey bars, n=6), NOD (white bars, n=5). Open bars = absence of LPS, hatched bars = presence of LPS. p value < 0.05. ^ p value indicates <0.05 NOD vs. all other experimental groups in the absence of LPS. One

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79 Figure 2 8. NOD derived soluble mediators do not inhibit LPS deactivation of OCs. BM OCs were stimulated with RANK L in the presence or absence of E. coli LPS and either NOD conditioned media [NOD c.s.] or C57BL/6 conditioned media [ C57BL/6 c.s.] for 72hrs on bovi ne bone slices. Supernatants were collected after 1% Triton X 100 solubilizati on and ELISA used to quantify: A) collagen I telopeptide B) cathepsin K and C) MMP 9. C57BL/6 (black bars, n=5), BALB/c (dark grey bars, n=5), NOR (light grey bars, n=6), NOD (white bars, n=5). Open bars = absence of LPS, hatched bars = presence of LPS. p value < 0.05 NOD c.s vs C57BL/6 c.s.; ^ p value < 0.05 no LPS vs LPS NOD c.s p value < 0.05 no LPS vs. LPS C57BL/6 c.s. One way ANOVA with

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80 F igure 2 9. NOD derived BM OCs are from pre diabetic/euglycemic mice A) Blood glucose was measured at time of sacrifice. C57BL/6 (black circles, n=10), BALB/c (dark grey circles, n=10), NOR (light grey circles, n= 19), NOD (white circles, n=23) B) Pancrea ta from NOR (n=5) and NOD (n=5) were fixed, embedded, sectioned, H&E stained and scored using light microscopy based on percentage of lymphocyte infiltration (insulitis). N o insulitis = black, peri insulitis = hat ched, intra insulitis = white. C) Represent ative sections of scoring tech nique. i) no insulitis ii) peri insulitis iii) intra insulitis.

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81 CHAPTER 3 EXACERBATED RESPONSE TO MBSA INDUCED INFLAMMATORY ARTHRITIS IN NOD MICE Introduction Inflammatory b one and joint destruction are frequent co morbidities of type 1 diabetes [T1D]. These pathologies may be caused by intrinsic genetic factors and/or from environmental triggers (24, 46) Inflammation resulting from autoimmune destruction of t he pancreas or hyperglycemia as a result of this autoimmunity may also lead to secondary joint pathologies (140, 311, 312) Bone remodeling is normally a tightly regulated process where cells such as osteoclasts and osteoblasts are responsible for bone resorption and format ion, respectively (68) Immune cells and soluble mediators secreted by these cells are important regulators of this process. Thus, aberrant immune cell activation and function have been implicated in many bone diseases, including inflammatory arthritis, where the balance of remodeling is tipped towards resorption (69, 288) Inflammatory arthritis is characterized by abnormal activation of bone resorbing osteoclasts by inflammatory mediators which are produced after an initial stimulus is sensed as foreign by the immune system. This activat ion leads to proliferation of synoviocytes and infiltration of immune cells into the joint with subsequent erosion of subchondral bone in advanced lesions (288, 298) Injection of methylated BSA [mBSA] induces an acute inflammatory arthritis which is transient in nature. Methylation of BSA causes a change in the molecule rendering it positively charged which binds the negatively charged cartilage and other avascular connective tissues in the joint. The retention of mBSA in the joint leads to immune complex deposition and inflammatory

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82 infiltration which creates an environment suitable for progressive cartilage destruction and ultimately bone erosion (334) Methylated BSA is efficiently processed and presented by antigen presenting cells compared to unmethylated BSA and can persist in the joint for up to 28 d ays thereby leading to a smoldering arthritis mimicking that seen in humans with inflammatory arthritis. The synovial lining containing synoviocytes also becomes enlarged and a pannus consisting of many cell types including neutrophils and mesenchymal cel ls then develops and eventually covers the femoral condyle (normally covered in cartilage). Cartilage destruction and subchondral bone erosion occur below this pannus with bone destroying osteoclasts at the front (335) It is important to note that this inflammatory arthritis is confined to the joint injected a nd does not progress to systemic inflammation (334) However, with systemic or local challenge with mBSA, a flare of arthritis can be induced again which is principally mediated by antigen specific T cells, mostly of the T H 17 lineage (334, 336) T H 17 cells are prolific producers of IL 17 which induces RANK L expression on other cell types to aid in the differentiation of osteoclast precursors into fully f unctional osteoclasts (69) This T helper type also expresses more RANK L than the other T helper subsets and thus can activate osteoclasts more eff iciently than other T cells (211) In both murine and human T1D, macrophages and dendritic cells have been shown to be hyperactive to stimulation resulting in excessive pro inflammatory c ytokine production (186, 190, 315) Osteoclasts arise from the same lineage as these innate immune cells (97) and have been shown to respo nd aberrantly to inflammatory stimuli in the NOD mouse. Pro inflammatory cytokines including TNF 6 have

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83 been known to stimulate differentiation and activation of osteoclasts with IL TNF ption, (91, 160) and these mediators are found in high abundance in arthritic joints (91, 159) While osteoblast maturation and function has been long known to be decreased in T1D (140, 239) we have recently shown that osteoclasts are also implicated in inflammation induced bone loss in the NOD mouse model. In addition, NOD mice are more susceptible to spontaneous and induced arthritis (317) Due to the hyper responsiveness of NOD derived osteoclasts to inflammatory stimuli and the susceptibility of this model to induced arthritis, we hypothesized that NOD mice would displ ay more severe inflammatory arthritis than controls due to increased osteoclast mediated bone resorption. Therefore in the present study, we induced an acute inflammatory arthritis in NOD mice and determined the extent of osteoclast function. Materials and Methods Mouse Models NOD/LtJ [NOD], NOR/LtJ [NOR], and C57BL/6 J [ C57BL/6 ] mice were maintained in a specific pathogen free environment at the breeding facilities of the University of Florida. The NOD mouse spontaneously develops an autoimmune mediated destruction of the insulin cells of the pancreas producing very similar pathology as seen in individuals with T1D (148) The NOR mouse model has a similar genetic background as the N OD mouse but does not develop insulitis and diabetes due to the dispersal of C57BL/6 genome within diabetes susceptibility loci (318) Thus the C57BL/6 strain serves as an additional genetic background control. All experimental procedures were conducted in accordance with the guidelines of the Univers ity of Florida Institutional Animal Care and Use Committee.

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84 Arthritis Induction Methylated BSA (Sigma) was resuspended in sterile phosphate buffered saline [PBS]. 20uL of mBSA (0pg, 300pg, 1ng) in PBS or PBS alone was injected intra articularly into the knee joints through the infrapatellar ligament with an insulin syringe (0.5cc with 28.5 gauge needle) (BD) Injections were repeated at 7 d Knee joint diameters were measured using a micrometer caliper (Ajax Scientific) to assess joint swelling at 0, 10 12, 14, and 16 d post first injection of mBSA. Knee joints were also lavaged with sterile PBS using an insulin syringe at time of sacrifice for collagen ELISAs. Collagen Telopeptide ELISA Collagen carboxy terminal telopeptides were detected using an ELISA according to manufacturer instructions (Immunodiagnostic Systems). Lavages were pre incubated with biotin conjugated anti telopeptide and horseradish peroxidase [HRP] conjugated anti telopeptide and added to an ELISA plate coated with streptavidin [ SAV]. Following five washes, tetramethylbenzidine [TMB] substrate was used to develop reactivity followed by quenching with H 2 SO 4 Colorimetric reactions were detected using an Epoch microplate spectrophotometer (Biotek) set at a dual wavelength reading of 450nm with a reference of 655nm. Gen5 Software (Biotek) and a standard curve were used to determine nM concentrations. Histological Analysis of Joints Knee capsules were fixed in 10% formalin (Fisher) and embedded in paraffin. 5m sections were mounted on glass slides and deparaffinized in xylenes and rehydrated using 100% ethanol (EtOH), 95% EtOH, 75% EtOH (Fisher), and dH 2 O. Sections were stained with Harris hematoxylin, incubated in a bluing solution [1.5%

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85 NH 4 OH (EMD Chemicals) in 70% EtOH] and stai ned with eosin [1% aqueous Eosin Y, 1% aqueous phloxyine B (Fisher), 100% EtOH and glacial acetic acid (Mallinckrodt Chemicals)]. Sections were dehydrated with 70% EtOH, 95% EtOH, 100% EtOH, and xylene (2 times), mounted with Permount (Fisher) and observe d under light microscopy. Results Intra articular Injection of mBSA Causes an Acute Inflammatory A rthritis in NOD, NOR and C57BL/6 M ice In our model, we observed synovial hyperplasia, extensive pannus formation, and erosion of subchondral bone with 1ng m BSA injections (Figure 3 1). PBS injections alone did not cause cartilage or bone destruction as evidenced by intact cartilage overlying the bone. The synovial lining does appear to be inflamed in the PBS treated joints; however, this may be due to the t rauma caused by intra articular injections. The disruption of the meniscus in PBS treated joints may also be an artifact of the sectioning process. While there does not appear to be a difference in the amount of infiltrate or subchondral bone erosion bet ween strains, this may be because of the plane that the joint was sectioned. Further studies would require multiple sections taken per joint to assure that the total infiltrate and joint destruction can be adequately visualized. NOD Mice do not have Incre ased Joint S welling After Induction of Arthritis Joint swelling is normally an acute process and peaks after 3 5 days post induction of the inflammatory arthritis. Swelling then begins to decline after day 7 but can be reactivated with boosters of antigen (334) We observed no large differences in joint swelling between strains or amo unt of mBSA injected (Figu re 3 2 ). It is interesting to note that the 300pg group had peak swelling earlier (day 10) than the 1ng group (day 12) which might indicate a more acute and transient arthritis. It may be that we did not

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86 capture the initial edema that is associated with the recruiting of immune cells to the area. Swelling should therefore be measured earlier, at days 3, 5, and 7 to capture the acute inflammation and edema from the initial insult. NOD Mice Display I ncreased B one D estruction via C ollagen D egradation in Arthritic J oints To measure bone destruction, we lavaged the injected joints at time of sacrifice and determined the amount of collagen degradation via ELISA for collagen I telopeptide concentration. As expected, collagen degradation increased with mB SA injections versus PBS alone (Figure 3 3 ). C57BL/6 and NOR behaved similarly in both the 300pg and 1ng groups with increased degradation at 1ng compared to 300pg suggesting that higher mBSA concentrations lead to increased joint damage. C57BL/6 showed the most pronounced increase with saline versus mBSA with increased collagen breakdown, especially in the 1ng group. NOD displayed more collagen degradation compared to C57BL/6 and NOR in both 300pg and 1ng mBSA treatments. These data suggest that NOD mi ce are more susceptible to mBSA induced arthritis and display increased bone destruction via collagen breakdown compared to controls. Moreover, these findings support our in vitro bone marrow derived osteoclast data where NOD derived osteoclasts degrade m ore bone than controls with inflammatory stimuli. Discussion Individuals with T1D have increased incidence of inflammatory arthritis as well as osteoporosis, two diseases principally mediated by a dysregulation in bone remodeling (17, 72) Bone formation by osteoblasts is notably d ecreased in individuals with T1D which tips the balance of bone remodeling towards that of less bone deposition (72) We previously demonstrated an osteoclast specific contribution to altered bone

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87 remodeling, where T1D derived osteoclasts are more responsive to RANK L and inflammatory stimuli than T1D free derived osteoclasts suggesting an additional tip in the balance to that of increased bone resorption. Here we show that the NOD mouse model displays increased collagen degradation by osteoclasts in an arthritic joint compared to NOR and C57BL/6 two T1D free mouse models. Our previous study showed that T1D derived os teoclasts secrete increased levels of cathepsin K and MMP 9, two enzymes necessary for collagen degradation which are directly affected by RANK signaling (8, 112) This heightened function was observed under baseline stimulation with RANK L and was further augmented in response to a pro inflammatory cytokine cocktail. The pr o inflammatory environment in the joints treated with mBSA can serve to activate osteoclasts to resorb bone. Since it has already been shown that NOD derived macrophages and monocytes secrete elevated levels of pro inflammatory cytokines in response to in flammatory stimuli (190 192) it stands to reason that the osteoclasts in this inflammatory environment where monocytes and macrophages are in high abu ndance would be activated even further, which indeed is the case in our mBSA induced arthritis model. NOD mice show increased collagen degradation in the inflammatory environment caused by mBSA intra articular injections which suggests more bone destructi on by osteoclasts. Inflammatory infiltrates in mBSA induced arthritis include neutrophils, eosinophils, and mononuclear leukocytes (monocytes and macrophages), all of which secrete pro inflammatory cytokines and chemokines which serve to recruit other im mune cells but also can activate osteoclasts (335) T H 17 cells, the main T cell subset in this arthritis model, also produce RANK L which increases differentiation of osteoclast precursors

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88 into fully functional osteoclasts (69) Increased expression of RANK L by T H 17 cells can also serve to over activate osteoclasts more efficiently than other T cells (211) The hyper reactive NOD derived osteoclast, which is already known to respond aberrantly to RANK L stimulation with further augmented function in response to inflammatory stimuli, is therefore more likely to be over activated by the pro inflammatory environment in the arthritic joint and destroy increased amounts of bone compared to T1D free hosts. Further studies are needed to determine statistical significance in our models along with extensive characterization of the location, amount, and size of osteoclasts in the arthritic joint. D elineating the causative cell type in inflammatory arthritis seen more often in participants with T1D can therefore lead to more targeted therapies to counteract the excessive joint destruction.

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89 Figure 3 1. Intra articular injection of mBSA causes an acute inflammatory arthritis in NOD, NOR, and C57BL/6 mice. Knee capsules from C57BL/6 (n=2), NOR (n=2) and NOD (n=2) were fixed, embedded, sectioned, H&E stained and imaged using light microscopy. Arrows indica te bone erosion. Representative sections of joints, shown imaged at 5x magnification.

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90 Figure 3 2. NOD mice do not have increased joint swelling after induction of arthritis. Knee diameters were measured by micrometer caliper at day 0, 10, 12, 14, an d 16 post injection of mBSA or PBS. C57BL/6 (circles, n=2), NOR (squares, n=2), and NOD (diamonds, n=2). Arrows indicate mBSA injections. Red = 300pg, blue = 1ng, black = 0pg mBSA. Data shown as knee joint thickness in mm.

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91 Figure 3 3. NOD mice display increased bone destruction via collagen degradation in arthritic joints Joint lavages were collected with PBS and ELISA was used to quantify collagen I telopeptide. Data shown in nM concentration in lavages. C57BL/6 (circles, n=2), NOR (squares, n=2), and NOD (diamonds, n=2). (Left) 300pg mBSA group (Right) 1ng mBSA group.

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92 CHAPTER 4 HYPERGLYCEMIA INDUCE D AND INTRINSIC ALTE RATIONS IN TYPE 2 DI ABETES DERIVED OSTEOCLAST F UNCTION Introduction Periodontitis is a cum ulative inflammatory condition, initiated by bacteria but hard tissues of the periodontium and, if left untreated, can lead to tooth loss (276) Susceptibility to periodontitis is enhanced by the interaction between acquired, environmental and genetic factors which modify the host response toward the subgingival biofilm (337) Many systemic diseases including type 2 diabetes [T2D] modify the progression of periodontal disease contributing to increased prevalence, incidence and/or seve rity of disease (173, 277, 282, 316, 338) T2D, previously referred to as non insulin dependent diabetes or adult onset diabetes, accounts for 90 95% of diabetes cases, where the development of insulin resistance in target tissues leads to hyperglycemia and if left u nchecked can result in the loss of insulin secretion by the islets of Langerhans in the pancreas (156) Just as T2D is a major ris k factor for periodontitis, periodontitis is now classified as the sixth complication of diabetes where the level of glycemic control is an important determinant in the relationship (175, 339, 340) The periodontium is comprised of gingiva, periodontal ligament, and alveolar bone w hich work together to anchor the teeth where the alveolar bone lines the tooth socket and serves as an attachment site of the periodontal ligament fibers (173) Increased alveolar bone loss is observed in periodontitis participants wit h T2D when compared to diabetes free individuals (Nelson Shlossman 1990). This progressive periodontal bone loss in T2D is related to changes that alter the balance between resorption and

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93 deposition phases of bone metabolism (90, 281) Traditional bone cells including osteoclasts [OC] and os teoblasts [OB] are responsible for bone resorption and formation, respectively, although immune cells have also been implicated in their regulation (68) While changes in impaired osseous healing, OB proliferation and function as well as collagen function and deposition have been described in T2D associated periodontitis, the effect of T2D on OC diff erentiation and function has not been reported (341) OC differentiation is regulated by macrophage colony stimulating factor [M CSF] and the receptor activator of nuclear factor kappa B ligand [RANK L]. Resorption of bone is initiated by binding of the OCs to the mineralized bone surface via alpha v beta (313) Vesicles containing osteoclastic enzymes such as tartrate resistant acid phos phatase [TRAP], the serine protease cathepsin K, and matrix metalloproteinase 9 [MMP 9] induce collagen degradation after bone demineralization through acidification of the lacunae by the vacuolar H+ ATPase (126, 133) Activation of OC mediated bone resorption can be augmented by infection and inflammatio n, as well as hormonal alterations (69) Lipopolysaccharide [LPS], a cell wall component of gram negative bacteria, has been found to be highly immu nogenic and induces the production of pro inflammatory cytokines by various immune cells. OCs and their precursors, which share the same lineage as macrophages and dendritic cells, express many innate immune receptors including toll like receptors [TLRs] and thus can respond to bacterial components (71, 199, 314) In a co culture of osteoclasts and osteoblasts, LPS, the ligand for TLR4, augments bone resorption (199) However,

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94 when supporting cells such as osteoblasts or other immune cells are absent, LPS inhibits bone resorption in OC pure cultures, although the exact mechanism(s) is not known (200) In addition, pro inflammatory cytokines including TNF 6 stimulate differentiation and activa tion of OCs with IL involved in activating resorption (91, 160) Importantl y, inflammatory cytokines and bacterial components are found in abundance within the periodontal lesion where they can directly affect OC differentiation and function thereby shaping the bone remodeling process. T2D leads to alterations of the function of innate immune cells such as monocytes and macrophages resulting in the production of increased levels of pro inflammatory cytokines, namely IL (20, 21, 149, 316, 342, 343) In addition, T2D associated neutrophil defects lead to decreased clearance of microbes, further amplifying the inflammatory process (22, 150) (207) Thus, since a similar heightened inflammatory response is evident in the periodontium of T2D individuals which is exacerbated in poorly controlled diabetics (60) it is entirely possible that these individuals would be more susceptible to severe alveolar bone loss than T2D f ree individuals given the effects of these soluble mediators on OC activation and function. Similarly, given the phylogenetic relationship of OCs to macrophages and dendritic cells and the aberrant TLR responsiveness of these cell types in T2D, we postula te that T2D derived OCs have heightened sensitivity to stimulation resulting in augmented differentiation and activation. Therefore in the present study, we characterized T2D associated OC specific differentiation, activation and function in the presence a nd absence of inflammatory stimuli utilizing both a murine

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95 model of T2D, namely the db/db mouse model, and human primary osteoclasts derived from T2D individuals. Materials and Methods Participant Population The Institutional Review Board [IRB] for protection of human subjects at the University of Florida approved this protocol in accordance with the World Medical Association Declaration of Helsinki. All data and samples were obtained under informed consent. Participants were recruited from the University of Florida, College of Dentistry from January 2009 until January 2012. Inclusion criteria: aged 13 to 75 years old, diagnosed with type 2 diabetes [T2D] without diabetic ketoacidosis, currently under the ca re of a physician or diabetes free. Exclusion criteria: diabetic complications that would affect the safety or compliance or that could influence the course of either periodontal disease or diabetes care and glucose control; received immunosuppressive, ant ibiotic, glucocorticoid, or bisphophonate therapy over the last 6 months; and current tobacco use. A single venous blood sample of 30mL was collected from all participants, where non fasting blood glucose levels and glycated hemoglobin [HbA1c] were measur ed with the Ascensia Contour blood glucose meter (Bayer, Tarry Town, NY) and A1CNow meter (Bayer), respectively. Peripheral Mononuclear Cell [PBMC] Isolation, Osteoclast [OC] Differentiation and A ctivation Whole blood samples were divided equally and diluted 1:1 with balanced working salt solution [1 volume salt solution A:9 volumes salt solution B]. Salt solution A: 1g/L anhydrous D glucose, 0.0074g/L CaCl 2 0.1992g/L MgCl 2 0.4026g/L KCl and 17.565g/L Tris in dH 2 O. Salt solution B: 8.19g/L NaCl in dH 2 O. Diluted blood was overlaid onto

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96 15mL of Ficoll Paque (GE Healthcare Life Sciences, Waukesha, WI) and centrifuged for 40 min at 1200rpm. The interface layer containing PBMCs was removed and washed 3 times with 6mL of working salt solution. The resu lting PBMC cell pellet was MEM complete media (Sigma MEM with 10% fetal bovine serum (Mediatech Inc., Manassas, VA), 1% L glutamine (Thermo Fisher Sc ientific Inc., Waltham, MA), and 1% penicillin/streptomycin/amphotericin B (Thermo Fisher Scientific Inc.)] supplemented with 10ng/mL recombinant human M CSF [rhM CSF] (Peprotech, Rocky Hill, NJ)] at 37 C and 5% CO 2 for 14 d with media refreshed every 3 d. Non adherent cells were removed and 5.9x10 5 cells/mL of adherent cells were seeded in 24 well plates on either glass coverslips (Thermo Fisher Scientific Inc.) or 1 cm 2 bovine bone slices cut with an Isomet Low Speed Saw (Buehler, Lake Bluff, IL). All c ultures were supplemented with 10ng/mL rhM CSF and 50ng/mL recombinant human soluble RANK L [rhsRANK L] (Peprotech) and allowed to culture for 12 d with complete media refreshed every 3 d. After 12 MEM compl ete media supplemented with 10ng/mL rhM CSF and 50ng/mL rhsRANK L. Cells were allowed to resorb bone for 72 h in the presence or absence of 1ug/mL Escherichia coli LPS [LPS] (Sigma Aldrich). Cultures were permeablized with 1% Triton X 100 for 10 min. Sup ernatants were stored at 80 C until cathepsin K ELISA, collagen type I telopeptide ELISA, and cyto/chemokine multi plex analysis were performed. All outcome measures were normalized to total number of TRAP+ cells. Mouse Models All experimental procedures were conducted in accordance with the guidelines of the University of Florida Institutional Animal Care and Use Committee [IACUC] where

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97 measures were taken to minimize pain or discomfort. B6.BKS(D) Lepr db /J [db/db] and C57BL/6 J [B6] mice were maintained i n a specific pathogen free environment at the breeding facilities of the University of Florida. Non fasting blood glucose levels and glycated hemoglobin [HbA1c] were measured with the Ascensia Contour blood glucose meter and A1CNow meter, respectively, at the time of sacrifice. Bone Marrow Osteoclast [BMOC] Differentiation and Activation Bone marrow was harvested from 10 12 wk old db/db and B6 mice. Femora and MEM complete media (Sigma MEM with 10% fetal bovine serum (Mediatech Inc., 1% L glutamine (Thermo Fisher Scientific Inc.), and 1% penicillin/streptomycin/amphotericin B (Thermo Fisher Scientific Inc.)]. Cells were seeded in T75 flasks at a concentration of 1.5x10 6 cells /mL supplemented with 5ng/mL recombinant murine M CSF [rmM CSF] (Peprotech) and allowed to culture for 24 h at 37 C and 5% CO 2 Non adherent cells were removed and 5.9x10 5 cells/mL of adherent cells were seeded in 24 well plates on either glass coverslips (Thermo Fisher Scientific) or 1 cm 2 bovine bone slices cut with an Isomet Low Speed Saw (Buehler). All cultures were supplemented with 10ng/mL rmM CSF and 50ng/mL recombinant murine soluble RANK L [rmsRANK L] (Peprotech) and allowed to culture for 6 d wit h complete media refreshed every 3 d. After 6 d of MEM complete media supplemented with 10ng/mL rmM CSF and 50ng/mL rmsRANK L. Cells were allowed to resorb bone for 72 h in the presence or absence of 1ug/mL Esch erichia coli LPS (Sigma Aldrich). Cultures were permeablized with 1% Triton X 100 for 10mins. Supernatants were stored at 8 0 C until cathepsin K ELISA, collagen type I telopeptide ELISA, and cyto/chemokine

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98 multi plex analysis were performed. All outcome measures were normalized to total number of TRAP+ cells. TRAP Staining After differentiation, human and murine OCs [hOCs and mOCs, respectively] plated on glass coverslips were fixed with 2% paraformaldehyde/PBS (Thermo Fisher Scientific Inc.). C ells were washed with PBS and permeablized in 0.5% Triton X 100/PBS (Thermo Fisher Scientific Inc.). Cells were washed and probed for leukocyte acid phosphatase [TRAP] [1:1:1:2:4 Fast Garnet GBC Base Solution:Sodium Nitrite Solution:Napthol AS BI Phosphat e Solution:Tartrate Solution:Acetate Solution] (Sigma Aldrich) after which cells were washed and mounted on glass slides with MOWIOL 4 88 solution (Calbiochem, San Diego, CA). TRAP positive cells [purple in color] were counted according to number of nucle i present: mononuclear cells [1 nucleus], multinucleated osteoclasts [2 10 nuclei], and giant osteoclasts [11+ nuclei] using light microscopy at 40x magnification. Whole coverslips were counted for each cell type. Collagen Telopeptide ELISA Collagen carboxy terminal telopeptides were detected using an ELISA according to manufacturer instructions (Immunodiagnostic Systems, Scottsdale, AZ). Supernatants were pre incubated with biotin conjugated anti telopeptide and horseradish peroxidase [HRP] conjugat ed anti telopeptide and added to an ELISA plate coated with streptavidin [SAV]. Following five washes, tetramethylbenzidine [TMB] substrate was used to develop reactivity followed by quenching with H 2 SO 4 Colorimetric reactions were detected using an Epo ch microplate spectrophotometer (Biotek, Broadview, IL) set at a dual wavelength reading of 450nm with a reference of

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99 655nm. Gen5 Software (Biotek) and a standard curve were used to determine nM concentrations. Cathepsin K ELISA Active cathepsin K was det ected using an ELISA according to manufacturer instructions (Alpco Diagnostics, Salem, NH). Supernatants pre incubated with HRP conjugated anti cathepsin K were added to an ELISA plate pre coated with polyclonal sheep anti cathepsin K. Following five was hes, TMB substrate was used to develop reactivity followed by quenching with STOP solution. Colorimetric reactions were detected using an Epoch microplate spectrophotometer (Biotek) set at a dual wavelength reading of 450nm with a reference of 655nm. Ge n5 Software (Biotek) and a standard curve were used to determine pM/L concentrations of active cathespin K. Soluble Mediator Analysis Cytokines and chemokines from resorption supernatants were detected and quantified using a mouse 22 cyto/chemokine multipl ex or a human 14 cyto/chemokine Supernatant and antibody coated beads were allowed to incubate overnight at 4 C in a 96 well primed plate. Following two washes, reactivity was probed with biotinylated detection antibodies and SAV phycoerythrin [PE]. All incubations occurred while gently shaking in the dark. Following two washes, beads were resuspended in sheath fluid and reactivity acquired using a Luminex 200 IS system w ith Xponent software (Millipore). Milliplex analyst software (Viagene, Beverly Hills, CA), 5 parameter logistics and a standard curve were used to determine pg/ml concentrations.

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100 Statistical Analysis One re used to analyze and determine statistical significance (p<0.05). Results Hyperglycemia Enhances Differentiation of T2D derived OCs Db/db mice on the C57BL/6 genetic background provide an animal model of obese, insulin resistant type 2 diabetes (344, 345) whereby plasma insulin begins to rise around 10 to 14 days of age resulting in obvious obesity around 3 to 4 weeks after which hyperglycemia sets in around four to eight weeks of age. Osteoclasts [mOCs] were derived from the bone marrow of 10 12 week old db/db and C57BL/6 mice and the differentiation potential evaluated. TRAP staining was used to determine the number of multinucleated osteoclasts defined as having 2 10 nuclei and giant cells containing > 11 nuclei (Fig. 4 2A). As expected, at the time of bone marrow harvest, db/db mice had significantly higher non fasting glucose and %HbA1c compared to C57BL/6 control mice (Fig. 4 1A, B). Here following long term and severe hyperglycemia, a signi ficantly higher number of multi nucleated and giant mOCs in db/db derived cultures were observed compared to C57BL/6 controls (Fig. 4 2C, D). In order to determine if similar phenomenon occurred in human disease, oste oclasts [hOCs] were derived from peripheral blood monocytes of individuals with and without T2D (Fig. 4 2D). Although the T2D cohort presented with a statistically higher non fasting blood glucose and %HbA1c, when compared to the diabetes free cohort (Figu re 4 1C, D), the levels of non fasting glucose and %HbA1c present within the T2D cohort indicate that the cohort was under glycemic control as described by the American Diabetes Association (346, 347) Here in the absence of long term

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101 hypergly cemia, similar numbers of multi nucleated and giant hOCs were observed in T2D derived cultures compared to those derived from diabetes free individuals (Fig 4 2E, F). Enhanced Differentiation Results in Exacerbated Bone Resorption Although the differentiation data suggests that hyperglycemia and n ot the metabolic disease of T2D results in enhanced differentiation with an overall larger osteoclast size, this data does not address the bone resorbing function of these cells. In order to determine the bone resorbing capabilities of T2D derived osteocl asts, mOCs and hOCs were seeded onto bovine bone slices and stimulated with RANK L to promote bone resorption. During bone resorption, after demineralization by the H+ ATPase, serine proteases such as cathespin K in the resorption lacunae degrade the organ ic portion of bone containing type I collagen, where vesicles containing collagen telopeptides are transcytosed out of the osteoclast and released into the extracellular milieu (322) Therefore, in order to quantify bone resorption, levels of intra and extra cellular cathepsin K and collagen telopeptides were evaluated (Fig. 4 3). While mOCs derived from db/db mice produced signif icantly more cathepsin K and resorbed significantly more bone than control mice (Fig. 4 3A, B), no significant difference in cathepsin K expression nor bone resorption was observed in hOC derived cultures (Fig. 4 3E, F). Due to the increased numbers of mu ltinucleated and giant mOCs in db/db cultures, it would be expected for these cultures to have both increased cathepsin K and bone resorption capabilities. Thus cathepsin K expression and collagen degradation was normalized on a per cell basis where the bone resorption was now similar between db/db and C57BL/6 mOCs (Fig. 4 3C, D), suggesting that the increased resorption observed in mOC cultures was simply du e to increased numbers of multi nucleated and

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102 giant mOCs in the culture (Fig. 4 2), rather than enh anced function due to the metabolic disease. T2D derived Osteoc lasts Respond Aberrantly to LPS The inflammatory pathology of periodontal diseases includes a large bacterial burden, where activation of bone resorption can be induced by bacterial components Bacterial components such as LPS act on osteoblasts and activated T cells to produce more RANK L to stimulate osteoclasts to differentiate and resorb bone (105, 278) However, LPS can also act on osteoclasts directly by inhibiting resorption by mature osteoclasts (200, 202) Thus the affect of LPS on T2D derived osteoclast bone resorbing capacity was evaluated, whereby mOCs and hOCs were seeded onto bovine bone slices and stimulated to resorb bone with RANK L in the presence of LPS. LPS induced deactivation of OC function was observed in C57BL/6 mOCs and diabetes free hOC derived cultures indicated by a decrease in the cathepsin K secretion and collagen degradation (Fig. 4 3). However, this LPS induced deactivation did not occur in the db/db derived mOCs cu ltures nor the T2D derived hOC cultures (Fig. 4 3). Interestingly, while db/db derived mOCs also displayed an increase in collagen degradation and cathepsin K secretion in the presence of LPS compared to RANK L stimulation alone (Fig. 4 3C, D), T2D derive d hOCs did not (Fig. 4 3E, F). Elevated Pro Osteoclastic Milieu in T2D derived OC Cultures is Augmented by LPS Because osteoclast function is regulated by many soluble immune mediators, the cytokine and chemokine profile within mOC and hOC cultures were evaluated. Following RANK L stimulation, db/db mOC derived cultures had elevated levels of the chemokines IL 6, IP 10, MIP osteoclastic mediator IL

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103 10 when compared to C57BL/6 mOC derived cultures (Fig. 4 4). Similarly, T2D hOC derived cultures had elevated levels of IL 6, MIP 10 nor the anti osteoclastic mediator IL 10 when compared to those derived from the diabetes free cohort (Fig. 4 5). LPS stimulation of OC cultures resulted in an upregulation of all cyto/chemokines measured in all mOC and hOC cultures. Significantly higher levels of MCP 1, IP 10 and MIP derived mOC and hOC cultures (Fig. 4 4, 4 5), while hOC cultures also had significantly higher levels of IL 6 and TNF 4 5). Inter e stingly, both T2D derived mOC and hOC cultures presented with a significantly lower amount of the anti osteoclastic cytokine IL 10 (Fig. 4 4C, 4 5C). Discussion Individuals with T2D have a greater risk of developing periodontal disease where the degree of hyperglycemia is a confounding factor (175, 339, 340) Specifically, gingival inflammation as well as soft and hard periodontal tissue destruction are more severe in individuals with T2D, where the risk of progressive alveolar bone loss increases over time al though the exact mechanism is unclear (12, 28, 174, 282, 316, 338) Importantly, other bone metabolism pathologies are prevalent in individuals with T2D, including osteopenia and delayed fracture healing (239) Previous studies have demonstrated both defective bone fo rmation and osteoblast maturation under hyperglycemic conditions in murine and rat models of T2D. Specifically, Graves and colleagues have demonstrated increased osteoblast apoptosis and less osteoid formation in the Goto Kakizaki rat model of periodontal disease in T2D (12) This result was also observed by Liu and colleagues in the Zucker diabetic fatty rat, where by bone formation did not occur after resolution of inflammation as was

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104 observed in T2D free controls (4) Hyperglycemia also affects osteoblast differentiation and mineralization capabilities whereby the production of osteocalcin and calcium deposition are known to be decreased in the presence of high blood glucose le vels (239) thus s hunting mesenchymal stem cells [MSCs] which serve as precursors for osteoblasts, towards the adipocyte lineage leading to adipose tissue formation in bone marrow (274) Conversely, the role of osteoclasts in T2D associated bone loss are less defined, although literature suggests that hyperglycemia may lead to augmented OC function (4, 7, 8, 13, 223, 253) For instance, bone mineral density was found to be decreased in individuals with uncontrolled T2D compared to T2D free controls, whereby urinary levels of collagen degradation products from bone resorption such as CT x, DPD, and NTx as well as serum TRAP levels were found to be significantly elevated (7) In addition, augmented osteoclastogenesis and resorptive capabilities from peripheral blood monon uclear cells were found in a subset of individuals with diabetes suffering from and bone degradation in the foot (8) To date, the li terature describing the direct e ffect of hyperglycemia on osteoclast differentiation and function vary with some models suggesting high glucose leads to increased function (247, 248, 333) while others suggest decreased resorptive capabilities under hyperglycemic conditions (249) Here we report that in a murine model of T2D, recent long term hyper glycemia enhances the differentiation/fusion of osteoclast precursors resulting in more resorptive cultures. On the other hand, human osteoclasts from participants with well controlled T2D, differentiated similarly to those

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105 derived from diabetes free part icipants. Cell surface receptors such as DC STAMP are critical for fusion from mononuclear precursors into multinucleated osteoclasts. Thus, it is plausible that the expression of these fusion receptors is altered by hyperglycemia allowing for more effic ient fusion (117) Mechanisms associated with hypergly cemia induced augmented differentiation are currently being investigated in our laboratory. While initially our data also suggested a hyper responsiveness of db/db derived osteoclasts to RANK L, when resorption data was normalized to cell number, the obse rved increased resorption could only be attributed to a significant increase in larger osteoclasts. Interestingly, both human and murine osteoclasts derived from T2D sources displayed altered responses to LPS where deactivation did not occur as was observ ed in T2D free cultures. This alteration in function appears to be independent of hyperglycemia considering that hOCs derived from well controlled hyperglycemic environments also displayed this phenotype. Aberrant LPS responsiveness in innate immune cell s such as monocytes and macrophages, relatives of osteoclasts, have already been described in both type 1 and type 2 diabetes independent of hyperglycemia, resulting in augmented pro inflammatory cytokine secretion (41) In conjunction with a lack of inactivation, osteoclasts derived from T2D individuals and db/db mice displayed LPS induced augm ented pro inflammatory cytokine and chemokine secretion compared to T2D free controls. Here chemokines such as MCP 1, IP 10, and MIP (85, 101, 211) were elevated. Similarly, the cytokines IL 6 and TNF activators and can aid in osteoclastogenesis (85, 101, 21 1) were also elevated. On the other hand, the anti osteoclastic cytokine IL 10 (85, 101, 211) was significantly lower in

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106 T2D derived cultures, suggesting a lack of re gulatory function in T2D derived osteoclasts. Indeed, others have demonstrated that peripheral blood derived osteoclasts from T2D individuals are less sensitive to the soluble decoy receptor for RANK L, osteoprotegrin [OPG] than those derived from diabet es free controls again resulting in increased bone resorption (8, 323) Data presented here de scribe environmental and intrinsic mechanisms associated with the increased alveolar bone loss observed in periodontal participants with T2D. Specifically, hyperglycemia augmented osteoclast differentiation/fusion resulting in a more resorptive environment In addition, independent of hyperglycemia, T2D derived osteoclasts were less responsive to regulation whereby they were refractive to LPS induced deactivation and secreted lower anti osteoclastic soluble mediators. Finally, T2D derived osteoclast cultu res were more osteoclastic in response to LPS perpetuating the uncontrolled bone resorption. Thus, one can imagine that the presence of LPS during infections such as periodontal disease would lead to a vicious cycle of uncontrolled osteoclast differentiati on and activation that would lead to excessive alveolar bone loss in periodontal participants with T2D.

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107 Figure 4 1. Glycemic indices of murine and h uman cohorts. A, C) Blood glucose and B, D) HbA1c were measured at time of A, B) bone marrow harvest or C, D) peripheral blood collection. Red line is indicative of the high limit of normal indices. Non fasting blood glucose = 250mg/dL; glycated hemoglobin (HbA1c) = 7.5% *p value < 0.05. Unpaired t diabetes free participan ts, T2D = participants with type 2 diabetes.

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108 Figure 4 2. Db/db bone marrow derives increased numbers of larger osteoclasts. A C) 6d post differentiation, bone marrow derived mOCs were imaged with light mi croscopy at 10x magnification. A) Representativ e images of TRAP positive [purple in color] i) mononuclear osteoclasts, ii) m ultinucleated osteoclasts and iii) giant osteoclasts. Nu mber of B) multinucleated and C) giant mOCs per coverslip were enume rated using 40x magnification. D F) 12d post different iation, peripheral blood derived hOCs were imaged with light microscopy at i) 20x and ii) 40x magnification. TRAP positive [purple in color] E) multinucleated and F) giant hOCs were enumerated using 40x magnification. p value < 0.05. Unpaired t test with diabetes free participants, T2D = participants with type 2 diabetes.

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109 Figure 4 3. Type 2 diabetes derived osteoclasts are less responsiv e to LPS induced deactivation. A D) mOCs and E, F) hOCs were stimulated with RANK L in the presence and absence of E. coli LPS for 72hrs on bovine bone slices. Supernatants collected after 1% Triton X 100 sol ubilization were evaluated for A, C, E) cathepsin K and B, D, F) collagen I telopeptide by E LISA. Data shown as pM and nM respectively. C, D) mOC collagen and cathesin K expression normalized to number of multinucleated cells. Normalized outcome measure = [raw value x cell number corrective ratio]. Cell number corrective ratio = [lowest average c ell number within sample set] / [average

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110 cell number for group of interest]. *p value < 0.05. One way ANOVA with free participants, T2D = participants with type 2 diabetes.

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111 Figure 4 4. LPS induced elevation of pro inflammatory and pro osteoclastic soluble mediators in db/db osteoclast cultures. mOCs were stimulated with RANK L in the presence or absence of E. coli LPS for 72hrs on bovine bone slices. A) IL 6, B) MCP 1, C) IL 10, D) IP 10, E) M IP and F) TNF evaluated in the supernatants and using Milliplex technology. Data shown as pg/mL. *p value < 0.05. One comparison correction.

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112 Figure 4 5. LPS induced elevatio n of pro inflammatory and pro osteoclastic soluble mediators in human type 2 diabetes osteoclast cultures. hOCs were stimulated with RANK L in the presence or absence of E. coli LPS for 72hrs on bovine bone slices. A) IL 6, B) MCP 1, C) IL 10, D) IP 10, E ) MIP F) TNF levels were evaluated in the supernatants and using Milliplex technology. Data shown as pg/mL. *p value < 0.05. One way ANOVA with

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11 3 CHAPTER 5 DISCUSSION D ifferentiation Type 1 Diabetes Derived Osteoclasts Type 1 diabetes derived osteoclasts from the NOD mouse model show altered differentiation where fusion is decreased leading to fewer multinucleated and giant cells than the diabetes free NOR, C57BL/6 and BALB/c strains. This altered differentiation is also seen in NOD derived cells from the same myeloid lineage as osteoclasts, namely macrophages and DCs. Macrophages derived from NOD mice are more immature in phenotype with lower MHC class I expression than their diabetes free counter parts yet have increased response to inflammatory stimuli with pro inflammatory cytokine secretion (190 192) DCs from NOD mice also display lower lev els of differentiation markers such as MHC class II and co stimulatory molecules (184, 185) This findin g is recapitulated in humans with T1D where DCs have lowered co stimulatory molecule B7.1/2 expression and have impaired T cell activation ability (182) It is interes ting that while these cells do appear less mature in the NOD mouse, there are more circulating precursors for macrophages and DCs and NOD marrow can derive more of these cell types than other strains (348) However, there is a preference for NOD marrow to shunt these precursors to that of the macrophage lineage (349) which may explain why more NOD osteoclasts resemble mononuclear macrophages than multinucleated osteoclasts in their ability to secrete high levels of pro inflammatory c ytokines. It should also be mentioned that mononuclear cells can have resorptive function (350) which may explain why even with fewer multinucleated ce lls, T1D derived mononuclear osteoclasts can still resorb more than diabetes free cells.

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114 Type 2 Diabetes Derived Osteoclasts Type 2 diabetes derived osteoclasts from the db/db mouse model show enhancement of differentiation contrary to what was seen in T1D derived osteoclasts from the NOD mouse. The db/db mouse derives more multinucleated and giant osteoclasts and fewer mononuc leated precursors compared to the diabetes free C57BL/6 mouse suggesting augmented fusion capability. This phenomenon may be caused by increased pro osteoclastic cytokine release from db/db derived osteoclasts with RANK L stimulation which can lead to IL 1 and TNF enhancers of osteoclastogenesis (101) Human derived osteoclasts from participants with T2D, however, do not show this augmented differentiation. In fact, T2D participants derive similar numbers of multinucleated and giant osteoclasts to a diabetes free cohort. It is important to note that the glycemic state of the human T2D subjects was considered well controlled while the db/db were severely hyperglycemic at the time of sacrifice. This may account for the differences seen in differentiation where hyperglycemia could enhance osteoclastogenesis. RANK L Activation Normal Osteoclasts RANK L will induce diff erentiation and fusion of M CSF dependent osteoclast precursors. The binding of RANK L to its receptor RANK on these precursors induces NF specific genes (351, 352) Calcitonin receptor, beta 3 integrin, cathepsin K, and MMP 9, and the transcription factor c src are all turned on by RANK L and aid in osteocla st function (112, 113) C src allows for the fusion of these committed mononuclear precursors to become multinucleated osteoclasts (351) RANK L will induce the mature cell to start the

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115 resorption process where the cell will attach to the bone matrix, reorganize the actin cytoskeleton to form the actin ring and sealing zone, and eventually form the ruffled border. Targeted acid secretion and subsequent demineralization of the bone then occurs after which the organic portion is degraded by enzymes such as cathepsin K (322) Diabetic Osteoclasts Both T1D derived osteoclasts from the NOD mouse and T2D derived osteoclasts from the db/db mouse display enhanced bone resor ption with RANK L stimulation. They produce elevated levels of MMP 9 and cathepsin K and subsequently degrade more collagen than non diabetic controls. Since RANK L stimulation leads to NF activation to elicit osteoclast function, it may be that this pathway has defects where the translocation of the transcription factor to the nucleus to turn on osteoclast specific genes is enhanced. DCs have been found to have defects in the NF lead to a hyperactive phenotype whereby they are render ed more sensitive to inflammatory stimuli, release increased amounts of pro inflammatory cytokines, and have augmented T cell activation capabilities (186 188) Macrophages from NOD mice also display defects similar to DCs where cells are hype r responsive to inflammatory stimuli and have increased NF (190 192) Monocytes and macrophages in T2D participants are also constitutiv ely activated and respond even more so to inflammatory stimuli (41) which ma y indicate a similar defect in the NF Thus, the diabetes derived osteoclast may have aberrant NF RANK ligation which could explain the increased sensitivity to RANK L and subsequent increased resorptive capability.

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116 LPS induced Deactivation Normal Osteoclasts While LPS can act on supporting cell types such as stromal cells, osteoblasts, and activated T cells to produce RANK L which stimulates osteoclastogenesis and initiates resorption (70) LPS stimulation on osteoclasts direc tly leads to deactivation and inhibits differentiation of precursors. This inhibition of differentiation and function of osteoclasts is thought to be a way to switch the lineage of the cell back to that of a macrophage or immune cell to help clear the sup posed infection. Osteoclast precursors in the absence of RANK L treated with LPS will not differentiate into multinucleated osteoclasts. With co treatment of LPS and RANK L, LPS still inhibits osteoclastogenesis. However, once RANK L has led to the comm itment of the precursor to the osteoclast lineage, LPS treatment can enhance differentiation (200, 201) Mature osteoclasts pre treated with RANK L stimulated with LPS shut down resorptive processes (200) We have also found that osteoclasts stimulated with LPS instead secrete pro inflammatory cytokines and chemokines which may serve to recruit other immune cells to the site of infection. Thus, in the presence of LPS, osteoclast mediated bone resorption is inhibited and cellular function switches to that of an immune cell recruiter. Diabetic Osteoclasts Osteoclasts derived from hosts with diabetes do not display LPS induced deactivation of bone resorption. NOD derived osteoclasts continue to secrete cathepsin K and MMP 9 to degrade collagen and have similar capability to form resorption pits in the presence of LPS. Osteoclasts derived from the db/db T2D mouse model also show this inhibition of LPS induced deactivation. Surprisingly, even though the T2D human derived osteoclasts had simila r differentiation potential to diabetes free humans, LPS

PAGE 117

117 induced deactivation did not occur. Thus, regardless of the glycemic control of the host at the time of precursor harvest, osteoclasts derived from either T1D or T2D hosts do not respond correctly to LPS and instead continue to resorb bone. Monocytes and macrophages from T2D participants secrete increased levels of multiple pro inflammatory cytokines in response to LPS compared to diabetes free parti cipants Interestingly, these cells are constitutively activated in T2D hosts and thus are not tolerized to the presence of TLR ligands which are constantly circulating due to poor control of infections. Presence of low levels of TLR ligands such as LPS normally induce tolerance to a second challenge, yet the response seen in T2D participants is exacerbated and never brought under control (41) This phenomenon of hyper reactivity also seems to be true for osteoclasts derived from T2D hosts, both murine and human. LPS induces an exaggerated response by osteoclasts via increased secretion of pro inflammatory mediators compared to diabetes free sources. The continuation of resorption also suggests lack of control of the inflammatory response. Not surprisingly, IL 10, a regulatory cytokine which decreases osteoclast function (212) is secreted in s ub optimal amounts in the db/db and T2D human derived osteoclast cultures which may account for this lack of control. This defective IL 10 production has also been noted in Breg cells (also of the myeloid lineage) isolated from T2D participants when stimu lated with TLR ligands (41) Thus, there seems to be a shared defect in IL 10 secretion in T2D myeloid cells, including osteoclasts, which may lead to exaggerated pro inflammatory and pro osteoclastic responses and dampening of IL 10 mediated osteoclast deactivation.

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118 Osteoclast Regulation by Inflammatory Mediators Precursor Mobil ization Osteoclast precursors from bone marrow can be mobilized out to peripheral sites of bone remodeling by hematopoietic growth factors such as GM CSF and G CSF. Chemokines such as SDF 1 induce MMP 9 expression to aid in the mobilization of precursors to bone (212) and are important for precursor survival (353) Interestingly, lower SDF 1 expression leads to precursor cell release from the bone marrow where cells follow the decreasing SDF 1 gradient to bone remodeling sites (2) Pro inflammatory cytokines such as IL 15 act to increase precursor numbers (212) while chemokines such as MIP 3, RANTES, and MCP 3 exert chemotactic effects on these osteoclast precursor populations (353) GM CSF and RANTES were found to be elevated in NOD derived osteoclast cultures treated with LPS suggesting increased capability to recruit T1D osteoclast precursors from bone ma rrow to the site of resorption. T2D derived osteoclasts secrete elevated MIP of resorption where they can differentiate into mature osteoclasts. Differentiation Osteoclasts themselves can secrete pro inflammatory mediators such as TNF IL 6, and IL (324) TNF osteoclastogenesis even in the absence of RANK L, yet can augment RANK L dependent differentiation. This pro inflammatory cytokine acts to prime precursor cells in the bone m arrow to become osteoclast precursors by up regulating receptors for M CSF and RANK L which can then respond to peripheral TNF (87, 310) TNF L expression on supporting cell types (159)

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119 Chemokines such as IL 8, RANTES, MCP 1, and MIP f osteoclast precursors (212) RANTES, MIP 3 also stimulate chemotaxis of precursors and serve to augment osteoclastogenesis by increasing osteoclast number and size (354) Pro inflammatory cytokines such as IL 6, IL 11, IL 17, and IL 18 also promote osteoclastogenesis but do so by inducing RANK L expression on supporting cell types (69) Interestingly, TNF 6, IL 11, and IL 18 can substitute for RANK L when in the presence of M CSF to promote non canonical osteoclastogenesis (101) TNF stimulation in NOD derived cultures which would indicate a more favorable environment for osteoclastogenesis in a T1D host. Increased TNF regulate the expression of RANK on osteoclast precursors rendering these cells more sensitive to RANK L stimulation. RANTES was also up regul ated in NOD derived cultures which would allow for more efficient chemotaxis of osteoclast precursors to the site where they can be activated to differentiate. Alternatively, T2D derived osteoclasts secrete elevated levels of IL 6, MCP 1, and MIP would increase osteoclastogenesis. MCP 1 and MIP augment osteoclast size, which is the case in murine T2D derived osteoclasts. Activation TNF 1, and IL 6 are important potent stimulators of bone resorption. These pro inflammatory cytokines activate mature osteoclasts directly by enhancing RANK L activity and subsequent RANK signaling and indirectly by up regulating RANK L on other cells. RANTES, IL 8, and MIP late motility of mature osteoclasts (212) yet RANTES and MIP (355)

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120 IL 8, however, is also considered a resorption stimulator and does so independently of RANK L (212) Elevated RANTES would allow for increased motility of mature osteoclasts to the site of resorption while TNF 1 derived cultures, would increase osteoclast activation to resorb bone in a T1D environment. This highly pro resorptive environment seen in NOD derived cultures would lead to the perfect storm of increased precursor mobilization, o steoclastogenesis, and activation to destroy increased quantities of bone. Decreased anti osteoclastic IL 10 in NOD derived cultures would further exacerbate this response. It is also important to remember that with pro inflammatory cytokine cocktail (TN F 6) stimulation, NOD derived osteoclasts resorb almost double the amount of bone than controls which would indicate an increased sensitivity to these inflammatory mediators. Coupled with increased mediator secretion, T1D derived osteocla sts would therefore be even more sensitive to this inflammatory environment leading to a highly destructive response that cannot be controlled. Similarly, T2D derived osteoclasts produce elevated MIP increase mature osteoclast motility after which i ncreased IL 6 can activate these cells to resorb more bone. Decreased IL 10 secretion by T2D derived osteoclasts would further amplify this response by lack of this crucial anti osteoclastic signal. Therefore, T2D derived osteoclasts would also display i ncreased precursor mobilization, osteoclastogenesis, and resorption induction similar to T1D derived osteoclasts, albeit by different inflammatory mediators.

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121 Hyperglycemic Effects on Osteoclasts Differentiation Hyperglycemic conditions have been shown to h ave varying effects on osteoclast differentiation (240, 246, 249) Glucose utilization during differentiation is critical for this process to occur and it is assumed that osteoclasts should differentiate more efficient ly in hyperglycemic environments. Osteoclasts derived from the murine macrophage immortalized cell line RAW264.7 display optimal differentiation at 5mM glucose yet show similar potential between 2 and 20mM glucose, equivalent to hypoglycemic to hyperglyce mic conditions (36 360mg/dL). Differentiation does decrease with 30mM+ glucose concentrations, equivalent to extreme hyperglycemia (540mg/dL), which would most likely be due to toxicity from the hyperosmotic environment (246) Another study found that murine RAW 264.7 osteoclasts in hyperglycemic environments (10 or 25mM equivalent to 180 or 450mg/dL) have decreased differentiation, especially at 25mM glucose (249) While both of these studies do assess osteoclast specific effects of hyperglycemia, there are performed on immortalized cell lines that may not recapitulate what is se en in primary bone marrow derived osteoclasts. Studies performed in vivo utilizing a bacterial induced bone loss model have shown decreased osteoclastogenesis in T2D db/db mice (400 450mg/dL blood glucose levels). Fewer osteoclasts were found in db/db c alvarial sections and bone loss was decreased in these animals after P. gingivalis inoculation, presumably due to less osteoclasts able to resorb. Baseline resorption capabilities of these osteoclasts in the db/db mouse, however, were not assessed in this study (240) We have shown that after long term and severe hyperg lycemia, osteoclasts derived from db/db mice have augmented differentiation with signif icantly higher numbers of multi nucleated and giant

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122 cells compared to C57BL/6 controls. Conversely, in the absence of long term hypergly cemia, similar numbers of multi nu cleated and giant osteoclasts were formed in human T2D derived cultures compared to diabetes free individuals. These data may suggest augmented RANK signaling in db/db derived osteoclasts, or a host that has overt hyperglycemia, that may have been caused by irreversible alterations to the precursor population due to a high glucose environment. Activation Osteoclasts derived from avian medullary bone utilize glucose as the principal energy source during resorptive function and consume more glucose when cult ured on bone. These osteoclasts resorb optimal amounts of bone between 7mM and 25mM glucose, suggesting that hyperglycemia would augment resorptive function (247) Glucose also has been found to up regulate the expression of V ATPase which is critical for demineralization of hydroxyapatite (248) High glucose also increases ATP production and increases intracellular calcium levels in osteoclasts, both necessary for the initiation of resorption (333) These findings were recapitulated in mouse RAW 264.7 osteoclasts where maximum cell growth occurred at 20mM glucose (246) However, another study showed that calcitonin receptor, cathepsin K, and MMP 14 were down regulated in RAW 264.7 osteoclasts while MMP 9 showed little change at 25mM glucose. ROS production, NF this hyp erglycemic environment (249) Again, primary osteoclasts have not been studied in hyperglycemic environments, therefore actual effects of hyp erglycemia on function of these cells remains to be determined. Hyperglycemic T1D NOD mice (~480mg/dL blood glucose) display no change in bone loss measured by serum collagen I telopeptide levels after tibial distraction (273)

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123 nor have increased cathepsin K expression suggesting no increase in osteoclast activi ty (274) T1D human subjects with hyperglyc emia also show no change compared to diabetes free subjects in regards to plasma collagen telopeptide levels (83) although other studies show increased urine resorption markers in similar T1D subjects compared to controls (9, 137) Conversely, T2D human subjects with hyperglycemia show increased osteoclast function measured by serum TRAP levels and urine bone resorption markers (collagen telopeptides and deoxypyridinoline) (7) We have found that osteoclasts derived from db/db mice resorb increased amounts of bone via cathepsin K than control mice, however this phenomenon was not observed in human T2D osteoclast cultures. This was found to be due to the increased numbers of multi nucleated and giant cells in db/db cultures when cathepsin K expression and collagen degradation was normalized on a per cell basis. Similarly, human T2D derived osteoclasts do not resorb more bone than diabetes free cont rols, which correlates with their similar size distribution. Therefore, increased resorption in db/db osteoclasts is not due to increased activity, but rather to increased numbers of cell s able to resorb. Interestingly, LPS induced deactivation of osteocl ast function is inhibited in both human T2D and murine db/db osteoclast cultures, regardless of the glycemic state of the host. This was also observed in T1D derived osteoclasts from the NOD mouse, which were normoglycemic at the time of marrow harvest. Pro inflammatory cytokine and chemokine secretion is also increased in both T1D and T2D derived osteoclast cultures. While the mechanisms of diabetes differ between these models, it seems that the inflammatory environment that is present in diabetes melli tus alters osteoclast

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124 precursors rendering them unable to deactivate with LPS and more pro inflammatory in nature. Possible Adjunct Therapies Addition of anti osteoclastic drugs: Anti RANK L Anti RANK L, or denosumab, is a high affinity human monoclonal a ntibody to RANK L that acts similarly to OPG that is normally secreted by osteoblasts to dampen osteoclastogenesis and bone resorption (356) RANK L is also important in secondary lymphoid tissue organization and development and when absent, leads to lack of lymph nodes and abnormal spleens (357) Immunodeficiency however, is not observed in RANK L deficient mice due to the redundancy of other T cell and DC interactions and administration of anti RANK L in humans does not cause immune defects or deficiencies (69) However, reports of cellulitis have been documented in humans undergoing denosumab treatm ent (358) RANK L produced by activated T cells can also activate dendritic cells and stimulate autoimmunity as in inflammatory bowel disease (69) Conversely, RANK L produced by keratinocytes in the skin after UV exposure activates Langerhans cells and may lead to the expansion of Tregs. Therefore, RANK L not only can activate osteoclas ts, but also has effects on the immune system, however these secondary activities may be compensated by other ligands such as CD40 (3) Anti RANK L treatment would therefore keep osteoblasts and activated T cells from activating osteoclast precursors to differentiate and mature cells from resorbing bone. Since T1D derived osteoclasts display augmented function in the presence of RANK L, it follows that blockade of this osteoclastogenic molecule would dampen this aberrant function and prevent exacerbated bone loss, especially in the case of inflammatory arthritis or periodontal disease.

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125 Addition of anti osteoclastic drugs: Bisphosphonates Current anti resorptive therapies mainly include bisphosphonates such as alendronate (359) Two classes of bisphosphonates are currently used, nitrogen containing bisphosphonates such as alendronate and zoledronate are more potent anti resorptives than the simple class containing drugs such as clodronate. Bisphosphonates act to inhibit osteoclast function either by reducing differentiation, chemotaxis, and resorptive activity or by inducing apoptosis in osteoclasts. Once bound to bone minerals, namely hydroxyapat ite, bisphosphonates are then taken up by osteoclasts during the resorption process. Ruffled borders begin to disappear after endocytosis and the cytoskeleton becomes disrupted. Simple bisphosphonates are thought to act as ATP analogues and ultimately le ad to cell death due to accumulation of these non hydrolyzable metabolites. Nitrogen containing compounds disrupt mevalonate biosynthesis which leads to prenylation of proteins, namely cholesterol and small GTPases. These important signaling proteins reg ulate cytoskeletal architecture and subsequent cell motility, ruffling of membranes, and transportation of vesicles. Thus, osteoclast differentiation, resorptive function, and survival are all affected by this highly potent class of anti resorptives (360) While these drugs are ideal for keeping osteoclasts from resorbing bone, normal wear and tear such as microfractures may not be remodeled and thus healed thereby leading to increased frac ture risk. However, low dose administration of these drugs has shown to be efficacious in reducing fracture risk and increasing bone mineralization (360) Therefore, use of bisphos ph onates may be a useful adjunct therapy in inflammatory bone pathologies in T1D and T2D, where osteoclasts are aberrantly activated.

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126 Future Directions Mechanism of inhibition of LPS induced deactivation While it is clear that LPS leads to deactivation of resorpt ion by osteoclasts, the exact mechanism of how this occurs is unknown (200, 201) We have shown that IL 10, a regulatory cytokine that leads to inh ibition of osteoclasts, is decreased after LPS stimulation in T2D derived cultures. IL 10 has been known to be produced in decreased quantities by Breg cells derived from participants with T2D (41) as well as macrophages derived from diabetic NOD mice stimulated with LPS (180) It may be that the entire myeloid lineage secretes decreased IL 10 in response to inflammatory stimuli and therefore cannot keep exacerbated responses in check. Determining the response of T1D and T2D derived osteoclasts to IL 10 is currently being studied in our laboratory where exogenous IL 10 will be added to osteoclast cultures and resorptive capabilities after LPS treatment assessed. The NF (186 188) and T2D (41, 206) rendering myeloid lineage cells such as macrophages and dendritic cells inherently more responsive to inflammatory stimuli. the inhibitor of NF translocation, is phosphorylated more efficiently in T1D and thus NF enhanced (186 188) It may be that this enhanced nuclear translocation of NF to increased resorption due to the production of pro inflammatory cytokines. Further work to determine defects in the NF mechanism. TLR 4, the receptor for LPS, has also been found to be increased in expression on diabetic NOD bone marrow derived macrophages. LPS treatment leads to down regulation of TLR 4 on diabetes free macrophages however this is not observed on

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127 diabetic NOD macrophages where expression does not change (180) This lack of TLR 4 down regulation may be a mechanism of aberrant LPS induced deactivation occurring in our osteoclasts derived from T1D and T2D hosts. Therefore, determining TLR 4 expression before and after LPS treatment on T1D and T2D der ived osteoclasts may lead to a better understanding of the aberrant deactivation. Mechanism of decreased fusion in T1D osteoclasts T1D derived osteoclasts display altered differentiation where fusion is decreased and fewer multinucleated and giant cells are formed. Osteoclast fusion is regulated by many molecules, one of particular interest being DC STAMP. A T1D mouse model STAMP expression on osteoclasts along with decreased function yet i ncreased numbers (259) While this model does not correlate as well to human T1D as the NOD mouse, investigation into the expression of this fusigen is warranted and is expected to be decreased on NOD derived osteoclast precursors. Regulating receptor expression: Activating (RANK, TNF R, and IL 1R) Activating receptors on osteoclasts allow for the initiation as well as propagation of resorption. Because T1D derived osteoclasts respond to RANK L and pro inflammatory cytokines in an exacerbated manner, activating receptors such as RANK, TNF R, and IL 1R may be altered in expression rendering the cells more easily activated. It has been observed that osteoclas participants are also more sensitive to RANK L stimulated differentiation, have higher bone resorption capability, and are less able to be deactivated by OPG administration compared to diabetes free an d participants with diabetes (8) These findings suggest increased RANK signaling or increased receptor expression in

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128 diabetes derived osteoclasts. Another prominent member of the same tr imeric cytokine receptor family, the TNF receptor, leads to the activation of the NF ultimately activates the cell when bound by its ligand TNF (325) TNFR1 is found on osteoclasts, and when stimulated w ith its ligand, leads to fusion of precursors (87) Similarly, IL 1R bound to its ligands IL precursors and activation of resorption (74) Due to the exacerbated response of NOD derive d osteoclasts to the RANK L with further activation in the presence of a pro inflammatory cytokine cocktail containing TNF these cytokine receptors or increased intracellular signaling may explain this phenomenon. Regulating receptor expression: Deactivating (Calcitonin Receptor) Blood calcium homeostasis is partly regulated by calcitonin which binds directly to osteoclast calcitonin receptors [CTR] and inhibits resorption (350) CTR ligation causes osteoclasts to cease motility and disrupts the ruffled borders leading to cell retraction and loss of resorptive function. Calcitonin also inhibits RANK signali ng which leads to decreased osteoclastogenesis and activation (313) While cal citonin is not present in our ex vivo osteoclast cultures, determining the true extent of dysregulation in T1D and T2D derived osteoclasts requires study of both activating and deactivating receptors. Constant CTR ligation over time leads to down regulati on of these receptors where the cell becomes unresponsive to calcitonin treatment (361) Calcitonin levels in participants with diabetes are similar to those without diabetes and therefore would not lead to CTR ligation induced down regulation of CTRs (362) Nonetheless, with d ecreased CTR exp ression, one would expect less opportunity for calcitonin to bind and therefore dampened deactivation.

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129 LIST OF REFERENCES 1. Abu Amer, Y., I. Darwech, and J. Otero. 2008. Role of the NF kappaB axis in immune modulation of osteoclasts an d bone loss. Autoimmunity 41:204 211. 2. Boyce, B. F., E. M. Schwarz, and L. Xing. 2006. Osteoclast precursors: cytokine stimulated immunomodulators of inflammatory bone disease. Curr Opin Rheumatol 18:427 432. 3. Nakashima, T., and H. Takayanagi. 2008. The dynamic interplay between osteoclasts and the immune system. Arch Biochem Biophys 473:166 171. 4. Liu, R., H. S. Bal, T. Desta, N. Krothapalli, M. Alyassi, Q. Luan, and D. T. Graves. 2006. Diabetes enhances periodontal bone loss through enhanced resorption and diminished bone formation. Journal of dental research 85:510 514. 5. Taylor, G. W., B. A. Bu rt, M. P. Becker, R. J. Genco, and M. Shlossman. 1998. Glycemic control and alveolar bone loss progression in type 2 diabetes. Ann Periodontol 3:30 39. 6. Motyl, K. J., S. Botolin, R. Irwin, D. M. Appledorn, T. Kadakia, A. Amalfitano, R. C. Schwartz, and L. R. McCabe. 2009. Bone inflammation and altered gene expression with type I diabetes early onset. J Cell Physiol 218:575 583. 7. Suzuki, K., T. Kurose, M. Takizawa, M. Maruyama, K. Ushikawa, M. Kikuyama, C. Sugimoto, Y. Seino, S. Nagamatsu, and H. Ishid a. 2005. Osteoclastic function is accelerated in male patients with type 2 diabetes mellitus: the preventive role of osteoclastogenesis inhibitory factor/osteoprotegerin (OCIF/OPG) on the decrease of bone mineral density. Diabetes Res Clin Pract 68:117 125 8. Mabilleau, G., N. L. Petrova, M. E. Edmonds, and A. Sabokbar. 2008. Increased osteoclastic activity in acute Charcot's osteoarthropathy: the role of receptor activator of nuclear factor kappaB ligand. Diabetologia 51:1035 1040. 9. Mathiassen, B., S. Nielsen, J. S. Johansen, D. Hartwell, J. Ditzel, P. Rodbro, and C. Christiansen. 1990. Long term bone loss in insulin dependent diabetic patients with microvascular complications. J Diabet Complications 4:145 149. 10. Kemink, S. A., A. R. Hermus, L. M. Swinkels, J. A. Lutterman, and A. G. Smals. 2000. Osteopenia in insulin dependent diabetes mellitus; prevalence and aspects of pathophysiology. J Endocrinol Invest 23:295 303. 11. Galluzzi, F., S. Stagi, R. Salti, S. Toni, E. Piscitelli, G. Simonini, F. Falcini, and F. Chiarelli. 2005. Osteoprotegerin serum levels in children with type 1 diabetes: a potential modulating role in bone status. Eur J Endocrinol 153:879 885.

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130 12. Pacios, S., J. Kang, J. Galicia, K. Gluck, H. Patel, A. Ovaydi Man del, S. Petrov, F. Alawi, and D. T. Graves. 2011. Diabetes aggravates periodontitis by limiting repair through enhanced inflammation. Faseb J 13. Gough, A., H. Abraha, F. Li, T. S. Purewal, A. V. Foster, P. J. Watkins, C. Moniz, and M. E. Edmonds. 1997. Measurement of markers of osteoclast and osteoblast activity in patients with acute and chronic diabetic Charcot neuroarthropathy. Diabet Med 14:527 531. 14. Lumachi, F., V. Camozzi, V. Tombolan, and G. Luisetto. 2009. Bone mineral density, osteocalcin, a nd bone specific alkaline phosphatase in patients with insulin dependent diabetes mellitus. Ann N Y Acad Sci 1173 Suppl 1:E64 67. 15. Moyer Mileur, L. J., H. Slater, K. C. Jordan, and M. A. Murray. 2008. IGF 1 and IGF binding proteins and bone mass, geome try, and strength: relation to metabolic control in adolescent girls with type 1 diabetes. J Bone Miner Res 23:1884 1891. 16. Coe, L. M., R. Irwin, D. Lippner, and L. R. McCabe. 2011. The bone marrow microenvironment contributes to type I diabetes induced osteoblast death. J Cell Physiol 226:477 483. 17. Somers, E. C., S. L. Thomas, L. Smeeth, and A. J. Hall. 2009. Are individuals with an autoimmune disease at higher risk of a second autoimmune disorder? Am J Epidemiol 169:749 755. 18. Salvi, G. E., J. D. Beck, and S. Offenbacher. 1998. PGE2, IL 1 beta, and TNF alpha responses in diabetics as modifiers of periodontal disease expression. Ann Periodontol 3:40 50. 19. Raunio, T., M. Knuuttila, L. Hiltunen, R. Karttunen, O. Vainio, and T. T ervonen. 2009. IL 6( 174) genotype associated with the extent of periodontal disease in type 1 diabetic subjects. J Clin Periodontol 36:11 17. 20. Salvi, G. E., J. G. Collins, B. Yalda, R. R. Arnold, N. P. Lang, and S. Offenbacher. 1997. Monocytic TNF alpha secretion patterns in IDDM patients with periodontal diseases. J Clin Periodontol 24:8 16. 21. Salvi, G. E., B. Yalda, J. G. Collins, B. H. Jone s, F. W. Smith, R. R. Arnold, and S. Offenbacher. 1997. Inflammatory mediator response as a potential risk marker for periodontal diseases in insulin dependent diabetes mellitus patients. J Periodontol 68:127 135. 22. McMullen, J. A., T. E. Van Dyke, H. U Horoszewicz, and R. J. Genco. 1981. Neutrophil chemotaxis in individuals with advanced periodontal disease and a genetic predisposition to diabetes mellitus. Journal of periodontology 52:167 173.

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131 23. Centers for Disease Control and Prevention Atlanta, G. U. S. D. o. H. a. H. S., Centers for Disease Control and Prevention. 2011. National Diabetes Fact Sheet: national estimates and general information on diabetes and prediabetes in the United States, 2011. 24. Burner, T. W., and A. K. Rosenthal. 2009. Diab etes and rheumatic diseases. Curr Opin Rheumatol 21:50 54. 25. Wasko, M. C., J. Kay, E. C. Hsia, and M. U. Rahman. 2011. Diabetes mellitus and insulin resistance in patients with rheumatoid arthritis: risk reduction in a chronic inflammatory disease. Arth ritis Care Res (Hoboken) 63:512 521. 26. Lalla, E., B. Cheng, S. Lal, S. Kaplan, B. Softness, E. Greenberg, R. S. Goland, and I. B. Lamster. 2007. Diabetes mellitus promotes periodontal destruction in children. J Clin Periodontol 34:294 298. 27. Taylor, G. W., B. A. Burt, M. P. Becker, R. J. Genco, M. Shlossman, W. C. Knowler, and D. J. Pettitt. 1996. Severe periodontitis and risk for poor glycemic control in patients with non insulin dependent diabetes mellitus. J Periodontol 67:1085 1093. 28. Sandberg, G. E., H. E. Sundberg, C. A. Fjellstrom, and K. F. Wikblad. 2000. Type 2 diabetes and oral health: a comparison between diabetic and non diabetic subjects. Diabetes Res Clin Pract 50:27 34. 29. Kaur, G., B. Holtfreter, W. Rathmann, C. Schwahn, H. Wallasc hofski, S. Schipf, M. Nauck, and T. Kocher. 2009. Association between type 1 and type 2 diabetes with periodontal disease and tooth loss. J Clin Periodontol 36:765 774. 30. Knip, M., and H. Siljander. 2008. Autoimmune mechanisms in type 1 diabetes. Autoim mun Rev 7:550 557. 31. Onengut Gumuscu, S., and P. Concannon. 2005. The genetics of type 1 diabetes: lessons learned and future challenges. J Autoimmun 25 Suppl:34 39. 32. Forlenza, G. P., and M. Rewers. 2011. The epidemic of type 1 diabetes: what is it telling us? Curr Opin Endocrinol Diabetes Obes 18:248 251. 33. D'Angeli, M. A., E. Merzon, L. F. Valbuena, D. Tirschwell, C. A. Paris, and B. A. Mueller. 2010. Environmental factors associated with childhood onset type 1 diabetes mellitus: an exploration of the hygiene and overload hypotheses. Arch Pediatr Adolesc Med 164:732 738. 34. Knip, M., and O. Simell. 2010. Environmental triggers of type 1 diabetes. Cold Spring Harb Perspect Med 2:a007690.

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132 35. Boettler, T., and M. von Herrath. 2011. Protection against or triggering of Type 1 diabetes? Different roles for viral infections. Expert Rev Clin Immunol 7:45 53. 36. EURODIAB. 2002. Rapid early growth is associated with increased risk of childhood type 1 diabetes in various European populations. Diabetes care 25:1755 1760. 37. Sepa, A., J. Wahlberg, O. Vaarala, A. Fro di, and J. Ludvigsson. 2005. Psychological stress may induce diabetes related autoimmunity in infancy. Diabetes care 28:290 295. 38. Toeller, M. 2002. Fibre consumption, metabolic effects and prevention of complications in diabetic patients: epidemiologic al evidence. Dig Liver Dis 34 Suppl 2:S145 149. 39. Hyoty, H. 2002. Enterovirus infections and type 1 diabetes. Ann Med 34:138 147. 40. Diaz Horta, O., M. Bello, E. Cabrera Rode, J. Suarez, P. Mas, I. Garcia, I. Abalos, R. Jofra, G. Molina, O. Diaz Diaz, and U. Dimario. 2001. Echovirus 4 and type 1 diabetes mellitus. Autoimmunity 34:275 281. 41. Nikolajczyk, B. S., M. Jagannathan Bogdan, H. Shin, and R. Gyurko. 2011. State of the union between metabolism and the immune system in type 2 diabetes. Genes Immun 12:239 250. 42. Hansen, L., and O. Pedersen. 2005. Genetics of type 2 diabetes mellitus: status and perspectives. Diabetes Obes Metab 7:122 135. 43. Dedoussis, G. V., A. C. Kaliora, and D. B. Panagiotakos. 2007. Genes, diet and type 2 diabetes mellitus: a review. Rev Diabet Stud 4:13 24. 44. Deeb, S. S., L. Fajas, M. Nemoto, J. Pihlajamaki, L. Mykkanen, J. Kuusisto, M. Laakso, W. Fuji moto, and J. Auwerx. 1998. A Pro12Ala substitution in PPARgamma2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity. Nat Genet 20:284 287. 45. Creely, S. J., P. G. McTernan, C. M. Kusminski, M. Fisher, N. F Da Silva, M. Khanolkar, M. Evans, A. L. Harte, and S. Kumar. 2007. Lipopolysaccharide activates an innate immune system response in human adipose tissue in obesity and type 2 diabetes. Am J Physiol Endocrinol Metab 292:E740 747. 46. Scott, I. C., S. Ste er, C. M. Lewis, and A. P. Cope. 2011. Precipitating and perpetuating factors of rheumatoid arthritis immunopathology: linking the triad of genetic predisposition, environmental risk factors and autoimmunity to disease pathogenesis. Best Pract Res Clin Rhe umatol 25:447 468.

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133 47. Guthrie, K. A., N. R. Tishkevich, and J. L. Nelson. 2009. Non inherited maternal human leukocyte antigen alleles in susceptibility to familial rheumatoid arthritis. Ann Rheum Dis 68:107 109. 48. Grant, S. F., G. Thorleifsson, M. L. Frigge, J. Thorsteinsson, B. Gunnlaugsdottir, A. J. Geirsson, M. Gudmundsson, A. Vikingsson, K. Erlendsson, J. Valsson, H. Jonsson, D. F. Gudbjartsson, K. Stefansson, J. R. Gulcher, and K. Steinsson. 2001. The inheritan ce of rheumatoid arthritis in Iceland. Arthritis Rheum 44:2247 2254. 49. Stahl, E. A., S. Raychaudhuri, E. F. Remmers, G. Xie, S. Eyre, B. P. Thomson, Y. Li, F. A. Kurreeman, A. Zhernakova, A. Hinks, C. Guiducci, R. Chen, L. Alfredsson, C. I. Amos, K. G. Ardlie, A. Barton, J. Bowes, E. Brouwer, N. P. Burtt, J. J. Catanese, J. Coblyn, M. J. Coenen, K. H. Costenbader, L. A. Criswell, J. B. Crusius, J. Cui, P. I. de Bakker, P. L. De Jager, B. Ding, P. Emery, E. Flynn, P. Harrison, L. J. Hocking, T. W. Huizing a, D. L. Kastner, X. Ke, A. T. Lee, X. Liu, P. Martin, A. W. Morgan, L. Padyukov, M. D. Posthumus, T. R. Radstake, D. M. Reid, M. Seielstad, M. F. Seldin, N. A. Shadick, S. Steer, P. P. Tak, W. Thomson, A. H. van der Helm van Mil, I. E. van der Horst Bruin sma, C. E. van der Schoot, P. L. van Riel, M. E. Weinblatt, A. G. Wilson, G. J. Wolbink, B. P. Wordsworth, C. Wijmenga, E. W. Karlson, R. E. Toes, N. de Vries, A. B. Begovich, J. Worthington, K. A. Siminovitch, P. K. Gregersen, L. Klareskog, and R. M. Plen ge. 2010. Genome wide association study meta analysis identifies seven new rheumatoid arthritis risk loci. Nat Genet 42:508 514. 50. Hinks, A., A. Barton, S. John, I. Bruce, C. Hawkins, C. E. Griffiths, R. Donn, W. Thomson, A. Silman, and J. Worthington. 2005. Association between the PTPN22 gene and rheumatoid arthritis and juvenile idiopathic arthritis in a UK population: further support that PTPN22 is an autoimmunity gene. Arthritis Rheum 52:1694 1699. 51. Daha, N. A., F. A. Kurreeman, R. B. Marques, G. Stoeken Rijsbergen, W. Verduijn, T. W. Huizinga, and R. E. Toes. 2009. Confirmation of STAT4, IL2/IL21, and CTLA4 polymorphisms in rheumatoid arthritis. Arthritis Rheum 60:1255 1260. 52. Ruiz Esquide, V., J. A. Gomez Puerta, J. D. Canete, E. Graell, I. Vazquez, M. G. Ercilla, O. Vinas, A. Gomez Centeno, I. Haro, and R. Sanmarti. 2011. Effects of smoking on disease activity and radiographic progression in early rheumatoid arthritis. The Jou rnal of rheumatology 38:2536 2539. 53. Mikuls, T. R., G. M. Thiele, K. D. Deane, J. B. Payne, J. R. O'Dell, F. Yu, H. Sayles, M. H. Weisman, P. K. Gregersen, J. H. Buckner, R. M. Keating, L. A. Derber, W. H. Robinson, V. Michael Holers, and J. M. Norris. 2012. Porphyromonas gingivalis and disease related autoantibodies in individuals at increased risk for future rheumatoid arthritis. Arthritis Rheum

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134 54. Joseph, R., S. Rajappan, S. G. Nath, and B. J. Paul. 2012. Association between chronic periodontitis an d rheumatoid arthritis: a hospital based case control study. Rheumatol Int 55. Wesley, A., C. Bengtsson, A. C. Elkan, L. Klareskog, L. Alfredsson, and S. Wedren. 2012. Association between overweight, obesity, ACPA positive and ACPA negative Rheumatoid Ar thritis results from the EIRA case control study. Arthritis Care Res (Hoboken) 56. Pedersen, M., S. Jacobsen, M. Klarlund, and M. Frisch. 2006. Socioeconomic status and risk of rheumatoid arthritis: a Danish case control study. The Journal of rheumatol ogy 33:1069 1074. 57. Zambon, J. J., H. Reynolds, J. G. Fisher, M. Shlossman, R. Dunford, and R. J. Genco. 1988. Microbiological and immunological studies of adult periodontitis in patients with noninsulin dependent diabetes mellitus. J Periodontol 59:23 31. 58. Mandell, R. L., J. Dirienzo, R. Kent, K. Joshipura, and J. Haber. 1992. Microbiology of healthy and diseased periodontal sites in poorly controlled insulin dependent diabetics. J Periodontol 63:274 279. 59. Verma, R. K., I. Bhattacharyya, A. Sevi lla, I. Lieberman, S. Pola, M. Nair, S. M. Wallet, I. Aukhil, and L. Kesavalu. 2010. Virulence of major periodontal pathogens and lack of humoral immune protection in a rat model of periodontal disease. Oral Dis 16:686 695. 60. Engebretson, S. P., J. Hey Hadavi, F. J. Ehrhardt, D. Hsu, R. S. Celenti, J. T. Grbic, and I. B. Lamster. 2004. Gingival crevicular fluid levels of interleukin 1beta and glycemic control in patients with chronic periodontitis and type 2 diabetes. J Periodontol 75:1203 1208. 61. Dah aghin, S., S. M. Bierma Zeinstra, B. W. Koes, J. M. Hazes, and H. A. Pols. 2007. Do metabolic factors add to the effect of overweight on hand osteoarthritis? The Rotterdam Study. Ann Rheum Dis 66:916 920. 62. Yoo, H. G., S. I. Lee, H. J. Chae, S. J. Park, Y. C. Lee, and W. H. Yoo. 2011. Prevalence of insulin resistance and metabolic syndrome in patients with gouty arthritis. Rheumatol Int 31:485 491. 63. Dessein, P. H., B. I. Joffe, and A. E. Stanwix. 2002. Ef fects of disease modifying agents and dietary intervention on insulin resistance and dyslipidemia in inflammatory arthritis: a pilot study. Arthritis Res 4:R12. 64. Nelson, R. G., M. Shlossman, L. M. Budding, D. J. Pettitt, M. F. Saad, R. J. Genco, and W. C. Knowler. 1990. Periodontal disease and NIDDM in Pima Indians. Diabetes care 13:836 840.

PAGE 135

135 65. Morran, M. P., L. A. Alexander, B. D. Slotterbeck, and M. F. McInerney. 2009. Dysfunctional innate immune responsiveness to Porphyromonas gingivalis lipopolysac charide in diabetes. Oral Microbiol Immunol 24:331 339. 66. Marcus, R. 1987. Normal and abnormal bone remodeling in man. Annu Rev Med 38:129 141. 67. Lazner, F., M. Gowen, D. Pavasovic, and I. Kola. 1999. Osteopetrosis and osteoporosis: two sides of the same coin. Human molecular genetics 8:1839 1846. 68. Raggatt, L. J., and N. C. Partridge. 2010. Cellular and molecular mechanisms of bone remodeling. The Journal of biological chemistry 285:25103 25108. 69. Takayanagi, H. 2007. Osteoimmunology: shared me chanisms and crosstalk between the immune and bone systems. Nat Rev Immunol 7:292 304. 70. Bar Shavit, Z. 2008. Taking a toll on the bones: regulation of bone metabolism by innate immune regulators. Autoimmunity 41:195 203. 71. Matsuo, K., and N. Irie. 2 008. Osteoclast osteoblast communication. Arch Biochem Biophys 473:201 209. 72. Thrailkill, K. M., C. K. Lumpkin, Jr., R. C. Bunn, S. F. Kemp, and J. L. Fowlkes. 2005. Is insulin an anabolic agent in bone? Dissecting the diabetic bone for clues. Am J Phys iol Endocrinol Metab 289:E735 745. 73. Ducy, P., M. Amling, S. Takeda, M. Priemel, A. F. Schilling, F. T. Beil, J. Shen, C. Vinson, J. M. Rueger, and G. Karsenty. 2000. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell 100:197 207. 74. Lee, Y. M. N. Fujikado, H. Manaka, H. Yasuda, and Y. Iwakura. 2010. IL 1 plays an important role in the bone metabolism under physiological conditions. Int Immunol 22:805 816. 75. Yang, C. M., C. S. Chien, C. C. Yao, L. D. Hsiao, Y. C. Huang, and C. B. Wu. 2004. M echanical strain induces collagenase 3 (MMP 13) expression in MC3T3 E1 osteoblastic cells. The Journal of biological chemistry 279:22158 22165. 76. Heino, T. J., T. A. Hentunen, and H. K. Vaananen. 2002. Osteocytes inhibit osteoclastic bone resorption thr ough transforming growth factor beta: enhancement by estrogen. Journal of cellular biochemistry 85:185 197.

PAGE 136

136 77. Granholm, S., P. Lundberg, and U. H. Lerner. 2007. Calcitonin inhibits osteoclast formation in mouse haematopoetic cells independently of transcriptional regulation by receptor activator of NF {kappa}B and c Fms. J Endocrinol 195:415 427. 78. Naot, D., an d J. Cornish. 2008. The role of peptides and receptors of the calcitonin family in the regulation of bone metabolism. Bone 43:813 818. 79. Yasuda, H., N. Shima, N. Nakagawa, K. Yamaguchi, M. Kinosaki, S. Mochizuki, A. Tomoyasu, K. Yano, M. Goto, A. Muraka mi, E. Tsuda, T. Morinaga, K. Higashio, N. Udagawa, N. Takahashi, and T. Suda. 1998. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 95:3597 3 602. 80. Kong, Y. Y., H. Yoshida, I. Sarosi, H. L. Tan, E. Timms, C. Capparelli, S. Morony, A. J. Oliveira dos Santos, G. Van, A. Itie, W. Khoo, A. Wakeham, C. R. Dunstan, D. L. Lacey, T. W. Mak, W. J. Boyle, and J. M. Penninger. 1999. OPGL is a key regul ator of osteoclastogenesis, lymphocyte development and lymph node organogenesis. Nature 397:315 323. 81. Mancini, L., N. Moradi Bidhendi, M. L. Brandi, M. Perretti, and I. MacIntyre. 2000. Modulation of the effects of osteoprotegerin (OPG) ligand in a hum an leukemic cell line by OPG and calcitonin. Biochem Biophys Res Commun 279:391 397. 82. Srivastava, S., G. Toraldo, M. N. Weitzmann, S. Cenci, F. P. Ross, and R. Pacifici. 2001. Estrogen decreases osteoclast formation by down regulating receptor activato r of NF kappa B ligand (RANKL) induced JNK activation. The Journal of biological chemistry 276:8836 8840. 83. Lappin, D. F., B. Eapen, D. Robertson, J. Young, and P. J. Hodge. 2009. Markers of bone destruction and formation and periodontitis in type 1 dia betes mellitus. J Clin Periodontol 36:634 641. 84. Mathiassen, B., S. Nielsen, J. Ditzel, and P. Rodbro. 1990. Long term bone loss in insulin dependent diabetes mellitus. J Intern Med 227:325 327. 85. Gillespie, M. T. 2007. Impact of cytokines and T lymp hocytes upon osteoclast differentiation and function. Arthritis Res Ther 9:103. 86. Zou, W., I. Hakim, K. Tschoep, S. Endres, and Z. Bar Shavit. 2001. Tumor necrosis factor alpha mediates RANK ligand stimulation of osteoclast differentiation by an autocri ne mechanism. Journal of cellular biochemistry 83:70 83.

PAGE 137

137 87. Zhang, Y. H., A. Heulsmann, M. M. Tondravi, A. Mukherjee, and Y. Abu Amer. 2001. Tumor necrosis factor alpha (TNF) stimulates RANKL induced osteoclastogenesis via coupling of TNF type 1 receptor and RANK signaling pathways. The Journal of biological chemistry 276:563 568. 88. Kotake, S., N. Udagawa, N. Takahashi, K. Matsuzaki, K. Itoh, S. Ishiyama, S. Saito, K. Inoue, N. Kamatani, M. T. Gillespie, T. J. Martin, and T. Suda. 1999. IL 17 in synovia l fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis. The Journal of clinical investigation 103:1345 1352. 89. Redlich, K., S. Hayer, R. Ricci, J. P. David, M. Tohidast Akrad, G. Kollias, G. Steiner, J. S. Smolen, E. F. Wagner, and G. Schett. 2002. Osteoclasts are essential for TNF alpha mediated joint destruction. The Journal of clinical investigation 110:1419 1427. 90. Graves, D. 2008. Cytokines that promote periodontal tissue destruction. J Periodontol 79:1585 1 591. 91. D, O. G., D. Ireland, S. Bord, and J. E. Compston. 2004. Joint erosion in rheumatoid arthritis: interactions between tumour necrosis factor alpha, interleukin 1, and receptor activator of nuclear factor kappaB ligand (RANKL) regulate osteoclasts. Ann Rheum Dis 63:354 359. 92. Theill, L. E., W. J. Boyle, and J. M. Penninger. 2002. RANK L and RANK: T cells, bone loss, and mammalian evolution. Annu Rev Immunol 20:795 823. 93. Josien, R., B. R. Wong, H. L. Li, R. M. Steinman, and Y. Choi. 1999. TRANC E, a TNF family member, is differentially expressed on T cell subsets and induces cytokine production in dendritic cells. J Immunol 162:2562 2568. 94. Neve, A., A. Corrado, and F. P. Cantatore. 2011. Osteoblast physiology in normal and pathological condit ions. Cell Tissue Res 343:289 302. 95. Ishimi, Y., C. Miyaura, C. H. Jin, T. Akatsu, E. Abe, Y. Nakamura, A. Yamaguchi, S. Yoshiki, T. Matsuda, T. Hirano, and et al. 1990. IL 6 is produced by osteoblasts and induces bone resorption. J Immunol 145:3297 330 3. 96. O'Brien, C. A., I. Gubrij, S. C. Lin, R. L. Saylors, and S. C. Manolagas. 1999. STAT3 activation in stromal/osteoblastic cells is required for induction of the receptor activator of NF kappaB ligand and stimulation of osteoclastogenesis by gp130 ut ilizing cytokines or interleukin 1 but not 1,25 dihydroxyvitamin D3 or parathyroid hormone. The Journal of biological chemistry 274:19301 19308. 97. Vaananen, H. K., and T. Laitala Leinonen. 2008. Osteoclast lineage and function. Arch Biochem Biophys 473: 132 138.

PAGE 138

138 98. Fuller, K., J. M. Owens, C. J. Jagger, A. Wilson, R. Moss, and T. J. Chambers. 1993. Macrophage colony stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts. The Journal of experimental medicine 178:1733 1744. 99. Tanaka, S., N. Takahashi, N. Udagawa, T. Tamura, T. Akatsu, E. R. Stanley, T. Kurokawa, and T. Suda. 1993. Macrophage colony stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors. The Journal of clin ical investigation 91:257 263. 100. Shiotani, A., M. Takami, K. Itoh, Y. Shibasaki, and T. Sasaki. 2002. Regulation of osteoclast differentiation and function by receptor activator of NFkB ligand and osteoprotegerin. Anat Rec 268:137 146. 101. Knowles, H J., and N. A. Athanasou. 2009. Canonical and non canonical pathways of osteoclast formation. Histol Histopathol 24:337 346. 102. Muto, A., T. Mizoguchi, N. Udagawa, S. Ito, I. Kawahara, Y. Abiko, A. Arai, S. Harada, Y. Kobayashi, Y. Nakamichi, J. M. Pen ninger, T. Noguchi, and N. Takahashi. 2011. Lineage committed osteoclast precursors circulate in blood and settle down into bone. J Bone Miner Res 26:2978 2990. 103. Lari, R., P. D. Kitchener, and J. A. Hamilton. 2009. The proliferative human monocyte sub population contains osteoclast precursors. Arthritis Res Ther 11:R23. 104. Hume, D. A., J. F. Loutit, and S. Gordon. 1984. The mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80: macrophages of bone and associated connective tissue. J Cell Sci 66:189 194. 105. Takahashi, N., N. Udagawa, S. Tanaka, H. Murakami, I. Owan, T. Tamura, and T. Suda. 1994. Postmitotic osteoclast precursors are mononuclear cells which express macrophage associated phenotypes. Dev Biol 163:212 221. 106. Sharma, S. M., A. Bronisz, R. Hu, K. Patel, K. C. Mansky, S. Sif, and M. C. Ostrowski. 2007. MITF and PU.1 recruit p38 MAPK and NFATc1 to target genes during osteoclast differentiation. The Journal of biological chemistry 282:15921 15929. 107. Chen, C., R. W. Clarkson, Y. Xie, D. A. Hume, and M. J. Waters. 1995. Growth hormone and colony stimulating factor 1 share multiple response elements in the c fos promoter. Endocrinology 136:4505 4516.

PAGE 139

139 108. Grigoriadis, A. E., Z. Q. Wang, M. G. Cecchini, W. Hofstetter, R. Felix, H. A. Fleisch, and E. F. Wagner. 1994. c Fos: a key regulator of osteoclast macrophage lineage determination and bone remodeling. Science 266:443 448. 109. Luchin, A., G. Purdom, K. Murphy, M. Y. Clark, N. A ngel, A. I. Cassady, D. A. Hume, and M. C. Ostrowski. 2000. The microphthalmia transcription factor regulates expression of the tartrate resistant acid phosphatase gene during terminal differentiation of osteoclasts. J Bone Miner Res 15:451 460. 110. Luch in, A., S. Suchting, T. Merson, T. J. Rosol, D. A. Hume, A. I. Cassady, and M. C. Ostrowski. 2001. Genetic and physical interactions between Microphthalmia transcription factor and PU.1 are necessary for osteoclast gene expression and differentiation. The Journal of biological chemistry 276:36703 36710. 111. Gohda, J., T. Akiyama, T. Koga, H. Takayanagi, S. Tanaka, and J. Inoue. 2005. RANK mediated amplification of TRAF6 signaling leads to NFATc1 induction during osteoclastogenesis. Embo J 24:790 799. 112 Cappellen, D., N. H. Luong Nguyen, S. Bongiovanni, O. Grenet, C. Wanke, and M. Susa. 2002. Transcriptional program of mouse osteoclast differentiation governed by the macrophage colony stimulating factor and the ligand for the receptor activator of NFkap pa B. The Journal of biological chemistry 277:21971 21982. 113. Miyamoto, T., and T. Suda. 2003. Differentiation and function of osteoclasts. Keio J Med 52:1 7. 114. Yao, G. Q., B. H. Sun, E. C. Weir, and K. L. Insogna. 2002. A role for cell surface CSF 1 in osteoblast mediated osteoclastogenesis. Calcif Tissue Int 70:339 346. 115. Fan, X., D. M. Biskobing, D. Fan, W. Hofstetter, and J. Rubin. 1997. Macrophage colony stimulating factor down regulates MCSF receptor expression and entry of progenitors into the osteoclast lineage. J Bone Miner Res 12:1387 1395. 116. Lum, L., B. R. Wong, R. Josien, J. D. Becherer, H. Erdjument Bromage, J. Schlondor ff, P. Tempst, Y. Choi, and C. P. Blobel. 1999. Evidence for a role of a tumor necrosis factor alpha (TNF alpha) converting enzyme like protease in shedding of TRANCE, a TNF family member involved in osteoclastogenesis and dendritic cell survival. The Jour nal of biological chemistry 274:13613 13618. 117. Oursler, M. J. 2010. Recent advances in understanding the mechanisms of osteoclast precursor fusion. Journal of cellular biochemistry 110:1058 1062.

PAGE 140

140 118. Cui, W., E. Cuartas, J. Ke, Q. Zhang, H. B. Einars son, J. D. Sedgwick, J. Li, and A. Vignery. 2007. CD200 and its receptor, CD200R, modulate bone mass via the differentiation of osteoclasts. Proc Natl Acad Sci U S A 104:14436 14441. 119. Lundberg, P., C. Koskinen, P. A. Baldock, H. Lothgren, A. Stenberg, U. H. Lerner, and P. A. Oldenborg. 2007. Osteoclast formation is strongly reduced both in vivo and in vitro in the absence of CD47/SIRPalpha interaction. Biochem Biophys Res Commun 352:444 448. 120. Mensah, K. A., C. T. Ritchlin, and E. M. Schwarz. 2010. RANKL induces heterogeneous DC STAMP(lo) and DC STAMP(hi) osteoclast precursors of which the DC STAMP(lo) precursors are the master fusogens. J Cell Physiol 223:76 83. 121. Yang, M., M. J. Birnbaum, C. A. MacKay, A. Mason Savas, B. Thompson, and P. R. Odgren. 2008. Osteoclast stimulatory transmembrane protein (OC STAMP), a novel protein induced by RANKL that promotes osteoclast differentiation. J Cell Physiol 215:497 505. 122. Akisaka, T., H. Yoshida S. Inoue, and K. Shimizu. 2001. Organization of cytoskeletal F actin, G actin, and gelsolin in the adhesion structures in cultured osteoclast. J Bone Miner Res 16:1248 1255. 123. Nakamura, I., J. Gailit, and T. Sasaki. 1996. Osteoclast integrin alphaVbe ta3 is present in the clear zone and contributes to cellular polarization. Cell Tissue Res 286:507 515. 124. Yamaki, M., H. Nakamura, N. Takahashi, N. Udagawa, and H. Ozawa. 2005. Transcytosis of calcium from bone by osteoclast like cells evidenced by dir ect visualization of calcium in cells. Arch Biochem Biophys 440:10 17. 125. Lee, B. S., S. L. Gluck, and L. S. Holliday. 1999. Interaction between vacuolar H(+) ATPase and microfilaments during osteoclast activation. The Journal of biological chemistry 27 4:29164 29171. 126. Blair, H. C., S. L. Teitelbaum, R. Ghiselli, and S. Gluck. 1989. Osteoclastic bone resorption by a polarized vacuolar proton pump. Science 245:855 857. 127. Forgac, M. 2007. Vacuolar ATPases: rotary proton pumps in physiology and path ophysiology. Nat Rev Mol Cell Biol 8:917 929. 128. Toyomura, T., Y. Murata, A. Yamamoto, T. Oka, G. H. Sun Wada, Y. Wada, and M. Futai. 2003. From lysosomes to the plasma membrane: localization of vacuolar type H+ ATPase with the a3 isoform during osteoc last differentiation. The Journal of biological chemistry 278:22023 22030.

PAGE 141

141 129. Laitala, T., and K. Vaananen. 1993. Proton channel part of vacuolar H(+) ATPase and carbonic anhydrase II expression is stimulated in resorbing osteoclasts. J Bone Miner Res 8: 119 126. 130. Blair, H. C., S. L. Teitelbaum, H. L. Tan, C. M. Koziol, and P. H. Schlesinger. 1991. Passive chloride permeability charge coupled to H(+) ATPase of avian osteoclast ruffled membrane. Am J Physiol 260:C1315 1324. 131. Sassi, M. L., H. Eriks en, L. Risteli, S. Niemi, J. Mansell, M. Gowen, and J. Risteli. 2000. Immunochemical characterization of assay for carboxyterminal telopeptide of human type I collagen: loss of antigenicity by treatment with cathepsin K. Bone 26:367 373. 132. Okada, Y., K Naka, K. Kawamura, T. Matsumoto, I. Nakanishi, N. Fujimoto, H. Sato, and M. Seiki. 1995. Localization of matrix metalloproteinase 9 (92 kilodalton gelatinase/type IV collagenase = gelatinase B) in osteoclasts: implications for bone resorption. Laboratory investigation; a journal of technical methods and pathology 72:311 322. 133. Hayman, A. R. 2008. Tartrate resistant acid phosphatase (TRAP) and the osteoclast/immune cell dichotomy. Autoimmunity 41:218 223. 134. Vaaraniemi, J., J. M. Halleen, K. Kaarlonen, H. Ylipahkala, S. L. Alatalo, G. Andersson, H. Kaija, P. Vihko, and H. K. Vaananen. 2004. Intracellular machinery for matrix degradation in bone resorbing osteoclasts. J Bone Miner Res 19:1432 1440. 135. Anders son, G., B. Ek Rylander, K. Hollberg, J. Ljusberg Sjolander, P. Lang, M. Norgard, Y. Wang, and S. J. Zhang. 2003. TRACP as an osteopontin phosphatase. J Bone Miner Res 18:1912 1915. 136. Ek Rylander, B., and G. Andersson. 2010. Osteoclast migration on phosphorylated osteopontin is regulated by endogenous tartrate resistant acid phosphatase. Exp Cell Res 316:443 451. 137. Bjorgaas, M., E. Haug, and H. J. Johnsen. 1999. The urinary excretio n of deoxypyridinium cross links is higher in diabetic than in nondiabetic adolescents. Calcif Tissue Int 65:121 124. 138. Ma, L., L. Oei, L. Jiang, K. Estrada, H. Chen, Z. Wang, Q. Yu, M. C. Zillikens, X. Gao, and F. Rivadeneira. 2012. Association betwee n bone mineral density and type 2 diabetes mellitus: a meta analysis of observational studies. Eur J Epidemiol 27:319 332.

PAGE 142

142 139. Schwartz, A. V., E. Vittinghoff, D. C. Bauer, T. A. Hillier, E. S. Strotmeyer, K. E. Ensrud, M. G. Donaldson, J. A. Cauley, T. B Harris, A. Koster, C. R. Womack, L. Palermo, and D. M. Black. 2011. Association of BMD and FRAX score with risk of fracture in older adults with type 2 diabetes. Jama 305:2184 2192. 140. McCabe, L. R. 2007. Understanding the pathology and mechanisms of type I diabetic bone loss. Journal of cellular biochemistry 102:1343 1357. 141. Maritim, A. C., R. A. Sanders, and J. B. Watkins, 3rd. 2003. Diabetes, oxidative stress, and antioxidants: a review. J Biochem Mol Toxicol 17:24 38. 142. Gale, E. A. 2001. Th e discovery of type 1 diabetes. Diabetes 50:217 226. 143. Steele, C., W. A. Hagopian, S. Gitelman, U. Masharani, M. Cavaghan, K. I. Rother, D. Donaldson, D. M. Harlan, J. Bluestone, and K. C. Herold. 2004. Insulin secretion in type 1 diabetes. Diabetes 53 :426 433. 144. Lieberman, S. M., and T. P. DiLorenzo. 2003. A comprehensive guide to antibody and T cell responses in type 1 diabetes. Tissue Antigens 62:359 377. 145. Aguilera, E., R. Casamitjana, G. Ercilla, J. Oriola, R. Gomis, and I. Conget. 2004. Ad ult onset atypical (type 1) diabetes: additional insights and differences with type 1A diabetes in a European Mediterranean population. Diabetes care 27:1108 1114. 146. Riley, W. J., N. K. Maclaren, J. Krischer, R. P. Spillar, J. H. Silverstein, D. A. Sch atz, S. Schwartz, J. Malone, S. Shah, C. Vadheim, and et al. 1990. A prospective study of the development of diabetes in relatives of patients with insulin dependent diabetes. The New England journal of medicine 323:1167 1172. 147. Imagawa, A., T. Hanafus a, J. Miyagawa, and Y. Matsuzawa. 2000. A novel subtype of type 1 diabetes mellitus characterized by a rapid onset and an absence of diabetes related antibodies. Osaka IDDM Study Group. The New England journal of medicine 342:301 307. 148. Anderson, M. S. and J. A. Bluestone. 2005. The NOD mouse: a model of immune dysregulation. Annu Rev Immunol 23:447 485. 149. Naguib, G., H. Al Mashat, T. Desta, and D. T. Graves. 2004. Diabetes prolongs the inflammatory response to a bacterial stimulus through cytokine dysregulation. J Invest Dermatol 123:87 92.

PAGE 143

143 150. Ayilavarapu, S., A. Kantarci, G. Fredman, O. Turkoglu, K. Omo ri, H. Liu, T. Iwata, M. Yagi, H. Hasturk, and T. E. Van Dyke. 2010. Diabetes induced oxidative stress is mediated by Ca2+ independent phospholipase A2 in neutrophils. J Immunol 184:1507 1515. 151. Goh, S. Y., and M. E. Cooper. 2008. Clinical review: The role of advanced glycation end products in progression and complications of diabetes. J Clin Endocrinol Metab 93:1143 1152. 152. Shanmugam, N., M. A. Reddy, M. Guha, and R. Natarajan. 2003. High glucose induced expression of proinflammatory cytokine and c hemokine genes in monocytic cells. Diabetes 52:1256 1264. 153. Inaba, M., M. Terada, H. Koyama, O. Yoshida, E. Ishimura, T. Kawagishi, Y. Okuno, Y. Nishizawa, S. Otani, and H. Morii. 1995. Influence of high glucose on 1,25 dihydroxyvitamin D3 induced effe ct on human osteoblast like MG 63 cells. J Bone Miner Res 10:1050 1056. 154. Nolan, C. J., P. Damm, and M. Prentki. 2011. Type 2 diabetes across generations: from pathophysiology to prevention and management. Lancet 378:169 181. 155. Poitout, V., and R. P. Robertson. 2008. Glucolipotoxicity: fuel excess and beta cell dysfunction. Endocrine reviews 29:351 366. 156. Fernandez Real, J. M., and J. C. Pickup. 2008. Innate immunity, insulin resistance and type 2 diabetes. Trends Endocrinol Metab 19:10 16. 157. Graves, D. T., and R. A. Kayal. 2008. Diabetic complications and dysregulated innate immunity. Front Biosci 13:1227 1239. 158. Sell, H., and J. Eckel. 2009. Chemotactic cytokines, obesity and type 2 diabetes: in vivo and in vitro evi dence for a possible causal correlation? Proc Nutr Soc 68:378 384. 159. Boyce, B. F., P. Li, Z. Yao, Q. Zhang, I. R. Badell, E. M. Schwarz, R. J. O'Keefe, and L. Xing. 2005. TNF alpha and pathologic bone resorption. Keio J Med 54:127 131. 160. Grey, A., M. A. Mitnick, U. Masiukiewicz, B. H. Sun, S. Rudikoff, R. L. Jilka, S. C. Manolagas, and K. Insogna. 1999. A role for interleukin 6 in parathyroid hormone induced bone resorption in vivo. Endocrinology 140:4683 4690. 161. Gingery, A., E. W. Bradley, L. P ederson, M. Ruan, N. J. Horwood, and M. J. Oursler. 2008. TGF beta coordinately activates TAK1/MEK/AKT/NFkB and SMAD pathways to promote osteoclast survival. Exp Cell Res 314:2725 2738.

PAGE 144

144 162. Zheng, M. H., Y. Fan, A. Smith, S. Wysocki, J. M. Papadimitriou, and D. J. Wood. 1998. Gene expression of monocyte chemoattractant protein 1 in giant cell tumors of bone osteoclastoma: possible involvement in CD68+ macrophage like cell migration. Journal of cellular biochemistry 70:121 129. 163. Clee, S. M., and A. D. Attie. 2007. The genetic landscape of type 2 diabetes in mice. Endocrine reviews 28:48 83. 164. Tesch, G. H., and A. K. Lim. 2011. Recent insights into diabetic renal injury from the db/db mouse model of type 2 diabetic nephropathy. Am J Physiol Renal Physiol 300:F301 310. 165. Rosenbloom, A. L., D. C. Lezotte, F. T. Weber, J. Gudat, D. R. Heller, M. L. Weber, S. Klein, and B. B. Kennedy. 1977. Diminution of bone mass in childhood diabetes. Diabetes 26:1052 1055. 166. Lettgen, B., B. Hauffa, C. Mohlmann, C. Jeken, and C. Reiners. 1995. Bone mineral density in children and adolescents with juvenile diabetes: selective measurement of bone mineral density of trabecular and cortical bone using peripheral quantitative computed tomography Hormone research 43:173 175. 167. Raskin, P., M. R. Stevenson, D. E. Barilla, and C. Y. Pak. 1978. The hypercalciuria of diabetes mellitus: its amelioration with insulin. Clinical endocrinology 9:329 335. 168. Yang, J., X. Zhang, W. Wang, and J. Liu. 2 010. Insulin stimulates osteoblast proliferation and differentiation through ERK and PI3K in MG 63 cells. Cell Biochem Funct 28:334 341. 169. Zhang, M., S. Xuan, M. L. Bouxsein, D. von Stechow, N. Akeno, M. C. Faugere, H. Malluche, G. Zhao, C. J. Rosen, A Efstratiadis, and T. L. Clemens. 2002. Osteoblast specific knockout of the insulin like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization. The Journal of biological chemistry 277:44005 44012. 170. Stolk, R. P., P. L. Van Daele, H. A. Pols, H. Burger, A. Hofman, J. C. Birkenhager, S. W. Lamberts, and D. E. Grobbee. 1996. Hyperinsulinemia and bone mineral density in an elderly population: The Rotterdam Study. Bone 18:545 549. 171. Saremi, A., R. G. Nelson, M. Tulloch Reid, R. L. Hanson, M. L. Sievers, G. W. Taylor, M. Shlossman, P. H. Bennett, R. Genco, and W. C. Knowler. 2005. Periodontal disease and mortality in type 2 diabetes. Diabetes care 28:27 32.

PAGE 145

145 172. Oren, T. W., S. Botolin, A. Williams, A. Bucknell, and K. B. King. 2011. Arthroplasty in veterans: analysis of cartilage, bone, serum, and synovial fluid reveals differences and similarities in osteoarthritis with and without comorbid diabetes. Journal of rehabilitation research and development 4 8:1195 1210. 173. Nanci, A., and D. D. Bosshardt. 2006. Structure of periodontal tissues in health and disease. Periodontol 2000 40:11 28. 174. Tsai, C., C. Hayes, and G. W. Taylor. 2002. Glycemic control of type 2 diabetes and severe periodontal disease in the US adult population. Community Dent Oral Epidemiol 30:182 192. 175. Lalla, E., B. Cheng, S. Lal, S. Tucker, E. Greenberg, R. Goland, and I. B. Lamster. 2006. Periodontal changes in children and adolescents with diabetes: a case control study. Diab etes Care 29:295 299. 176. Koromantzos, P. A., K. Makrilakis, X. Dereka, S. Offenbacher, N. Katsilambros, I. A. Vrotsos, and P. N. Madianos. 2012. Effect of non surgical periodontal therapy on C reactive protein, oxidative stress, and matrix metalloprotei nase (MMP) 9 and MMP 2 levels in patients with type 2 diabetes: a randomized controlled study. J Periodontol 83:3 10. 177. Commisso, L., M. Monami, and E. Mannucci. 2011. Periodontal disease and oral hygiene habits in a type 2 diabetic population. Int J Dent Hyg 9:68 73. 178. Schmidt, A. M., E. Weidman, E. Lalla, S. D. Yan, O. Hori, R. Cao, J. G. Brett, and I. B. Lamster. 1996. Advanced glycation endproducts (AGEs) induce oxidant stress in the gingiva: a potential mechanism underlying accelerated periodontal disease associated with diabetes. J Periodontal Res 31:508 515. 179. Dasu, M. R., S. Devaraj, S. Park, and I. Jialal. 2010. Increased toll like receptor (TLR) activation and TLR ligands in recently diagnosed type 2 diabetic subjects. Diabetes care 33:861 868. 180. Mohammad M. K., M. Morran, B. Slotterbeck, D. W. Leaman, Y. Sun, H. Grafenstein, S. C. Hong, and M. F. McInerney. 2006. Dysregulated Toll like receptor expression and signaling in bone marrow derived macrophages at the onset of diabetes in the non obese diabetic mouse. Int Immunol 18:1101 1113. 181. Plesner, A., C. J. Greenbaum, L. K. Gaur, R. K. Ernst, and A. Lernmark. 2002. Macrophages from high risk HLA DQB1*0201/*0302 type 1 diabetes mellitus patients are hypersensitive to lipopolysaccharide stimulation. Scan dinavian journal of immunology 56:522 529.

PAGE 146

146 182. Angelini, F., E. Del Duca, S. Piccinini, V. Pacciani, P. Rossi, and M. L. Manca Bitti. 2005. Altered phenotype and function of dendritic cells in children with type 1 diabetes. Clin Exp Immunol 142:341 346. 183. Takahashi, K., M. C. Honeyman, and L. C. Harrison. 1998. Impaired yield, phenotype, and function of monocyte derived dendritic cells in humans at risk for insulin dependent diabetes. J Immunol 161:2629 2635. 184. Lee, M., A. Y. Kim, and Y. Kang. 200 0. Defects in the differentiation and function of bone marrow derived dendritic cells in non obese diabetic mice. J Korean Med Sci 15:217 223. 185. Strid, J., L. Lopes, J. Marcinkiewicz, L. Petrovska, B. Nowak, B. M. Chain, and T. Lund. 2001. A defect in bone marrow derived dendritic cell maturation in the nonobesediabetic mouse. Clin Exp Immunol 123:375 381. 186. Poligone, B., D. J. Weaver, Jr., P. Sen, A. S. Baldwin, Jr., and R. Tisch. 2002. Elevated NF kappaB activation in nonobese diabetic mouse dendr itic cells results in enhanced APC function. J Immunol 168:188 196. 187. Weaver, D. J., Jr., B. Poligone, T. Bui, U. M. Abdel Motal, A. S. Baldwin, Jr., and R. Tisch. 2001. Dendritic cells from nonobese diabetic mice exhibit a defect in NF kappa B regulat ion due to a hyperactive I kappa B kinase. J Immunol 167:1461 1468. 188. Wheat, W., R. Kupfer, D. G. Gutches, G. R. Rayat, J. Beilke, R. I. Scheinman, and D. R. Wegmann. 2004. Increased NF kappa B activity in B cells and bone marrow derived dendritic cell s from NOD mice. European journal of immunology 34:1395 1404. 189. Bradshaw, E. M., K. Raddassi, W. Elyaman, T. Orban, P. A. Gottlieb, S. C. Kent, and D. A. Hafler. 2009. Monocytes from patients with type 1 diabetes spontaneously secrete proinflammatory c ytokines inducing Th17 cells. J Immunol 183:4432 4439. 190. Sen, P., S. Bhattacharyya, M. Wallet, C. P. Wong, B. Poligone, M. Sen, A. S. Baldwin, Jr., and R. Tisch. 2003. NF kappa B hyperactivation has differential effects on the APC function of nonobese diabetic mouse macrophages. J Immunol 170:1770 1780. 191. Stoffels, K., L. Overbergh, A. Giulietti, A. Kasran, R. Bouillon, C. Gysemans, and C. Mathieu. 2004. NOD macrophages produce high levels of inflammatory cytokines upon encounter of apoptotic or nec rotic cells. J Autoimmun 23:9 15.

PAGE 147

147 192. Alleva, D. G., R. P. Pavlovich, C. Grant, S. B. Kaser, and D. I. Beller. 2000. Aberrant macrophage cytokine production is a conserved feature among autoimmune prone mouse strains: elevated interleukin (IL) 12 and an imbalance in tumor necrosis factor alpha and IL 10 define a unique cytokine profile in macrophages from young nonobese diabetic mice. Diabetes 49:1106 1115. 193. Soysa, N. S., and N. Alles. 2009. NF kappaB functions in osteoclasts. Biochem Biophys Res Commun 378:1 5. 194. Biswas, S. K., and E. Lopez Collazo. 2009. Endotoxin tolerance: new mechanisms, molecules and clinical significance. Trends Immunol 30:475 487. 195. West, M. A., and W. Heagy. 2002. Endotoxin tolerance: a review. Critical care medicine 30:S64 73. 196. Kobayashi, K. S., and R A. Flavell. 2004. Shielding the double edged sword: negative regulation of the innate immune system. Journal of leukocyte biology 75:428 433. 197. Nomura, F., S. Akashi, Y. Sakao, S. Sato, T. Kawai, M. Matsumoto, K. Nakanishi, M. Kimoto, K. Miyake, K. T akeda, and S. Akira. 2000. Cutting edge: endotoxin tolerance in mouse peritoneal macrophages correlates with down regulation of surface toll like receptor 4 expression. J Immunol 164:3476 3479. 198. Jiang, J., H. Li, F. S. Fahid, E. Filbert, K. E. Safavi, L. S. Spangberg, and Q. Zhu. 2006. Quantitative analysis of osteoclast specific gene markers stimulated by lipopolysaccharide. J Endod 32:742 746. 199. Jiang, J., J. Zuo, I. R. Hurst, and L. S. Holliday. 2003. The synergistic effect of peptidoglycan and lipopolysaccaride on osteoclast formation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 96:738 743. 200. Liu, J., S. Wang, P. Zhang, N. Said Al Naief, S. M. Michalek, and X. Feng. 2009. Molecular mechanism of the bifunctional role of lipopolysaccharid e in osteoclastogenesis. The Journal of biological chemistry 284:12512 12523. 201. Zou, W., and Z. Bar Shavit. 2002. Dual modulation of osteoclast differentiation by lipopolysaccharide. J Bone Miner Res 17:1211 1218. 202. Takami, M., N. Kim, J. Rho, and Y. Choi. 2002. Stimulation by toll like receptors inhibits osteoclast differentiation. J Immunol 169:1516 1523.

PAGE 148

148 203. Simonet, W. S., D. L. Lacey, C. R. Dunstan, M. Kelley, M. S. Chang, R. Luthy, H. Q. Nguyen, S. Wooden, L. B ennett, T. Boone, G. Shimamoto, M. DeRose, R. Elliott, A. Colombero, H. L. Tan, G. Trail, J. Sullivan, E. Davy, N. Bucay, L. Renshaw Gegg, T. M. Hughes, D. Hill, W. Pattison, P. Campbell, S. Sander, G. Van, J. Tarpley, P. Derby, R. Lee, and W. J. Boyle. 19 97. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309 319. 204. Lacey, D. L., E. Timms, H. L. Tan, M. J. Kelley, C. R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, H. Hsu, J. Sulliva n, N. Hawkins, E. Davy, C. Capparelli, A. Eli, Y. X. Qian, S. Kaufman, I. Sarosi, V. Shalhoub, G. Senaldi, J. Guo, J. Delaney, and W. J. Boyle. 1998. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165 176. 205. Takeda, K., and S. Akira. 2004. TLR signaling pathways. Seminars in immunology 16:3 9. 206. He, L., M. He, X. Lv, D. Pu, P. Su, and Z. Liu. 2010. NF kappaB binding activity and pro inflammatory cytokines expression correlate with body mass ind ex but not glycosylated hemoglobin in Chinese population. Diabetes Res Clin Pract 90:73 80. 207. Karima, M., A. Kantarci, T. Ohira, H. Hasturk, V. L. Jones, B. H. Nam, A. Malabanan, P. C. Trackman, J. A. Badwey, and T. E. Van Dyke. 2005. Enhanced superoxide release and elevated protein kinase C activity in neutrophils from diabetic patients: associati on with periodontitis. Journal of leukocyte biology 78:862 870. 208. Foss Freitas, M. C., N. T. Foss, D. M. Rassi, E. A. Donadi, and M. C. Foss. 2008. Evaluation of cytokine production from peripheral blood mononuclear cells of type 1 diabetic patients. A nn N Y Acad Sci 1150:290 296. 209. Arif, S., T. I. Tree, T. P. Astill, J. M. Tremble, A. J. Bishop, C. M. Dayan, B. O. Roep, and M. Peakman. 2004. Autoreactive T cell responses show proinflammatory polarization in diabetes but a regulatory phenotype in he alth. The Journal of clinical investigation 113:451 463. 210. Matsuo, K., and N. Ray. 2004. Osteoclasts, mononuclear phagocytes, and c Fos: new insight into osteoimmunology. Keio J Med 53:78 84. 211. Takayanagi, H. 2009. Osteoimmunology and the effects o f the immune system on bone. Nat Rev Rheumatol 5:667 676. 212. Lee, S. H., T. S. Kim, Y. Choi, and J. Lorenzo. 2008. Osteoimmunology: cytokines and the skeletal system. BMB Rep 41:495 510.

PAGE 149

149 213. Kobayashi, K., N. Takahashi, E. Jimi, N. Udagawa, M. Takami, S. Kotake, N. Nakagawa, M. Kinosaki, K. Yamaguchi, N. Shima, H. Yasuda, T. Morinaga, K. Higashio, T. J. Martin, and T. Suda. 2000. Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL RANK interactio n. The Journal of experimental medicine 191:275 286. 214. Kitajima, I., T. Nakajima, T. Imamura, I. Takasaki, K. Kawahara, T. Okano, T. Tokioka, Y. Soejima, K. Abeyama, and I. Maruyama. 1996. Induction of apoptosis in murine clonal osteoblasts expressed b y human T cell leukemia virus type I tax by NF kappa B and TNF alpha. J Bone Miner Res 11:200 210. 215. Centrella, M., T. L. McCarthy, and E. Canalis. 1988. Tumor necrosis factor alpha inhibits collagen synthesis and alkaline phosphatase activity independ ently of its effect on deoxyribonucleic acid synthesis in osteoblast enriched bone cell cultures. Endocrinology 123:1442 1448. 216. Nakase, T., K. Takaoka, K. Masuhara, K. Shimizu, H. Yoshikawa, and T. Ochi. 1997. Interleukin 1 beta enhances and tumor nec rosis factor alpha inhibits bone morphogenetic protein 2 induced alkaline phosphatase activity in MC3T3 E1 osteoblastic cells. Bone 21:17 21. 217. Kitaura, H., P. Zhou, H. J. Kim, D. V. Novack, F. P. Ross, and S. L. Teitelbaum. 2005. M CSF mediates TNF in duced inflammatory osteolysis. The Journal of clinical investigation 115:3418 3427. 218. Takaoka, Y., S. Niwa, and H. Nagai. 1999. Interleukin 1beta induces interleukin 6 production through the production of prostaglandin E(2) in human osteoblasts, MG 63 cells. Journal of biochemistry 126:553 558. 219. Park, Y. G., S. K. Kang, W. J. Kim, Y. C. Lee, and C. H. Kim. 2004. Effects of TGF beta, TNF alpha, IL beta and IL 6 alone or in combination, and tyrosine kinase inhibitor on cyclooxygenase expression, pros taglandin E2 production and bone resorption in mouse calvarial bone cells. The international journal of biochemistry & cell biology 36:2270 2280. 220. Ma, T., K. Miyanishi, A. Suen, N. J. Epstein, T. Tomita, R. L. Smith, and S. B. Goodman. 2004. Human int erleukin 1 induced murine osteoclastogenesis is dependent on RANKL, but independent of TNF alpha. Cytokine 26:138 144. 221. Sato, N., N. Takahashi, K. Suda, M. Nakamura, M. Yamaki, T. Ninomiya, Y. Kobayashi, H. Takada, K. Shibata, M. Yamamoto, K. Takeda, S. Akira, T. Noguchi, and N. Udagawa. 2004. MyD88 but not TRIF is essential for osteoclastogenesis induced by lipopolysacchar ide, diacyl lipopeptide, and IL 1alpha. The Journal of experimental medicine 200:601 611.

PAGE 150

150 222. De Benedetti, F., P. Pignatti, V. Gerloni, M. Massa, P. Sartirana, R. Caporali, C. M. Montecucco, A. Corti, F. Fantini, and A. Martini. 1997. Differences in syno vial fluid cytokine levels between juvenile and adult rheumatoid arthritis. The Journal of rheumatology 24:1403 1409. 223. Baumhauer, J. F., R. J. O'Keefe, L. C. Schon, and M. S. Pinzur. 2006. Cytokine induced osteoclastic bone resorption in charcot arthr opathy: an immunohistochemical study. Foot Ankle Int 27:797 800. 224. Houssiau, F. A., J. P. Devogelaer, J. Van Damme, C. N. de Deuxchaisnes, and J. Van Snick. 1988. Interleukin 6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis and rheumatism 31:784 788. 225. U son, J., A. Balsa, D. Pascual Salcedo, J. A. Cabezas, J. M. Gonzalez Tarrio, E. Martin Mola, and G. Fontan. 1997. Soluble interleukin 6 (IL 6) receptor and IL 6 levels in serum and synovial fluid of patients with different arthropathies. The Journal of rhe umatology 24:2069 2075. 226. Bozec, A., L. Bakiri, A. Hoebertz, R. Eferl, A. F. Schilling, V. Komnenovic, H. Scheuch, M. Priemel, C. L. Stewart, M. Amling, and E. F. Wagner. 2008. Osteoclast size is controlled by Fra 2 through LIF/LIF receptor signalling and hypoxia. Nature 454:221 225. 227. Poulton, I. J., N. E. McGregor, S. Pompolo, E. C. Walker, and N. A. Sims. 2012. Contrasting roles of leukemia inhibitory factor in murine bone development and remodeling involve region specific changes in vascularizat ion. J Bone Miner Res 27:586 595. 228. Walker, E. C., N. E. McGregor, I. J. Poulton, M. Solano, S. Pompolo, T. J. Fernandes, M. J. Constable, G. C. Nicholson, J. G. Zhang, N. A. Nicola, M. T. Gillespie, T. J. Martin, and N. A. Sims. 2010. Oncostatin M pro motes bone formation independently of resorption when signaling through leukemia inhibitory factor receptor in mice. The Journal of clinical investigation 120:582 592. 229. Palmqvist, P., E. Persson, H. H. Conaway, and U. H. Lerner. 2002. IL 6, leukemia i nhibitory factor, and oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF kappa B ligand, osteoprotegerin, and receptor activator of NF kappa B in mouse calvariae. J Immunol 169:3353 3362. 230. Mohamed, S. G., E. Sugiyama, K. Shinoda, H. Taki, H. Hounoki, H. O. Abdel Aziz, M. Maruyama, M. Kobayashi, H. Ogawa, and T. Miyahara. 2007. Interleukin 10 inhibits RANKL mediated expression of NFATc1 in part via suppression of c Fos and c Jun in RAW264.7 cells and mouse bon e marrow cells. Bone 41:592 602.

PAGE 151

151 231. Claudino, M., A. P. Trombone, C. R. Cardoso, S. B. Ferreira, Jr., W. Martins, Jr., G. F. Assis, C. F. Santos, P. C. Trevilatto, A. P. Campanelli, J. S. Silva, and G. P. Garlet. 2008. The broad effects of the functional IL 10 promoter 592 polymorphism: modulation of IL 10, TIMP 3, and OPG expression and their association with periodontal disease outcome. Journal of leukocyte biology 84:1565 1573. 232. Liu, D., S. Yao, and G. E. Wise. 2006. Effect of interleukin 10 on ge ne expression of osteoclastogenic regulatory molecules in the rat dental follicle. European journal of oral sciences 114:42 49. 233. Takayanagi, H., K. Ogasawara, S. Hida, T. Chiba, S. Murata, K. Sato, A. Takaoka, T. Yokochi, H. Oda, K. Tanaka, K. Nakamur a, and T. Taniguchi. 2000. T cell mediated regulation of osteoclastogenesis by signalling cross talk between RANKL and IFN gamma. Nature 408:600 605. 234. Kotake, S., Y. Nanke, M. Mogi, M. Kawamoto, T. Furuya, T. Yago, T. Kobashigawa, A. Togari, and N. Ka matani. 2005. IFN gamma producing human T cells directly induce osteoclastogenesis from human monocytes via the expression of RANKL. European journal of immunology 35:3353 3363. 235. Philip, R., and L. B. Epstein. 1986. Tumour necrosis factor as immunomod ulator and mediator of monocyte cytotoxicity induced by itself, gamma interferon and interleukin 1. Nature 323:86 89. 236. Kim, H. S., and M. S. Lee. 2009. Role of innate immunity in triggering and tuning of autoimmune diabetes. Curr Mol Med 9:30 44. 237. von Herrath, M. G., and M. B. Oldstone. 1997. Interferon gamma is essential for destruction of beta cells and development of insulin dependent diabetes mellitus. The Journal of experimental medicine 185:531 539. 238. Tsiavou, A., E. Hatziagelaki, A. Chaidaroglou, K. Koniavitou, D. Degiannis, and S. A. Raptis. 2005. Correlation between intracellular interferon gamma (IFN gamma) production by CD4+ and CD8+ lymphocytes and IFN gamma gene polymorphism in patients with type 2 diabetes mellitus and latent a utoimmune diabetes of adults (LADA). Cytokine 31:135 141. 239. Blakytny, R., M. Spraul, and E. B. Jude. 2011. Review: The diabetic bone: a cellular and molecular perspective. Int J Low Extrem Wounds 10:16 32. 240. He, H., R. Liu, T. Desta, C. Leone, L. C Gerstenfeld, and D. T. Graves. 2004. Diabetes causes decreased osteoclastogenesis, reduced bone formation, and enhanced apoptosis of osteoblastic cells in bacteria stimulated bone loss. Endocrinology 145:447 452.

PAGE 152

152 241. Verhaeghe, J., A. M. Suiker, B. L. N yomba, W. J. Visser, T. A. Einhorn, J. Dequeker, and R. Bouillon. 1989. Bone mineral homeostasis in spontaneously diabetic BB rats. II. Impaired bone turnover and decreased osteocalcin synthesis. Endocrinology 124:573 582. 242. Horcajada Molteni, M. N., B Chanteranne, P. Lebecque, M. J. Davicco, V. Coxam, A. Young, and J. P. Barlet. 2001. Amylin and bone metabolism in streptozotocin induced diabetic rats. J Bone Miner Res 16:958 965. 243. Lu, H., D. Kraut, L. C. Gerstenfeld, and D. T. Graves. 2003. Diabetes interferes with the bone formation by affecting the expression of transcription factors that regulate osteoblast differentiation. Endocrinology 144:346 352. 244. Botolin, S., and L. R. McCabe. 2006. Inhibition of PPARgamma prevents type I diabetic bone marrow adiposity but not bone loss. J Cell Physiol 209:967 976. 245. Botolin, S., M. C. Faugere, H. Malluche, M. Orth, R. Meyer, and L. R. McCabe. 2005. Increased bone adiposity and perox isomal proliferator activated receptor gamma2 expression in type I diabetic mice. Endocrinology 146:3622 3631. 246. Kim, J. M., D. Jeong, H. K. Kang, S. Y. Jung, S. S. Kang, and B. M. Min. 2007. Osteoclast precursors display dynamic metabolic shifts towar d accelerated glucose metabolism at an early stage of RANKL stimulated osteoclast differentiation. Cell Physiol Biochem 20:935 946. 247. Williams, J. P., H. C. Blair, J. M. McDonald, M. A. McKenna, S. E. Jordan, J. Williford, and R. W. Hardy. 1997. Regula tion of osteoclastic bone resorption by glucose. Biochem Biophys Res Commun 235:646 651. 248. Larsen, K. I., M. L. Falany, L. V. Ponomareva, W. Wang, and J. P. Williams. 2002. Glucose dependent regulation of osteoclast H(+) ATPase expression: potential ro le of p38 MAP kinase. Journal of cellular biochemistry 87:75 84. 249. Wittrant, Y., Y. Gorin, K. Woodruff, D. Horn, H. E. Abboud, S. Mohan, and S. L. Abboud Werner. 2008. High d(+)glucose concentration inhibits RANKL induced osteoclastogenesis. Bone 42:11 22 1130. 250. Suzuki, K., N. Miyakoshi, T. Tsuchida, Y. Kasukawa, K. Sato, and E. Itoi. 2003. Effects of combined treatment of insulin and human parathyroid hormone(1 34) on cancellous bone mass and structure in streptozotocin induced diabetic rats. Bone 33:108 114. 251. Hie, M., M. Yamazaki, and I. Tsukamoto. 2009. Curcumin suppresses increased bone resorption by inhibiting osteoclastogenesis in rats with streptozotocin induced diabetes. European journal of pharmacology 621:1 9.

PAGE 153

153 252. Hie, M., M. Shimono, K. Fujii, and I. Tsukamoto. 2007. Increased cathepsin K and tartrate resistant acid phosphatase expression in bone of streptozotocin induced diabetic rats. Bone 41:1045 1050. 253. Tamasi, J. A., B. J. Arey, D. R. Bertolini, and J. H. Feyen. 2003. Characterization of bone structure in leptin receptor deficient Zucker (fa/fa) rats. J Bone Miner Res 18:1605 1611. 254. Fujii, H., Y. Hamada, and M. Fukagawa. 2008. Bone formation in spontaneously diabetic Torii newly established model of no n obese type 2 diabetes rats. Bone 42:372 379. 255. Shinohara, M., T. Masuyama, T. Shoda, T. Takahashi, Y. Katsuda, K. Komeda, M. Kuroki, A. Kakehashi, and Y. Kanazawa. 2000. A new spontaneously diabetic non obese Torii rat strain with severe ocular compl ications. International journal of experimental diabetes research 1:89 100. 256. Clark, J. B., C. J. Palmer, and W. N. Shaw. 1983. The diabetic Zucker fatty rat. Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Bi ology and Medicine (New York, N.Y 173:68 75. 257. Like, A. A., M. C. Appel, R. M. Williams, and A. A. Rossini. 1978. Streptozotocin induced pancreatic insulitis in mice. Morphologic and physiologic studies. Laboratory investigation; a journal of technical methods and pathology 38:470 486. 258. Hamada, Y., H. Fujii, R. Kitazawa, J. Yodoi, S. Kitazawa, and M. Fukagawa. 2009. Thioredoxin 1 overexpression in transgenic mice attenuates streptozotocin induced diabetic osteopenia: a novel role of oxidative stres s and therapeutic implications. Bone 44:936 941. 259. Kasahara, T., S. Imai, H. Kojima, M. Katagi, H. Kimura, L. Chan, and Y. Matsusue. 2010. Malfunction of bone marrow derived osteoclasts and the delay of bone fracture healing in diabetic mice. Bone 47:6 17 625. 260. Kayal, R. A., D. Tsatsas, M. A. Bauer, B. Allen, M. O. Al Sebaei, S. Kakar, C. W. Leone, E. F. Morgan, L. C. Gerstenfeld, T. A. Einhorn, and D. T. Graves. 2007. Diminished bone formation during diabetic fracture healing is related to the prem ature resorption of cartilage associated with increased osteoclast activity. J Bone Miner Res 22:560 568. 261. Kayal, R. A., J. Alblowi, E. McKenzie, N. Krothapalli, L. Silkman, L. Gerstenfeld, T. A. Einhorn, and D. T. Graves. 2009. Diabetes causes the accelerated loss of cartilage during fracture repair which is reversed by insulin treatment. Bone 44:357 363.

PAGE 154

154 262. Guha, M., W. Bai, J. L. Nadler, and R. Natarajan. 2000. Molecular mechanisms of tumor necrosis factor alpha gene expression in monocytic cells via hyperglycemia induced oxidant stress dependent and independent pathways. The Journal of biological chemistr y 275:17728 17739. 263. Calder, P. C., G. Dimitriadis, and P. Newsholme. 2007. Glucose metabolism in lymphoid and inflammatory cells and tissues. Curr Opin Clin Nutr Metab Care 10:531 540. 264. Jakus, V., and N. Rietbrock. 2004. Advanced glycation end pr oducts and the progress of diabetic vascular complications. Physiol Res 53:131 142. 265. Santana, R. B., L. Xu, H. B. Chase, S. Amar, D. T. Graves, and P. C. Trackman. 2003. A role for advanced glycation end products in diminished bone healing in type 1 di abetes. Diabetes 52:1502 1510. 266. Lin, L., S. Park, and E. G. Lakatta. 2009. RAGE signaling in inflammation and arterial aging. Front Biosci 14:1403 1413. 267. Miyata, T., K. Notoya, K. Yoshida, K. Horie, K. Maeda, K. Kurokawa, and S. Taketomi. 1997. Advanced glycation end products enhance osteoclast induced bone resorption in cultured mouse unfractionated bone cells and in rats implanted subcutaneously with devi talized bone particles. J Am Soc Nephrol 8:260 270. 268. Lalla, E., I. B. Lamster, M. Feit, L. Huang, A. Spessot, W. Qu, T. Kislinger, Y. Lu, D. M. Stern, and A. M. Schmidt. 2000. Blockade of RAGE suppresses periodontitis associated bone loss in diabetic mice. The Journal of clinical investigation 105:1117 1124. 269. Ding, K. H., Z. Z. Wang, M. W. Hamrick, Z. B. Deng, L. Zhou, B. Kang, S. L. Yan, J. X. She, D. M. Stern, C. M. Isales, and Q. S. Mi. 2006. Disordered osteoclast formation in RAGE deficient mo use establishes an essential role for RAGE in diabetes related bone loss. Biochem Biophys Res Commun 340:1091 1097. 270. Yoshida, T., A. Flegler, A. Kozlov, and P. H. Stern. 2009. Direct inhibitory and indirect stimulatory effects of RAGE ligand S100 on s RANKL induced osteoclastogenesis. Journal of cellular biochemistry 107:917 925. 271. Zhou, Z., and W. C. Xiong. 2011. RAGE and its ligands in bone metabolism. Frontiers in bioscience (Scholar edition) 3:768 776. 272. Thomas, D. M., N. Udagawa, D. K. Hard s, J. M. Quinn, J. M. Moseley, D. M. Findlay, and J. D. Best. 1998. Insulin receptor expression in primary and cultured osteoclast like cells. Bone 23:181 186.

PAGE 155

155 273. Thrailkill, K. M., L. Liu, E. C. Wahl, R. C. Bunn, D. S. Perrien, G. E. Cockrell, R. A. Ski nner, W. R. Hogue, A. A. Carver, J. L. Fowlkes, J. Aronson, and C. K. Lumpkin, Jr. 2005. Bone formation is impaired in a model of type 1 diabetes. Diabetes 54:2875 2881. 274. Botolin, S., and L. R. McCabe. 2007. Bone loss and increased bone adiposity in s pontaneous and pharmacologically induced diabetic mice. Endocrinology 148:198 205. 275. Preshaw, P. M. 2009. Periodontal disease and diabetes. J Dent 37:S575 577. 276. Kinane, D. F. 2001. Causation and pathogenesis of periodontal disease. Periodontol 200 0 25:8 20. 277. Hallmon, W. W., and B. L. Mealey. 1992. Implications of diabetes mellitus and periodontal disease. Diabetes Educ 18:310 315. 278. Teng, Y. T. 2006. Protective and destructive immunity in the periodontium: Part 1 -innate and humoral immuni ty and the periodontium. J Dent Res 85:198 208. 279. Teng, Y. T. 2006. Protective and destructive immunity in the periodontium: Part 2 -T cell mediated immunity in the periodontium. J Dent Res 85:209 219. 280. Fuller, K., B. Kirstein, and T. J. Chambers. 2006. Murine osteoclast formation and function: differential regulation by humoral agents. Endocrinology 147:1979 1985. 281. Taubman, M. A., P. Valverde, X. Han, and T. Kawai. 2005. Immune response: the key to bone resorption in periodontal disease. J Periodontol 76:2033 2041. 282. Ryan, M. E., O. Carnu, and A. Kamer. 2003. The influence of diabetes on the periodontal tissues. J Am Dent Assoc 134 Spec No:34S 40S. 283. Manouchehr Pour, M., P. J. Spagnuolo, H. M. Rodman, and N. F. Bissada. 1981. Impai red neutrophil chemotaxis in diabetic patients with severe periodontitis. J Dent Res 60:729 730. 284. Kumar, M. S., G. Vamsi, R. Sripriya, and P. K. Sehgal. 2006. Expression of matrix metalloproteinases (MMP 8 and 9) in chronic periodontitis patients wit h and without diabetes mellitus. J Periodontol 77:1803 1808. 285. Safkan Seppala, B., T. Sorsa, T. Tervahartiala, A. Beklen, and Y. T. Konttinen. 2006. Collagenases in gingival crevicular fluid in type 1 diabetes mellitus. J Periodontol 77:189 194.

PAGE 156

156 286. F icara, A. J., M. P. Levin, M. F. Grower, and G. D. Kramer. 1975. A comparison of the glucose and protein content of gingival fluid from diabetics and nondiabetics. J Periodontal Res 10:171 175. 287. Yamaguchi, M., R. Takada, S. Kambe, T. Hatakeyama, K. Na itoh, K. Yamazaki, and M. Kobayashi. 2005. Evaluation of time course changes of gingival crevicular fluid glucose levels in diabetics. Biomed Microdevices 7:53 58. 288. Schett, G. 2008. Review: Immune cells and mediators of inflammatory arthritis. Autoimm unity 41:224 229. 289. Myasoedova, E., C. S. Crowson, H. M. Kremers, T. M. Therneau, and S. E. Gabriel. 2010. Is the incidence of rheumatoid arthritis rising?: results from Olmsted County, Minnesota, 1955 2007. Arthritis Rheum 62:1576 1582. 290. Hitchon, C. A., P. Alex, L. B. Erdile, M. B. Frank, I. Dozmorov, Y. Tang, K. Wong, M. Centola, and H. S. El Gabalawy. 2004. A distinct multicytokine profile is associated with anti cyclical citrullinated peptide antibodies in patients with early untreated inflamma tory arthritis. The Journal of rheumatology 31:2336 2346. 291. Neidel, J., M. Schulze, and J. Lindschau. 1995. Association between degree of bone erosion and synovial fluid levels of tumor necrosis factor alpha in the knee joints of patients with rheumato id arthritis. Inflamm Res 44:217 221. 292. Sharif, S., J. M. Thomas, D. A. Donley, D. L. Gilleland, D. E. Bonner, J. L. McCrory, W. G. Hornsby, H. Zhao, M. W. Lively, J. A. Hornsby, and S. E. Alway. 2011. Resistance exercise reduces skeletal muscle cachex ia and improves muscle function in rheumatoid arthritis. Case Report Med 2011:205691. 293. Engvall, I. L., A. C. Elkan, B. Tengstrand, T. Cederholm, K. Brismar, and I. Hafstrom. 2008. Cachexia in rheumatoid arthritis is associated with inflammatory activi ty, physical disability, and low bioavailable insulin like growth factor. Scand J Rheumatol 37:321 328. 294. Shaw, N. E., and B. F. Martin. 1962. Histological and histochemical studies on mammalian knee joint tissues. J Anat 96:359 373. 295. Edwards, J. C. 1994. The nature and origins of synovium: experimental approaches to the study of synoviocyte differentiation. J Anat 184 ( Pt 3):493 501. 296. Kiener, H. P., and T. Karonitsch. 2011. The synovium as a privileged site in rheumatoid arthriti s: cadherin 11 as a dominant player in synovial pathology. Best Pract Res Clin Rheumatol 25:767 777.

PAGE 157

157 297. Tiwari, N., S. Chabra, S. Mehdi, P. Sweet, T. B. Krasieva, R. Pool, B. Andrews, and G. M. Peavy. 2010. Imaging of normal and pathologic joint synovium using nonlinear optical microscopy as a potential diagnostic tool. J Biomed Opt 15:056001. 298. Gorman, C. L., and A. P. Cope. 2008. Immune mediated pathways in chronic inflammatory arthritis. Best Pract Res Clin Rheumatol 22:221 238. 299. Burmester, G. R., B. Stuhlmuller, G. Keyszer, and R. W. Kinne. 1997. Mononuclear phagocytes and rheumatoid synovitis. Mastermind or workhorse in arthritis? Arthritis Rheum 40:5 18. 300. Bartok, B., and G. S. Firestein. 2010. Fibroblast like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev 233:233 255. 301. Firestein, G. S., J. M. Alvaro Gracia, and R. Maki. 1990. Quantitative analysis of cytokine gene expression in rheumatoid arthritis. J Immunol 144:3347 3353. 302. Bellatin, M. F., M. Han, M. Fallena, L. Fan, D. Xia, N. Olsen, V. Branch, D. Karp, and P. Stastny. 2012. Production of autoantibodies against citrullinated antigens/peptides by human B cells. J Immunol 188:3542 3550. 303. Asquith, D. L., A. M. Miller, I. B. McInnes, and F Y. Liew. 2009. Animal models of rheumatoid arthritis. European journal of immunology 39:2040 2044. 304. Nielen, M. M., D. van Schaardenburg, H. W. Reesink, R. J. van de Stadt, I. E. van der Horst Bruinsma, M. H. de Koning, M. R. Habibuw, J. P. Vandenbro ucke, and B. A. Dijkmans. 2004. Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. Arthritis Rheum 50:380 386. 305. Schett, G., M. Stolina, B. Bolon, S. Middleton, M. Adlam, H. Brown, L. Z hu, U. Feige, and D. J. Zack. 2005. Analysis of the kinetics of osteoclastogenesis in arthritic rats. Arthritis Rheum 52:3192 3201. 306. Hayer, S., K. Redlich, A. Korb, S. Hermann, J. Smolen, and G. Schett. 2007. Tenosynovitis and osteoclast formation as the initial preclinical changes in a murine model of inflammatory arthritis. Arthritis Rheum 56:79 88. 307. Machold, K. P., T. A. Stamm, V. P. Nell, S. Pflugbeil, D. Aletaha, G. Steiner, M. Uffmann, and J. S. Smolen. 2007. Very recent onset rheumatoid art hritis: clinical and serological patient characteristics associated with radiographic progression over the first years of disease. Rheumatology (Oxford) 46:342 349.

PAGE 158

158 308. Marron, M. P., L. J. Raffel, H. J. Garchon, C. O. Jacob, M. Serrano Rios, M. T. Martin ez Larrad, W. P. Teng, Y. Park, Z. X. Zhang, D. R. Goldstein, Y. W. Tao, G. Beaurain, J. F. Bach, H. S. Huang, D. F. Luo, A. Zeidler, J. I. Rotter, M. C. Yang, T. Modilevsky, N. K. Maclaren, and J. X. She. 1997. Insulin dependent diabetes mellitus (IDDM) i s associated with CTLA4 polymorphisms in multiple ethnic groups. Human molecular genetics 6:1275 1282. 309. Wallis, S. K., L. A. Cooney, J. L. Endres, M. J. Lee, J. Ryu, E. C. Somers, and D. A. Fox. 2011. A polymorphism in the interleukin 4 receptor affec ts the ability of interleukin 4 to regulate Th17 cells: a possible immunoregulatory mechanism for genetic control of the severity of rheumatoid arthritis. Arthritis Res Ther 13:R15. 310. Yao, Z., P. Li, Q. Zhang, E. M. Schwarz, P. Keng, A. Arbini, B. F. Boyce, and L. Xing. 2006. Tumor necrosis factor alpha increases circulating osteoclast precursor numbers by promoting their proliferation and differentiation in the bone marrow through up r egulation of c Fms expression. The Journal of biological chemistry 281:11846 11855. 311. Khazai, N. B., G. R. Beck, Jr., and G. E. Umpierrez. 2009. Diabetes and fractures: an overshadowed association. Current opinion in endocrinology, diabetes, and obesit y 16:435 445. 312. Paula, F. J., and C. J. Rosen. Obesity, diabetes mellitus and last but not least, osteoporosis. Arquivos brasileiros de endocrinologia e metabologia 54:150 157. 313. Del Fattore, A., A. Teti, and N. Rucci. 2008. Osteoclast receptors and signaling. Arch Biochem Biophys 473:147 160. 314. Dumitrescu, A. L., S. Abd El Aleem, B. Morales Aza, and L. F. Donaldson. 2004. A model of periodontitis in the rat: effect of lipopolysaccharide on bone resorption, osteoclas t activity, and local peptidergic innervation. J Clin Periodontol 31:596 603. 315. Ghosh, S., M. J. May, and E. B. Kopp. 1998. NF kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 16:225 260. 316. Mealey, B. L., and L. F. Rose. 2008. Diabetes mellitus and inflammatory periodontal diseases. Curr Opin Endocrinol Diabetes Obes 15:135 141. 317. Lawlor, K. E., I. K. Campbell, K. O'Donnell, L. Wu, and I. P. Wicks. 2001. Molecular and cellular mediators of interl eukin 1 dependent acute inflammatory arthritis. Arthritis Rheum 44:442 450.

PAGE 159

159 318. Prochazka, M., D. V. Serreze, W. N. Frankel, and E. H. Leiter. 1992. NOR/Lt mice: MHC matched diabetes resistant control strain for NOD mice. Diabetes 41:98 106. 319. Lohoff M., A. Gessner, C. Bogdan, and M. Rollinghoff. 1998. The Th1/Th2 paradigm and experimental murine leishmaniasis. Int Arch Allergy Immunol 115:191 202. 320. Karel, Z. 1994. Contrast limited adaptive histogram equalization. In Graphics gems IV Academic P ress Professional, Inc. 474 485. 321. Sternberg, S. R. 1983. Biomedical Image Processing. Computer 16:22 34. 322. Bar Shavit, Z. 2007. The osteoclast: a multinucleated, hematopoietic origin, bone resorbing osteoimmune cell. Journal of cellular biochemist ry 102:1130 1139. 323. Poubelle, P. E., A. Chakravarti, M. J. Fernandes, K. Doiron, and A. A. Marceau. 2007. Differential expression of RANK, RANK L, and osteoprotegerin by synovial fluid neutrophils from patients with rheumatoid arthritis and by healthy human blood neutrophils. Arthritis Res Ther 9:R25. 324. Li, H., S. Hong, J. Qian, Y. Zheng, J. Yang, and Q. Yi. 2010. Crosstalk between the bone and immune systems: osteoclasts function as antigen presenting cells and activate CD4+ and CD8+ T cells. Blood 325. Karin, M., and E. Gallagher. 2009. TNFR signaling: ubiquitin conjugated TRAFfic signals control stop and go for MAPK signaling complexes. Immunol Rev 228:225 240. 326. Desta, T., J. Li, T. Chino, and D. T. Graves. Altered fibroblast proliferation and apoptosis in diabetic gingival wounds. Journal of dental research 89:609 614. 327. Siqueira, M. F., J. Li, L. Chehab, T. Desta, T. Chino, N. Krothpali, Y. Behl, M. Alikhani, J. Yang, C. Braasch, and D. T. Graves. Impaired wound healing in mouse models of diabetes is mediated by TNF alpha dysregulation and associated with enhanced activation of forkhead box O1 (FOXO1). Diabetologia 53:378 388. 328. Kayal, R. A., M. Siqueira, J. Alblowi, J. McLean, N. Krothapalli, D. Faibish, T. A. Einhorn, L. C. Gerste nfeld, and D. T. Graves. TNF alpha mediates diabetes enhanced chondrocyte apoptosis during fracture healing and stimulates chondrocyte apoptosis through FOXO1. J Bone Miner Res 25:1604 1615.

PAGE 160

160 329. Alblowi, J., R. A. Kayal, M. Siqueira, E. McKenzie, N. Kroth apalli, J. McLean, J. Conn, B. Nikolajczyk, T. A. Einhorn, L. Gerstenfeld, and D. T. Graves. 2009. High levels of tumor necrosis factor alpha contribute to accelerated loss of cartilage in diabetic fracture healing. The American journal of pathology 175:15 74 1585. 330. Alikhani, M., Z. Alikhani, C. Boyd, C. M. MacLellan, M. Raptis, R. Liu, N. Pischon, P. C. Trackman, L. Gerstenfeld, and D. T. Graves. 2007. Advanced glycation end products stimulate osteoblast apoptosis via the MAP kinase and cytosolic apopt otic pathways. Bone 40:345 353. 331. Graves, D. T., G. Naguib, H. Lu, C. Leone, H. Hsue, and E. Krall. 2005. Inflammation is more persistent in type 1 diabetic mice. Journal of dental research 84:324 328. 332. Liu, R., T. Desta, H. He, and D. T. Graves. 2004. Diabetes alters the response to bacteria by enhancing fibroblast apoptosis. Endocrinology 145:2997 3003. 333. Larsen, K. I., M. Falany, W. Wang, and J. P. Williams. 2005. Glucose is a key metabolic regulator of osteoclasts; glucose stimulated increa ses in ATP/ADP ratio and calmodulin kinase II activity. Biochem Cell Biol 83:667 673. 334. van den Berg, W. B., L. A. Joosten, and P. L. van Lent. 2007. Murine antigen induced arthritis. Methods Mol Med 136:243 253. 335. Staite, N. D., K. A. Richard, D. G. Aspar, K. A. Franz, L. A. Galinet, and C. J. Dunn. 1990. Induction of an acute erosive monarticular arthritis in mice by interleukin 1 and methylated bovine serum albumin. Arthritis Rheum 33:253 260. 336. Egan, P. J., A van Nieuwenhuijze, I. K. Campbell, and I. P. Wicks. 2008. Promotion of the local differentiation of murine Th17 cells by synovial macrophages during acute inflammatory arthritis. Arthritis Rheum 58:3720 3729. 337. Page, R. C., and K. S. Kornman. 1997. T he pathogenesis of human periodontitis: an introduction. Periodontol 2000 14:9 11. 338. Bascones Martinez, A., P. Matesanz Perez, M. Escribano Bermejo, M. A. Gonzalez Moles, J. Bascones Ilundain, and J. H. Meurman. 2011. Periodontal disease and diabetes R eview of the Literature. Med Oral Patol Oral Cir Bucal 339. Papapanou, P. N. 1996. Periodontal diseases: epidemiology. Ann Periodontol 1:1 36. 340. Loe, H. 1993. Periodontal disease. The sixth complication of diabetes mellitus. Diabetes Care 16:329 334.

PAGE 161

161 341. Mealey, B. L., and T. W. Oates. 2006. Diabetes mellitus and periodontal diseases. J Periodontol 77:1289 1303. 342. Mealey, B. L. 1999. Influence of periodontal infections on systemic health. Periodontology 2000 21:197 209. 343. Mealey, B. 1999. Di abetes and periodontal diseases. Journal of periodontology 70:935 949. 344. Coleman, D. L. 1982. Diabetes obesity syndromes in mice. Diabetes 31:1 6. 345. Leibel, R. L., W. K. Chung, and S. C. Chua, Jr. 1997. The molecular genetics of rodent single gene obesities. The Journal of biological chemistry 272:31937 31940. 346. Aguilar, R. B. 2011. Evaluating treatment algorithms for the management of patients with type 2 diabetes mellitus: a perspective on the definition of treatment success. Clinical therapeu tics 33:408 424. 347. Defronzo, R. A. 2009. Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus. Diabetes 58:773 795. 348. Steptoe, R. J., J. M. Ritchie, and L. C. Harrison. 2002. Incre ased generation of dendritic cells from myeloid progenitors in autoimmune prone nonobese diabetic mice. J Immunol 168:5032 5041. 349. Morin, J., A. Chimenes, C. Boitard, R. Berthier, and S. Boudaly. 2003. Granulocyte dendritic cell unbalance in the non ob ese diabetic mice. Cell Immunol 223:13 25. 350. Chambers, T. J. 1985. The pathobiology of the osteoclast. J Clin Pathol 38:241 252. 351. Teitelbaum, S. L., M. M. Tondravi, and F. P. Ross. 1997. Osteoclasts, macrophages, and the molecular mechanisms of bone resorption. Journal of leukocyte biology 61:381 388. 352. Edwards, J. R., and G. R. Mundy. 2011. Advances in osteoclast biology: old fi ndings and new insights from mouse models. Nat Rev Rheumatol 7:235 243. 353. Gronthos, S., and A. C. Zannettino. 2007. The role of the chemokine CXCL12 in osteoclastogenesis. Trends Endocrinol Metab 18:108 113.

PAGE 162

162 354. Yu, X., Y. Huang, P. Collin Osdoby, and P. Osdoby. 2004. CCR1 chemokines promote the chemotactic recruitment, RANKL development, and motility of osteoclasts and are induced by inflammatory cytokines in osteoblasts. J Bone Miner Res 19:2065 2077. 355. Yano S., R. Mentaverri, D. Kanuparthi, S. Bandyopadhyay, A. Rivera, E. M. Brown, and N. Chattopadhyay. 2005. Functional expression of beta chemokine receptors in osteoblasts: role of regulated upon activation, normal T cell expressed and secreted (RANTES) in osteoblasts and regulation of its secretion by osteoblasts and osteoclasts. Endocrinology 146:2324 2335. 356. Bai, Y. D., F. S. Yang, K. Xuan, Y. X. Bai, and B. L. Wu. 2008. Inhibition of RANK/RANKL signal transduction pathway: a promising approach for os teoporosis treatment. Med Hypotheses 71:256 258. 357. Dougall, W. C., M. Glaccum, K. Charrier, K. Rohrbach, K. Brasel, T. De Smedt, E. Daro, J. Smith, M. E. Tometsko, C. R. Maliszewski, A. Armstrong, V. Shen, S. Bain, D. Cosman, D. Anderson, P. J. Morriss ey, J. J. Peschon, and J. Schuh. 1999. RANK is essential for osteoclast and lymph node development. Genes Dev 13:2412 2424. 358. Iqbal, J., L. Sun, and M. Zaidi. 2010. Denosumab for the treatment of osteoporosis. Curr Osteoporos Rep 8:163 167. 359. Rehma n, Q., and N. E. Lane. 2001. Bone loss. Therapeutic approaches for preventing bone loss in inflammatory arthritis. Arthritis Res 3:221 227. 360. Russell, R. G. 2007. Bisphosphonates: mode of action and pharmacology. Pediatrics 119 Suppl 2:S150 162. 361. Karsdal, M. A., K. Henriksen, M. Arnold, and C. Christiansen. 2008. Calcitonin: a drug of the past or for the future? Physiologic inhibition of bone resorption while sustaining osteoclast numbers improves bone quality. BioDrugs 22:137 144. 362. Hegedus, L ., A. C. Moses, M. Zdravkovic, T. Le Thi, and G. H. Daniels. 2011. GLP 1 and calcitonin concentration in humans: lack of evidence of calcitonin release from sequential screening in over 5000 subjects with type 2 diabetes or nondiabetic obese subjects treat ed with the human GLP 1 analog, liraglutide. J Clin Endocrinol Metab 96:853 860.

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163 BIOGRAPHICAL SKETCH Dana Catalfamo was born and raised in Hammonton, New Jersey and graduated the salutatorian of her class in high school. Shortly after graduation, Dana moved down to Ft. Myers, Florida in 2003 to attend college at Florida Gulf Coast University where she majored in Biology. She graduated magna cum laude and began her graduate career at the University of Florida in August 2007. Dana originally wished to pursue microbiology during college, however her love for laboratory where she ultimately chose to pursue her dissertation research. Her research interests include innat e immunity, autoimmunity, and infectious disease. She plans to attend physician assistant school in the fall to aid in her ultimate career goal of physician scientist specializing in rheumatology.