Modulation of Immune Responses by Gut Resident Microbiota and Implications in Type 1 Diabetes Animal Models

Permanent Link: http://ufdc.ufl.edu/UFE0043594/00001

Material Information

Title: Modulation of Immune Responses by Gut Resident Microbiota and Implications in Type 1 Diabetes Animal Models
Physical Description: 1 online resource (109 p.)
Language: english
Creator: Lau, Kenneth Kit
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011


Subjects / Keywords: 1 -- apcs -- autoimmunity -- bacteria -- bmdcs -- defective -- diabetes -- environment -- gut -- johnsonii -- lactobacillus -- microbiota -- nod -- nor -- probiotics -- th17 -- type
Microbiology and Cell Science -- Dissertations, Academic -- UF
Genre: Microbiology and Cell Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation


Abstract: Type 1 Diabetes is an autoimmune disease that destroys insulin producing cells in the pancreas. Genetic factors are clearly important in disease outcome. However, environmental factors, like diet and gut bacteria, have also influenced onset of the disease. Recently, it has been shown that bacteria isolated from diabetes resistant rodents could prevent diabetes incidence in prone animals through oral feedings. We demonstrated that this resistance was correlated with a TH17 bias. TH17 cells are typically inflammatory, but can be protective in certain gut autoimmune diseases. Furthermore, their role in Type 1 Diabetes remains unclarified. We have shown that Lactobacillus johnsonii N6.2 (LjN6.2), a commensal bacterium isolated from diabetes resistant rodents, was particularly adept at promoting a TH17 bias in vitro, requiring T cell receptor stimulation and antigen presenting cells (APCs). As TH17 conversion to TH1 cells has been known to occur in vivo a few days following transfer, we ascertained whether the same could occur in our model. We isolated bone marrow derived dendritic cells from diabetes prone NOD mice. Following maturation and treatment with LjN6.2, the dendritic cells were footpad injected into 9-week old NOD mice. Even two months later, the lymph nodes of mice receiving LjN6.2-pulsed dendritic cells possessed a TH17 bias, as evidenced by their high output of IL-17 and IL-6, two vital TH17 cytokines. We next investigated the natural affinity of NOD and NOR (diabetes resistant) mice to develop TH17 cells. While NOR pancreas contained less infiltrating leukocytes, it housed higher levels of TH17-related factors compared to NOD mice. NOD lymphocytes produced virtually no IL-6 and lower levels of IL-17 compared to NOR lymphocytes upon in vitro stimulation. Moreover, NOD APCs are notoriously defective in antigen presentation and maturation. Therefore, we tested whether LjN6.2 could restore function to APCs. We discovered that APCs only responded to LjN6.2 by upregulating MHCs and decreasing DEC205, demonstrating a shift from antigen uptake to presentation. This data demonstrates that a shift in T Helper phenotype, away from the diabetogenic TH1 state, may be enough to mitigate symptoms of the disease, overriding genetic programming for autoimmunity.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kenneth Kit Lau.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Larkin Iii, Joseph.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043594:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043594/00001

Material Information

Title: Modulation of Immune Responses by Gut Resident Microbiota and Implications in Type 1 Diabetes Animal Models
Physical Description: 1 online resource (109 p.)
Language: english
Creator: Lau, Kenneth Kit
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011


Subjects / Keywords: 1 -- apcs -- autoimmunity -- bacteria -- bmdcs -- defective -- diabetes -- environment -- gut -- johnsonii -- lactobacillus -- microbiota -- nod -- nor -- probiotics -- th17 -- type
Microbiology and Cell Science -- Dissertations, Academic -- UF
Genre: Microbiology and Cell Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation


Abstract: Type 1 Diabetes is an autoimmune disease that destroys insulin producing cells in the pancreas. Genetic factors are clearly important in disease outcome. However, environmental factors, like diet and gut bacteria, have also influenced onset of the disease. Recently, it has been shown that bacteria isolated from diabetes resistant rodents could prevent diabetes incidence in prone animals through oral feedings. We demonstrated that this resistance was correlated with a TH17 bias. TH17 cells are typically inflammatory, but can be protective in certain gut autoimmune diseases. Furthermore, their role in Type 1 Diabetes remains unclarified. We have shown that Lactobacillus johnsonii N6.2 (LjN6.2), a commensal bacterium isolated from diabetes resistant rodents, was particularly adept at promoting a TH17 bias in vitro, requiring T cell receptor stimulation and antigen presenting cells (APCs). As TH17 conversion to TH1 cells has been known to occur in vivo a few days following transfer, we ascertained whether the same could occur in our model. We isolated bone marrow derived dendritic cells from diabetes prone NOD mice. Following maturation and treatment with LjN6.2, the dendritic cells were footpad injected into 9-week old NOD mice. Even two months later, the lymph nodes of mice receiving LjN6.2-pulsed dendritic cells possessed a TH17 bias, as evidenced by their high output of IL-17 and IL-6, two vital TH17 cytokines. We next investigated the natural affinity of NOD and NOR (diabetes resistant) mice to develop TH17 cells. While NOR pancreas contained less infiltrating leukocytes, it housed higher levels of TH17-related factors compared to NOD mice. NOD lymphocytes produced virtually no IL-6 and lower levels of IL-17 compared to NOR lymphocytes upon in vitro stimulation. Moreover, NOD APCs are notoriously defective in antigen presentation and maturation. Therefore, we tested whether LjN6.2 could restore function to APCs. We discovered that APCs only responded to LjN6.2 by upregulating MHCs and decreasing DEC205, demonstrating a shift from antigen uptake to presentation. This data demonstrates that a shift in T Helper phenotype, away from the diabetogenic TH1 state, may be enough to mitigate symptoms of the disease, overriding genetic programming for autoimmunity.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kenneth Kit Lau.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Larkin Iii, Joseph.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043594:00001

This item has the following downloads:

Full Text




2 2011 Kenneth Kit Lau


3 To my family, Patrick, Patricia, and Joanna Lau Your support is unending


4 ACKNOWLEDGMENTS I would like to thank my mentor, Dr. Joseph Larkin, III. Five years ago, you were willing to take a chance on a student without a specific focus. And, despite some conflict along the way, I feel we have established our fledgling laboratory and I look forward to seeing how it will expand onc e I have left. I would also like to thank the busy members of my committee, Drs. Joseph Larkin, III, Howard M. Johnson, Janet K. Yamamoto, Byung Ho Kang, Volker Mai, and Mark A. Atkinson for their insight and guidance in my project. I would also like to t hank many of my fellow graduate students in the Microbiology me from the very first day of graduate school, and we have fought through every bad test, every difficult c riticism, and every failed experiment together. Without her support, it is debatable whether my academic career would have been seen through to this point. Rea Dabelic has always been like a big sister to the Larkin lab. She has provided countless hours of support and a steady shoulder to lean on during difficult times. With insight into matters concerning the lab and life, Rea was indispensible during my entire academic life. Patrick Benitez was also instrumental in the establishment of several of the prot ocols being used in the Larkin lab and a necessary source of hilarity and laughter when situations became too serious. Despite being centered in Colorado these days, Patrick will always be valued for his technical prowess and uncensored sense of humor. Pa trick cannot be mentioned without Kelli Schoneck Benitez in the same breath. I would like to tha nk Kelli for keeping Patrick in line. Never be afraid to take him down a peg. Alexandria Ardissone has also been immensely helpful during my stay in the lab. I know she will be successful in whatever scientific endeavor she pursues I


5 would also like to express thanks to Tenisha Wilson and Roy Noon Song for their advice and opinions on critical matters. Outside of our labs, I would like to thank Algevis Wrench, J ohnathon Canton, Tyler Culpepper, and Cory Krediet. Each of them has provided a limitless amount of laughs and jokes. Without a doubt, each of you has contributed to creating one of the sharpest and socially outgoing group s of graduate students our departm ent will ever see. Finally, I would like to thank my family. My parents, Patrick and Patricia, and my sister, Joanna have pushed and supported me in every decision I will ever make. I would not be here today without their undying love, pushing me when I n eeded it most. Each and every person mentioned here has been irreplaceable and absolutely necessary for my growth in developmen t in graduate school and life.


6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ 4 LIST OF TABLES ................................ ................................ ................................ ........... 8 LIST OF FIGURES ................................ ................................ ................................ ........ 9 LIST OF ABBREVIATIONS ................................ ................................ .......................... 10 ABSTRACT ................................ ................................ ................................ .................. 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ... 14 Type 1 Diabetes ................................ ................................ ................................ .... 14 The NOD Mouse Model ................................ ................................ ................... 14 Genetic Factors in Type 1 Diabetes ................................ ................................ 15 APCs in T1D ................................ ................................ ................................ ... 18 Dendritic Cells in T1D ................................ ................................ ...................... 19 Macrophages in T1D ................................ ................................ ....................... 20 B cells in T1D ................................ ................................ ................................ .. 21 T cells in T1D ................................ ................................ ................................ .. 21 T Helper 17 Cells ................................ ................................ ................................ ... 24 TH17 Cell Differentiation ................................ ................................ ................. 25 TH17 Transcription Factors ................................ ................................ ............. 26 TH17 Associated Cytokines ................................ ................................ ............ 27 Plasticity of TH17 Cells ................................ ................................ ................... 31 The Role of TH17 Cells in Type 1 Diabetes ................................ ..................... 34 TH17 Cells, Humans, and T1D ................................ ................................ ........ 35 Environmental Factors in T1D ................................ ................................ ......... 36 Viruses in T1D ................................ ................................ ................................ 37 Die tary Factors ................................ ................................ ................................ 37 Other Environmental Factors ................................ ................................ ........... 38 Possible Treatments for T1D ................................ ................................ ........... 39 Modulation of the Immune Response by Bacteria ................................ ........... 41 Gut Bac teria and T1D ................................ ................................ ...................... 43 2 M ATERIALS AND METHODS ................................ ................................ ............... 49 Characterization of a TH17 Bias Induced by LjN6.2 ................................ ............... 49 Animals ................................ ................................ ................................ ........... 49 Bone Marrow Derived Dendritic Cell P reparation ................................ ............ 49 Dendritic Cell Vaccinations ................................ ................................ .............. 50 T Lymphocyte Purification ................................ ................................ ............... 50


7 Proliferation Assays ................................ ................................ ........................ 50 In Vitro Cytokine Secretion Analysis ................................ ................................ 51 Role of TH17 Cells in an Autoimmune Diabetes Setting ................................ ........ 51 Animals ................................ ................................ ................................ ........... 51 Flow Cytometry ................................ ................................ ............................... 52 Pancreas RNA Isolation/Histology ................................ ................................ ... 52 RNA Isolation and RT qPCR. ................................ ................................ .......... 53 Bacterial Enumeration and Cell Lysis ................................ .............................. 53 Statistical Calculations ................................ ................................ .................... 54 3 RESULTS ................................ ................................ ................................ .............. 55 Characterization of a LjN6.2 Mediated TH17 Bi as ................................ ................. 55 LjN6.2 Induces Apoptosis at High Concentrations ................................ ........... 55 T Cells Require TCR Stimulation to Create a TH17 Response to LjN6.2 ......... 56 APCs are Required to Create a LjN6.2 Mediated TH17 Bias. ......................... 57 BMDCs are Capable of Promoting a Long Term TH17 Bias ............................ 58 The Role of TH17 Cells in an Autoimmune Diabetic Setting ................................ .. 59 NOD Mice Display a TH17 Deficiency Compared to NOR Mice ...................... 59 NOD Lymph Nodes Contain Lower Quantity of APCs Compared to NOR ....... 61 LjN6.2 Increases NOD APC Numbers and Maturation ................................ .... 61 LjN6.2 Triggers BMDC Immunity Through a Surface Antigen .......................... 62 4 DISCUSSION ................................ ................................ ................................ ........ 82 LIST OF REFERENCES ................................ ................................ .............................. 89 BIOGRAPHICAL SKETCH ................................ ................................ ......................... 109


8 LIST OF TABLES Table pa ge 3 1 Primers used and/or discussed in this study. ................................ .................... 64


9 LIST OF FIGURES Figure page 1 1 Gut antigen sampling and presentation by dendritic cells. ................................ 46 1 2 T helper cell differentiation. ................................ ................................ ............... 47 1 3 IL 17 signalin g. ................................ ................................ ................................ .. 48 3 1 Lactobacillus johnsonii induces apoptosis at high end concentrations ............... 65 3 2 LjN6.2 mediation of a TH17 bias requires p roper TCR stimulation .................. 66 3 3 LjN6.2 boosts production of TH17 related cytokines in a time dependent manner. ................................ ................................ ................................ ............. 67 3 4 LjN6.2 medi ated TH17 bias requires the presence of APCs ............................. 68 3 5 B MDCs are sufficient to drive a LjN6.2 mediated TH17 bias ............................ 69 3 6 LjN6.2 pu lsed BMDCs mediate a TH17 bias in recipient NOD mice ................. 70 3 7 LjN6.2 pulsed BMDCs can mediate a longterm TH17 bias in recipient NOD mice ................................ ................................ ................................ .............. 71 3 8 Transfer of LjN6.2 pulsed BMDC does not p revent onset of T1D in NOD mice ................................ ................................ ................................ ................. 72 3 9 NOD lymphocytes display a reduced IL 6 and IL 17A production ..................... 73 3 10 NOD Pancreas display insulitis and higher infiltrating levels of T cells .. ............. 74 3 11 NOD Pancreas display reduced levels of TH17 related factors ........................ 75 3 12 NOD lymph nodes display reduced levels of APCs ................................ .......... 76 3 13 LjN6.2 increases CD11b+ and CD11c+ expression ................................ .......... 77 3 14 LjN6.2. enhances CD11b and CD11c expression of MHC I and II markers ...... 78 3 15 CD11c+ cells reduce expression of DEC205 in the presence of LjN6.2 ............ 79 3 16 LjN6.2 influences APCs via a membrane bound antigen. ................................ .. 80 3 17 A proposed mechanism for the action of LjN6.2 in a diabetes model. ................ 81


10 LIST OF ABBREVIATION S AHR Aryl Hydrocarbon Receptor AICD Antigen Induced Cell Death APCs Antigen Presenting Cells BBDP Bio Breeding Diabetes Prone BCG Bacillus Calmette Guerin BMDC Bone Marrow Derived Dendritic C ells CD Cluster of Differentiation CFA CM CTLA Cytotoxic T Lymphocyte Antigen DC Dendritic Cells EAE Experimental Autoimmune Encephalomyelitis GAD Glutamic Acid Decarboxylase GM CSF Granulocyte Macrophage Colony Stimula ting Factor GRAS Generally Regarded As Safe IDD Insulin Dependent Diabetes IFN Interferon IL Interleukin IRF Interferon Regulatory Factor JAK Janus Kinase LjN6.2 Lactobacillus johnsonii N6.2 LrTD1 Lactobacillus reuteri TD1 LPS Lipopolysaccharide MHC Major Histocompatibility Complex


11 MSC Mesenchymal Stem Cells NKT Natural Killer T NOD Non Obese Diabetic NOR Non Obese Resistant PLN Pancreatic Lymph Nodes ROR Retinoic Acid Receptor SCID Severe Combined Immuno Deficiency STAT Signal Transducer and Activator of Transcription T1D Type 1 Diabetes TCR T Cell Receptor TGF Transforming Growth Factor TH T Helper TLR Toll Like Receptor TNF Tumor Necrosis Factor Tregs Regulatory T cells


12 Abstract of Dissertation Presented to the Graduate School of the University of Flo rida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MODULATION OF IMMUNE RESPONSES BY GUT RESIDENT MICROBIOTA AND IMPLICATIONS IN TYPE 1 DIABETES ANIMAL MODELS By Kenneth Kit Lau December 2011 Chair: Jo seph Larkin, III Major: Microbiology and Cell Science Type 1 Diabetes is an autoimmune disease that destroys insulin producing cells in the pancreas. Genetic factors are clearly important in disease o u t come However, environmental factors, like diet and g ut bacteria, have also influenced onset of the disease. Recently, it has been shown that bacteria isolated from diabetes resistant rodents could prevent diabetes incidence in prone animals through oral feedings. We demonstrated that this resistance was cor related with a TH17 bias. TH17 cells are typically inflammat ory but can be protective in certain gut autoimmune diseases. Furthermore, their role in Type 1 Diabetes remains unclarified. We have shown that Lactobacillus johnsonii N6.2 (LjN6.2) a commensa l bac terium isolated from diabetes resistant rodents, was particularly adept at promoting a TH17 bias in vitro requir ing T cell receptor stimulation and antigen presenting cells (APCs) As TH17 conversion to TH1 cells has been known to occur in vivo a few days following transfer we ascertained whether the same c ould occur in our model. W e isolated bone marrow derived dendritic cells from diabetes prone NOD mice Following maturation and treatment with LjN6.2, the dendritic cells were footpad injected into 9 week old NOD mice E ven two months later, the lymph nodes of mice receiving LjN6.2 pulsed dendritic


13 cells possessed a TH17 bias, as evidenced by their high output of IL 17 and IL 6, two vital TH17 cytokines. We next investigated the natural affinity of NOD and NOR (diabetes resistant) mice to develop TH17 cells. W hile NOR pancreas contain ed less infiltrating leukocytes it housed higher levels of TH17 related factors compared to NOD mice. NOD lymphocytes produced virtually no IL 6 and lower levels of IL 17 compared to NOR lymphocytes upon in vitro s t imulation. Moreover, NOD APCs are notoriously defective in antigen presentation and maturation. T herefore we tested whether LjN6.2 could restore function to APCs. We discovered that APCs only responded to LjN 6.2 by upregulating MHC s and decreas ing DEC205, demonstrating a shift from antigen uptake to presentation. This data demonstrates that a shift in T Helper phenotype, away from the diabetogenic TH1 state, may be enough to mitigate symptoms of the disease o verriding genetic programm ing for autoimmunity.


14 CHAPTER 1 INTRODUCTION Type 1 Diabetes Currently, in the United States, one million Americans have been diagnosed with Type 1 Diabetes (T1D) and 15,000 children are diagnosed annually ( J uvenile Di abetes Research Foundation International, 2010). The average annual cost for a patient with T1D has been estimated at $14,900. Total costs from medical care and lost productivity are estimated at $15 billion for the U. S. annually (Dall, 2010). T1D is a T cell mediated autoimmune disease that tar g cells produce insulin, which tells cells to take up glucose from the blood and store it as glycogen. cells and insulin production, several complications arise. Excessive thirst and urination develop because of the high levels of blood sugar (hyperglycemia). Fluid is pulled from the tissue in response. At times, it can even be drawn from the eyes, causing blurred vision. The inability to take up glucose also causes increased hunger and fatigue Despite the need to eat more, patients may actually lose weight because muscle and fat cells shrink without glucose ( M ayo Clinic 2011). In order to gain an understanding of the mechanisms involved with T1D, researchers have used rodent mode ls. The Non Obese Diabetic mouse is one of the most well understood and studied animal models for T1D. The NOD Mouse Model The Non Obese Diabetic (NOD) mouse model was originally discovered by Makino et al. (Makin, 1980; Kikutani, 1992) in Japan following the selection of a cataract prone strain obtained from the JcI:ICR mouse line. Through repetitive inbreeding, it was found that the NOD strain spontaneously developed Type 1 Diabetes


15 (T1D). T1D is gender biased, as females are typically 80 90% diabetic by 24 weeks of age, while the incidence rate of males is roughly 40% by 30 weeks of age (Kikutani, 1992; Bach, 1994). NODs have become the standard in autoimmune diabetes models primarily because of their well studied genome. Transgenes or mutations are easil y introduced into the NOD model, allowing for the analysis of specific diabetogenic factors. Genetic Factors in Type 1 Diabetes In an effort to understand which chromosomes and genes are responsible for T1D, the N OD mouse strain has been bred with various diabetes resistant strains. Several genetic loci have been discovered and termed Idd for insulin dependent diabetes. To this point, over 30 loci on 15 different chromosomes have been isolated. Most loci also consist of multiple genes rather than one isolat ed gene, further broadening the range of possible underlying factors in this disease. The most prominent and well studied Idd gene loci will be discussed below. Idd1 is one of the most essential genes for T1D onset in NOD mice. Within Idd1 are several gene s associated with the Major Histocompatibility Complex (MHC). NOD mice possess a collective set of MHC mutations termed H2 g7 MHC are responsible for presenting peptide to T cells, activating them to carry out their specific function against the antigen. W hile nearly all nucleated cells express MHC Class I, mainly peptide to CD8+ cytotoxic T lymphocytes, while MHC II shows extracellular peptides to CD4+ Helper T cell s. The NOD mouse completely lacks MHC Class II E allele and contains mutations at two typically conserved sites, positions 56 and 57 of MHC II A g7 allele. These residues have been converted from proline and aspartic acid to histidine and


16 serine, respective ly (Acha Orbea, 1987). This exchange exposed positive residues in the MHC II complex, allowing negatively charged residues with larger side chains to bind. This expands the repertoire of peptides that the NOD MHC II can recognize. In addition, studies have shown that the NOD MHC II A g7 binds with less affinity to peptides compared to other MHC II complexes. The importance of the NOD MHC II A g7 was demonstrated when transgenic restoration of the MHC II E allele or a non MHC II A g7 prevented onset of T1D in N OD mice (Miyazaki, 1990; Lund, 1990, Slattery, 1990). Interestingly, this residue mutation is also seen in humans suffering from T1D in the DQ beta chain (Todd, 1988). Idd3 is associated with the cytokines IL 2 and IL 21. In the NOD mouse, it has been repo rted that there is unusually low levels of IL 2, but high expression of IL 21 (McGuire, 2009; Yamanouchi, 2007; Lyons, 2000). IL 2 is generally associated with promoting regulatory T cells (Tregs), which are cells associated with suppression of autoimmune diseases. Additionally, treating NOD mice with IL 2 prevented onset of disease. While IL 2 may be protective, IL 21 (detailed below) may be pathogenic. Genetic deletion of the IL21 receptor also prevented onset of T1D. Driver et al. have suggested that the se two factors may be directly linked, as the lack of IL 2 prevents proper function of Tregs, making it difficult to regulate the proliferative abilities of IL 21 on T effector cells. Idd5.1 is also affiliated with Tregs. The CTLA 4 gene is located within this loci. CTLA 4 can influence T1D by interacting with CD80 and CD86 on APCs. CTLA 4 is also required for Treg function and has been shown to inhibit function in T effector cells. In both NOD and human T1D, researchers have discovered


17 polymorphisms in the CTLA 4 gene, which leads to structural variance, affecting its proper function (Ueda, 2003). Idd9 reportedly promotes the development of natural killer T (NKT) cells, which have been known to be deficient in the NOD model. NKT cells recognize antigen thr ough a CD1d molecule, and not through the typical MHC process (Matsuda, 2008) In addition, these cells are unique because of their ability to quickly produce TH1 (inflammatory) and TH2 (anti inflammatory/humoral response) cytokines when activated. Several studies have shown NKT can be protective in T1D. Mechanisms include the induction of a TH2 response in the islets (Sharif, 2001; Hong, 2001), Tregs (Pillai, 2009), or tolerogenic dendritic cells (DCs) (Chen, 2005; Wang, 2008). The Idd9 region is also know n to add to abnormal development of B cells, causing them to express auto reactive antibodies. This may also support a group of B cells focused on promoting and activating pathogenic CD4 T cells (Silveira, 2006). Idd13 also exerts effects on NKT cell numbe rs (Esteban, 2003) microglobulin deficient NOD model, it was shown that reconstitution with an NOD or NOR (Non Obese microglobulin molecule led to diabetes susceptibility and resistance, respectively (Hamilton Williams, 2001). It has been proposed that the microglobulin molecule associated with it. In this case, the NOD conformation may allow APCs to more easily recognize CD8+ T cells during positive selection (a process that ensures that developing T cells properly recognize host MHC in the thymus).


18 APCs in T1D The first signs of T1D in NOD mice can be seen as early as 4 weeks of age when insulitis can be observed in the pancreas (Mathews, 2005) Termed peri insulitis, infiltrating lymphocytes begin by surrounding the islets. As the mice undergo puberty, increasing amounts of islets will show insulitis, losing roughly 25 cell mass. Of the infiltrating lymphocytes, a large percentage of the cells are CD4+ T helper cells. CD8+ cytotoxic T cells, B lymphocytes, macrophages, and DCs have also been found in the invading group (Jarpe, 19 90, 1991; Miyazaki, 1985; Faveeuw, 1995). A decrease in pancreatic insulin levels is observed in NOD females by 12 weeks of age. This drop in males is not observed until several weeks later (Gaskins, 1992). By the cell mass is obliterated hallmark symptoms of T1D can be observed, including: hyperglycemia, excess glucose in the blood; glycosuria, glucose in the urine; and polyuria, which is abnormal or excessive amounts of urine passage (Wilson, 1997). cells do not express MHC Class II or other costimulatory molecules, it is unlikely T cells are finding their antigen of choice within the pancreas. It is far more likely that APCs, like DCs, sample antigen from the pancreas and carry it to the pancreatic lymph node (PLN) to ac tivate T cells. This is supported by the fact that removal of PLN from young NOD mice prevents onset of T1D (Gagnerault, 2002; Hoglund, 1999; Zhang, 2002). Although T1D is a T cell mediated autoimmune disease, APCs are also vital. APCs can mediate the dest ruction of auto reactive T cells originating in the thymus and in the periphery (Walker, 2002) This typically occurs when T cells recognize self antigen presented on the MHC of APCs. Should they bind too strongly, they are eliminated to prevent autoimmuni ty. As noted earlier, NOD mice possess defective


19 MHC Class II markers that bind antigenic peptide loosely. It is also known that antigen induced cell death (AICD) requires stronger activation of T cells than normal effector T cell functions (Ucker, 1992). If APCs are unable to properly stimulate T cells, T cells may simply get activated instead of being deleted from a position of dangerous immune surveillance. Accordingly, several defects have been associated with the APCs of NOD mice. Dendritic Cells in T1 D Dendritic Cells (DCs) are important bridges between the innate and adaptive arms of the immune response. In an immature state, DCs are efficient antigen samplers. Upon maturation, markers for antigen uptake are decreased, while markers for antigen presen tation (i.e., MHC I and II) are upregulated to stimulate T cells (Figure 1 1). However, NOD DCs, like other NOD APCs, fail to upregulate markers associated with the costimulation of T cells (CD80, CD86) and MHC markers when properly stimulated with antigen like lipopolysaccharide (Strid, 2001). In addition, NOD bone marrow derived dendritic cells (BMDCs) are notoriously difficult to increase in culture showing lower proliferative ability (Nikolic, 2004). DCs are also known to promote different T Helper cel l phenotypes based on their maturation state. For instance, mature DCs are known to produce less IL 12, which skews T cells to a TH1 phenotype (Feili Hariri, 1999). The differentiation state of DCs also affects whether they can induce tolerance, anergy, de letion, or activation of T cells (Reis e Sousa, 2006). It has been shown that mature DCs isolated from the pancreatic lymph node of NOD mice can prevent onset of the disease when transferred into young NOD mice (Clare Salzler, 1992). Many of the tolerogeni c functions of DCs map to the Idd3 and Idd5 loci (Hamilton Williams, 2009).


20 Macrophages in T1D Macrophages, one of the most adept uptakers of antigen, are also defective in NOD mice. When stimulated with LPS and IL 1, NOD macrophages fail to activate CD8+ cytotoxic T cells in the same manner as non diabetic strains (Serreze and Gaedeke, 1993; Serreze and Gaskins, 1993). Similar to DCs, macrophages may not be able to properly stimulate T cells to induce tolerance through cell death. In addition, T cells req uire a stronger set of activation signals to convert to TH2 cells. The level of activation for TH1 cell conversion is much lower, possibly biasing the immune system toward an inflammatory response (Schweitzer, 1998). In addition, NOD macrophages have diffi cult ies properly clearing apoptotic bodies. When the apoptotic bodies are two inflammation associated cytokines. Macrophages from NOD mice also tend to express elevated levels of gran ulocyte/macrophage colony stimulating factor (GMCSF). STAT5 is consistently activated in these cells, leading to overproduction of prostag l a n din E2 (PGE2), which is associated with developing the chronic inflammatory environment seen in the NOD mouse (Lith erland, 2005). Apoptosis refers to programmed cell death, which occurs in a regulated, controlled manner. Following the proper stimuli (i.e., CD95 stimulation), a series of caspases are activated in cascade fashion. Caspases cleave and disrupt central comp onents of cell function. During apoptosis, the cell membrane is also warped, as the cytoskeleton degrades. In research, the early stages of apoptosis are measured by an Annexin V antibody, which binds phosphatidylserine (PS) ( Koopman, 1994). PS is restrict ed to the inside of the membrane, toward the cytoplasm. Upon apoptosis, the membrane loses its rigidity, and PS begins to get expressed on the surface of the cell, which can then be


21 bound by Annexin V antibody. Late stages of apoptosis are measured using 7 AAD, a compound that binds DNA ( Rabinovitch, 1986 ). When a cell has neared the end of apoptosis, the entire membrane is disrupted and 7 AAD, a nuclear staining dye, can bind to the cell. B cells in T1D B cells are the generators of antibodies. T1D is asso ciated with several autoimmune antibodies, the most well studied being for insulin, glutamic acid decarboxylase (GAD), protein tyrosin e phosphatase IA2, and Zinc Transporter ZnT8 (Lien and Zipris, 2009). Initial studies indicated B cells were essential in T1D, as suppression of B cells with antibodies or eliminating BAFF/BLyS, a B cell survival factor, ameliorated the disease (Hu, 2007; Fiorina, 2008). However, recent studies have show n that B cells alone are not sufficient to cause T1D. The transfer of ser um antibodies from diabetic NOD mice into a B cell deficient NOD model did not increase insulitis, nor did it cause T1D (Serreze, 1998). In addition, the simple addition of NOD diabetic T cells alone into a B cell deficient model can cause T1D. While autoa ntibodies undoubtedly aid in disease progression, it is more likely B cells act in an antigen presenting capacity. When MHC II was eliminated from B cells, but not from macrophages and DCs, T1D was suppressed in NOD mice (Noorchashm, 1999). While B cells a re not required for the initiation stages of T1D, their presence enhances reactivity agains cells. T cells in T1D As stated earlier, T cells are the main mediators of T1D. T cells recognize antigen through their T cell receptor (TCR). MHCs complex with the TCR. If the TCR is specific for the presented antigen, that T cell becomes activa ted if given the proper costimulation. T cells mature in the thymus after undergoing several checkpoints to


22 ensure proper MHC recognition and limited reactivity to self. If a T cell fails any of these tests, it is marked for deletion. In the NOD mouse, the re are underlying factors that are leading to the false maturation and export of self reactive T cells into the periphery (Sebzda, 1994) It has been shown that positive selection (MHC self restriction ) requires less T cell activation compared to negative selection (the elimination of self reactive cells). In other words, these pathogenic T cells bind with just enough strength to the MHC to be positively selected and recognized as MHC restricted, but weak enough that their self reactivity is not deemed dang erous, escaping negative selection (Ashton Rickardt, 1994). Even in a normal, non autoimmune model, some T cells will escape proper selection. In the periphery, these cells are typically removed from the body through AICD. However, NOD T cells display ele vated levels of c FLIP, Bclx anti apoptotic factors (Decallone, 2003; Arreaza, 2003) In addition, they display lower levels of IL 2, IL 4, FasL, and Caspase 8, which are pro apoptotic factors. By 6 weeks of age, NOD mice can express their full complement of T cell receptors. These T cells have been shown to become more pathogenic with age, as they increase in proliferative ability and production of IFN while decreasing generation of immunoregulatory factors like IL 4 and IL 10. This may l ead the T cells to resist regulation by Tregs. Combined with their penchant for eluding AICD, this may explain why T cells become pathogenic in T1D. It was discovered that both CD4+ and CD8+ T cells are required for disease progression in the NOD model. Th ese experiments used an NOD.SCID model. SCID mice lack catalytic polypeptide (Prkdc). Prkdc is involved with repairing double stranded


23 DNA breaks and recombining the variable (V), diversity (D), and joining (J) regions of immunoglobulin (antibody) and T ce ll receptor genes. Because of this defect, properly functioning T cells and B cells never develop in the mouse. Crossed onto an NOD NOD setting (Shultz, 1995). CD4+ c ells can transfer diabetes into an NOD.SCID if the donor cells were already diabetic. If CD4+ cells from young, prediabetic mice are transferred to an NOD.SCID mouse, they do not cause T1D. CD8+ cells alone cannot cause T1D in an NOD.SCID as they are unabl e to home to the islets to start destruction cells through IFN production (Suarez Pinzon, 1996). CD8+ cells are also thought to cause cell death through perforin and granzyme B (Estella, 2006). The phenotype of CD4+ T helper cells is dependent on the cytokine environment provided by the APC, typically DCs (Figure 1 2). CD4+ helper phenotype has also been implicated as important in T1D, as TH1 cells are typically associated with initiating disease onset, producing abundant amounts of Conversion of nave T cells from a TH1 phenotype to a TH2 phenotype is associated with protection from T1D (Falcone, 1999). However, the role of T Helper cells is not as simple as one would expect. IL 12 (TH1 differentiation factor) knockout mice actually show an accelerated disease incidence in NOD mice. In addition, IL 4 (TH2 promoting factor) knockouts develop T1D at a rate identical to control NOD mice (Wang, 1998). Despite some of these paradoxical findings, overwhelming evidenc e typically indicates TH1 related factors are detrimental in T1D.


24 Tregs are also vital in T1D, but in a protective role. Their depletion results in a greatly accelerated onset of disease (Salomon, 2000; Bour Jordan, 2004). Recent studies have proven that T regs do not decrease during the course of the disease, but they may lose their functionality (Mellanby, 2007; Tritt, 2008) Staining for Foxp3, the Treg transcription factor, in the thymus, peripheral lymphoid organs, and pancreas all indicated that Treg n umbers do not decline with age or as insulitis begins to occur. However, as the mice age, NOD Tregs lost suppressive effects. Furthermore, the adoptive transfer of Tregs and conventional T cells from young neonatal NOD mice but not mature NOD donors, inhi bited T1D (You, 2005; Gregori, 2003). The declining abilities of Tregs and increasing abilities of T effector cells with age may be two major factors in T1D disease development. T Helper 17 Cells T helper 17 (TH17) cells are a recently discovered subset of CD4+ Helper T cells. Much like other T helper subsets, TH17 cells are identified by their unique cytokine profile and set of transcription factors. Typically, TH17 cells are associated with the production of IL 17A IL17 F, IL 21, and IL 22. The TH17 line age specific transcription factors include ROR t, ROR IRF 4, and AHR. Th17 cells are thought to be intimately involved with host defense against pathogens, particularly extracellular fungi and bacteria (Ouyang, 2008). IL 17A deficient mice suffer from an inability to clear K. pneumonia and T. gondii infections. However, they do not show increased susceptibility to intracellular bacterial infection (Ouyang, 2008). Although several cell types have been implicated in T1D, the role of TH17 cells in T1D has no t been established yet.


25 TH17 Cell Differentiation T ypical dogma states that TH17 cell s are generated from nave CD4+ cells using 6, a cytokine typically associated with inflammation ( Bettelli, 2006; Mangan et al., 2006; Veldhoen et al., 2006 ) However, as more research is conducted on TH17 differe ntiation, several variations of TH17 differentiation have been suggested. Several studies have stated that a combination of IL 21 and TGF is sufficient to initiate TH17 differentiation (Kom, 2007). This is particularly intriguing because IL 21 drives the production of IL 17, itself, and the expression of IL 23R (Zhou, 2007; Nurieva, 2007; Kom, 2007). In addition, IL 6 may not be absolutely required for TH17 cell development (Elyaman, 2009). IL 6 / mice have reduced, but not completely absent levels of TH1 7 cells, suggesting other cytokines may supplement the loss of IL 6 (Bettelli, 2006). Recently, it has been suggested that IL 9 may also rep lace IL 6, IL 23, and IL te the controversy of creating TH17 cells, each proposed mechanism seems to center on various combinations of TH17 associated cytokines. IL 23 is composed of p40, a subunit also utilized by IL 12, and the p19 subunit. The receptor is composed of IL nd the unique IL 23R chain. IL 23 is generated by activated dendritic cells and phagocytes (Oppmann, 2000) and was initially believed to be a cytokine required for TH17 differentiation. However, it was demonstrated that IL 23R is not expressed on nave T c ells. Despite not being involved with the initial creation of TH17 cells, IL 23 was discovered to be essential in maintaining a TH17 population and expanding it (Bettelli, 2006; Veldhoen, 2006).


26 TH17 Transcription Factors Much like TH1 and TH 2 cells, TH17 cells have dedicated, lineage transcription factors responsible for master control of TH17 related genes. A majority of TH17 related cytokines do their signaling via Signal Transducer and Activator of Transcription 3 (STAT3). IL 17, IL 23, IL 6, IL 21, an d IL 22 all activate STAT3. STAT3 also regulates expression of IL 21R, IL 21, and IL 23R (Durant, 2010; Ghoreschi, 2010). STAT3 deficiency greatly reduced expression of ROR t and ROR two TH17 specific transcription factors (Yang, 2007, 2008). and ROR both belong to the r etinoic acid related o rphan hormone receptor family. 6 and its overexpression drives TH17 cell differentiation, while simultaneously blocking the development of TH1 and TH2 cells. It was also demonstrated that a deficiency in led to a lowered ability to create TH17 cells and limited IL 6, and IL 21 (Ivanov, 2006; Nuri eva, 2007). While deletion limited TH17 cell generation, it was not completely abolished. Mice lacking still develop Experimental Autoimmune Encephalomyelitis, an animal model of multiple sclerosis where TH17 cells are causative (Yang, 2008) Y ang et al., recently discovered TH17 cells also express ROR 6. ROR shares several of the properties of ROR and the two transcription factors likely share redundant function. Interferon Regulatory Factor 4 (IRF4) was a lso shown to be vital for TH17 cell differentiation (Brustle, 2007), as IRF4 deficiency completely inhibits TH17 differentiation and prevented disease onset in EAE. This was also correlated with a marked decrease in ROR expression, suggesting that IRF4 m ay operate upstream of ROR The precise functions and abilities of IRF 4 are still being investigated. Finally,


27 aryl hydrocarbon receptor (AHR) is also believed to play a crucial role in TH17 cell differentiation. While both T regulatory cells and TH17 c ells can express AHR, TH17 cells express this transcription factor at significantly higher levels compared to any other T cell subset. AHR deficient cells do not express IL 22 (Veldhoen, 2008). While less information is available on these two transcription factors, they are nonetheless very important in aiding TH17 cell development. TH17 Associated Cytokines Several TH17 associated cytokines are potent in the recruitment, activation, and migration of neutrophils, the most numerous and active members of the innate immune system. This recruitment and subsequent activation of neutrophils is mitigated by the function of IL 17, which stimulates the production of colony stimulating factors and CXCL8 by macrophages and other tissue resident cells. CXCL8 then induce s incoming immune cells to sample antigen through phagocytosis, triggering the activation of certain Toll like receptors (Lund, 2004). IL 17 consists of six family members, designated IL17A F. Among the family members, IL 17A and IL 17F share the most homo logy and are the most well studied. TH17 cells are capable of producing both IL 17A and IL 17F, while IL17B E can be produced by non T cells. IL17A and IL17F can exist as either a IL 17A homodimer, IL 17A and F heterodimer, or IL 17F homodimer, which have been listed in order of decreasing potency/efficacy (Liang, et al., 2007). There are several cell types besides TH17 cells that produce IL17A/F, including: Natural Killer Cells, which are involved in antiviral/anti tumor responses by destruction of target cells (Bryceson et al., 2011); invariant NKT cells, which recognize a limited repertoire of lipids and glycolipids presented by CD1d rather than the typical MHC peptide complex (Diana, 2011);


28 lymphoid tissue inducer(LTi) like cells, which are involved with lymphoid aggregate formation (Cua and Tato, 2010; Takatori, 2009) N eutrophils, and T cells, which are heavily prevalent in the gut mucosa and associated with gut mucosal barrier maintenance (Cua and Tato, 2010) have also been known to produce IL 17 The receptor for IL 17 spans IL17RA IL17RE. IL 17A and IL 17F signal through a hetero dimer receptor of IL17RA and IL17RC (Figure 1 3). IL 17RA is found ubiquitously on all cell types, particularly hematopoetic (areas that generate blood cells and leukocytes) tissue, while IL 17RC has low expression in hematopoetic tissue and high expressio n in the liver, thyroid, joints, and kidney. As IL 17A typically elicits a proinflammatory gene profile similar to those induced by innate immune receptors IL 1R and TLRs, studies were conducted on NF B, a classical transcriptional factor associated with inflammation. Accordingly, it has been found that several DNA elements bind NF B in the promoter of IL 17A and induce gene expression. Gel shift assays indicated IL 17A activates p50 and p65, two hall mark members of the canonical NF B pathway. There has been no confirmation on the use of non canonical NF B pathway as of yet (Ruddy, et al. 2004). Similar to IL 1R and TLRs, IL17 signaling requires TNFR associated factor 6 (TRAF6), as Traf6 / mice are cannot activate NF B through IL 17. In spite of all the similarity to innate signaling, IL17RA does not contain a typical TRAF 6 binding motif. And, it has been shown that common innate signaling proteins like MYD88, TRIF, IRAK4, and IRAK1 are not necessa ry for IL 17A signaling. Insight into IL 17 signaling was provided when SEFIR domains were discovered. A bioinformatics study revealed that IL17R all expressed a conserved motif homologous to the Toll/IL 1R domain (termed SEFIR) which provides docking sit es for intracellular adaptors like


29 MYD88. SEFIR then recruits ACT1, which has a TRAF6 binding domain that leads to NF B activation. IL 21 is another major product of TH17 cells. The IL 21R consists of the common c chain and the unique IL 21R chain and is expressed primarily in the spleen, thymus, peripheral blood, and lymph nodes (Ozaki, 2000; Parrish Novak, 2000). IL 2 1R is also constitutively expressed by B cells, dendritic cells, epithelial cells and NK cells. T cells will also express IL21R, but only upon activation (Brandt, 2003; Monteleone, 2006). IL 21 signals through Janus Kinase (JAK) 1 and JAK3, activating Sign al Transducer and Activator of Transcription (STAT) 3. It has also been known to activate STAT1 and weakly trigger STAT5 (Asao, 2001). IL 21 has broad immunological effects. It reduces the expression of MHC II, CD80, and CD86 in dendritic cells (Brandt, 20 03; Strengell, 2006). It also activates the phagocytic and proteolytic abilities of macrophages and contributes to inflammation and remodeling of extracellular matrix through the action of matrix metalloproteinases (MMPs) (Ruckert, 2008; Monteleone, 2006). IL 21 can induce death, proliferation, and antibody class switching in mature B cells (Mehta, 2003). CD4+ cells are the main producers of IL 21. This aids in TH17 development by inducing upregulation of IL 23R, which is involved with the long term sustain ment of a TH17 phenotype (Zhou, 2007). IL 21 can trigger CD8+ T cells to express lower levels of CD44, CD25, granzyme B, interferon gamma (IFN ), and decreased ability to kill target cells (Hinrichs, 2008). Additionally, IL 21 can induce IL 10 production, which limits CD8+ T cell activation and proliferation (Spolski, 2009). IL 21 has been implicated in other autoimmune models. In NOD mice, it caused increased T cell turnover (King, 2004). In addition, IL 21 is thought to play a role in the


30 MRL Fas lpr mouse model which spontaneously develops lupus ( Herber 200 7 ). As the mice aged and died of severe lupus, increased levels of IL 21 were observed. The CIA mouse model for rheumatoid arthritis showed alleviation of symptoms when IL 21 signaling was blockaded (Y oung, 2007). As IL 21 is closely associated with B cell proliferation and antibody class switching, it is plausible to believe that the promotion of auto antibodies may be detrimental in these autoimmune disease models. While T cells, LTi cells, LTi lik e cells, and NK cells have been known to make IL 22, it is another cytokine typically associated with the TH17 cell subset. Like IL 21, IL 22 signals primarily through the STAT3 pathway. The receptor for IL 22 consists of the unique IL 22R chain and the IL 22R expression is typically restricted to epithelial cells like keratinocytes and colonic epithelial cells (Lejeune, 2002). 6, this is not the ideal setup to make IL ually inhibits IL 22 production. IL 22 is generation is dependent on IL 23 and AHR expression, meaning that IL 22 is more heavily involved in the effector phase of this cell type (Siegemund, 2009; Munoz, 2009). Similar to IL 21, IL 22 has been involved i n different models of chronic inflammation like psoriasis and rheumatoid arthritis. However, the increase in IL 22 was a correlation, and not a direct cause, of these diseases (Wolk, 2006; Andoh, 2005). Mice overexpressing IL 22 display psoriasis and an ab errant skin phenotype (Wolk, 2009). In a model using collagen to generate joint autoimmunity, it was shown that an IL 22 deficiency was associated with decreased incidence of arthritis (Geboes, 2009). While IL 22 has been implicated in some inflammatory ac tivity, it has a dual nature. IL 22 has actually shown to be protective in hepatitis (Radaeva, 2004, Zenewicz, 2007).


31 IL 22 activates anti apoptotic and pro survival factors in hepatocytes, leading to their enhanced longevity (Radaeva, 2004). With IL 22, l iver tissue has also been shown to regenerate following a partial hepatectomy or alcohol induced damage (Ki, 2010; Ren, 2010). Patients suffering from inflammatory bowel disease have mutations in genes encoding IL 22 and the IL 09; Glocker, 2009). IL 22 has also been protective following the transfer of colitis inducing CD4+CD45RB high T cells. This protection may be correlated with the ability of IL 22 to induce expression of anti microbial molecules within the GI tract, which in defensins (Wolk, 2004; Zheng, 2008). In addition, IL 22 causes mucin to be produced. Mucin is a heavily glycosolated set of proteins that create a protective barrier lining the GI tract, which limits an immune response by separating both commensal and pathogenic bacteria from the epithelial layer (Sugimoto, 2008). Finally, IL 22 actually plays a role in healing and tissue repair. Mice deficient in IL 22 displayed delayed healing in colonic biopsies compared to controls (Pickert, 2009). Plasticity o f TH17 Cells T Helper cells are typically classified by their cytokine production profile and the expression of lineage specific transcription factors. Recent research has shown that TH17 cells demonstrate plasticity, blurring the lines between well define d T Helper classes. The phenotype of in vitro generated TH17 cells was shown to be unstable, as subsequent treatment with TH1 or TH2 polarizing conditions allowed the cells to be converted to TH1 like or TH2 like cells (Lexberg, 2008). And, it has been sho wn that IL 12 (a cytokine that favors TH1 cell development) and IL 23 are capable of inducing IFN production by TH 17 cells. Moreover, IL 12 suppresses TH17 related activities,


32 upregulating the TH1 lineage transcription factor T bet. In order to achieve the same type of efficacy, TH17 cells required several treatments of IL 23, demonstrating that IL 12 is a very potent inhibitor of TH17 differentiation and enhancer of the TH1 phenotype (Lee YK, 2009). Upon conversion to TH1 cells, the former TH17 cells also require the activity of STAT4 and T bet. The ability of TH17 cells to convert to TH1 cells was investigated by Bending et al. (2011). Bending studied epigenetic control of TH1 and TH17 related factors in Th17 cells created by both in vitro and in vivo methods. Epigenetics refers to the control of gene expression through factors unrelated to its DNA sequence. Methylation of amino acids is a common method of epigenetic control. For instance, Bending examined H3K4 (Histone 3, Lysine residue 4) and H3K27. The tri methylation of H3K4 is associated with the actively transcribed genes (Santos Rosa, 2003), while the tri methylation of H3K27 both ex vivo isolated TH17 cells and in vitro developed Th17 cells display bivalent marks for T bet, the TH1 transcription factor. Biva lency refers to genes containing epigenetic markers for both repression and activation. In this instance, the bivalency of T bet in TH17 cells may explain why they can be predisposed to changing to a TH1 phenotype so easily. Interestingly, Bending also sho wed that ex vivo isolated TH17 cells express lower levels of IL 12R) compared to TH17 cells made in vitro This lower expression was due to restrictions created by chromatin modifications, another method of epigenetic control. These restrictions could be lifted upon in vitro treat ment with IFN In addition, they showed that IL 12 in vitro treatment of freshly isolated ex vivo TH17 cells removed H3K27 tri methylation at T bet.


33 Epigenetic studies have provide d valuable insight as to why in vitro generated TH17 cells exhibit plastic ity. This ability was highlighted in studies by Martin Orozco et al. and Bending et al. (2009). Both groups transferred TH17 cells into an NOD.SCID model and noted an onset of T1D. However, they observed that these cells ceased to express TH17 related fact ors and began pumping out TH1 factors. Furthermore, only antibodies against IFN ameliorated disease, while anti IL 17 antibodies were ineffective at changing disease onset. Both groups provide evidence that TH17 cells first convert into TH1 cells before initiating disease. The NOD.SCID model represents a lymphopenic model, which lac ks properly functioning T and B cells. This type of environment may be especia lly conducive to TH1 conversion. This is particularly important because it can give researchers false indications on the role for a T cell population. For example, many believed TH17 cells could cause T1D upon transfer into a NOD.SCID mouse, but it was later revealed these cells convert to a TH1 phenotype before initiating T1D (Bending, 2009). Pakala et al. also demonstrated that the transfer of TH2 (T helper cells that promote a humoral and allergic response ) into NOD.SCIDs still caused T1D. In a lymphopenic environment, there may not be enough regulation from Tregs to prevent conversion to TH1 cells that attack the pancreas. Sujino et al. demonstrated a similar mechanism in a co litis model, as transferred TH17 cells converted into TH1 like cells before causing onset of the disease. R egulatory T cells stopped this conversion and prevented onset of colitis. There has even been work showing that a transitory Treg/TH17 cell type may exist. Tartar et al. demonstrated that a Foxp3/ROR t population exists naturally in NOD mice and can be expanded upon treatment with an antibody hybrid utilizing GAD protein. Upon transfer


34 with diabetogenic splenocytes into an NOD.SCID model, Foxp3+/ROR t+ cells prevented onset of disease by trafficking to the pancreas through CD62L expression. abilities. The Role of TH17 Cells in Type 1 Diabetes While TH17 cells have b een implicated as causal in other autoimmune models, their role in Type 1 Diabetes (T1D) is far more controversial. IL 21, one of the main products of TH17 cells, was shown to be critical for T1D onset in NOD mice. IL21R knockout mice did not display insul itis, with only 1 of 20 animals becoming diabetic (Spolski, 2008). For the one animal that did develop diabetes, it is possible that the infiltrating cells destroyed their target and left the pancreas, an event the histology snapshot could not capture. Ema maullee et al., suggested that TH17 cells may be responsible for causing T1D, as anti IL17 antibodies and recombinant IL 25 alleviated onset of disease in NOD mice. However, their method of inhibition revolves around a product not unique to TH17 cells. In addition, throughout onset of the disease, the group was unable to identify TH17 cells in the pancreas. Moreover, IL 25 is associated with biasing the immune system toward a TH2 phenotype. It may be more vital, then, to limit the abilities of a TH1 populat ion via conversion of nave CD4+ T cells to TH2 cells. Some studies involving TH17 cells and T1D revolved around the use of Complete Guerin (BCG). CFA utilizes killed pieces of Mycobacterium in mineral oil to el icit an immune response. It was shown that a single treatment of diabetic NOD mice with CFA could rever se diabetes onset (Ryu, 2001). Initial reports from Gao et al., implicated a reduction in IL 17 production following CFA


35 treatment of NOD mice. However, the paper does not specify a particular cell source, only citing that the IL 17 producer was not CD4+, CD8+, CD11b+, CD11c+ or TCR+ cells. The suppressed IL 17 producers were likely an unidentified myeloid population (Gao, 2010). On the other hand, BCG administration to NOD mice has shown a reduction in the proinflammatory cytokines TNF and IFN Nikoopour et al., also conduct ed cytokine analysis of NOD mice injected with CFA. Lymphocytes from the CFA treated mice showed increased production of IL 17, IL 22, IL 10, and IFN They also found that the adoptive transfer of CFA treated, TH17 polarized cells into an NOD.SCID mouse m odel delayed onset of T1D (it is likely that CFA treatment is able to maintain the TH17 phenotype and prevent conversion to TH1 cells which is in contrast to the studies performed by Bending and Martin Orozco listed above). The addition of anti IL 17 anti bodies restored onset of T1D in these mice. While the production of IFN 17, and anti inflammatory IL 10 is what proves vital in this model. It is also possible that the time frame could be responsible for the differences seen by Gao and Nikoopo ur, as they examined cytokine production 18 and 10 days post CFA administration respectively. Yang Hau et al. demonstrated that using all trans retinoic acid on NOD mice prevented producing CD4+ and CD8+ cells, while not affecting IL 17 producing CD4+ cells. To this point, the relationship between TH17 cells and T1D in NOD mice is still hotly contested. TH17 Cells, Humans, and T1D TH17 cells are less established in the human setting. Human TH17 cells do n ot match their mouse counterpart in cytokines required for differentiation, nor do they carry


36 the same types of surface markers (Crome, 2009). To this point, a few studies have been conducted on the relationship between T1D, TH17 cells, and humans. Upon tr eating peripheral blood mononuclear cells with protease resistant GAD peptide, it was observed that the cells produced less IL 17, IL limitations, as it does not represent an in vivo method. Honkanen et al. found that recently diagnosed children exhibited higher levels of TH17 related factors compared to healthy sibling controls. Stimulating their peripheral blood mononuclear cells cause d an increase in expression of IL 17, IL 22, and RORc. Bradshaw et al. found that human monocytes preferentially secreted IL 6, two cytokines capable of generating human TH17 cells. These monocytes elicited the development of TH17 cells. However, 17, suggesting a fluid phenotype. A separate study conducted by Chatziegeorgiou et al. demonstrated that patients suffering from T1D express higher levels of TH1 related factors within the first six months of di agnosis (as indicated in protein levels obtained from blood). In patients with disease duration over six months, they noticed an increase in TH17 related factors, suggesting they do not participate in initiation of the disease. Their role as an effector ce ll, however, is still plausible. While TH17 cells may seem to be pathogenic in an autoimmune diabetic human setting, evidence still points to TH1 cells as the initiators of the disease. The exact role of TH17 cells must still be established. Environmental Factors in T1D While genetic factors undoubtedly play a key role in T1D, recent studies have pushed environmental factors into the forefront. This has been supported by studies in humans, as the rate of T1D between monozygotic identical twins is only 30 50 %


37 (Barnett, 1981; Kumar, 1993). In addition, several nations have reported a 3 5% increase in T1D incidence annually over the last few decades (Gale, 2002). There are reports of geographic location influencing T1D development, as regions along the Mediterr anean Sea only report 5/100,000 children suffer from T1D (EURODIAB ACE Study Group, 2000). On the other hand, countries like Finland reported 50/100,000 T1D cases in children a ten fold difference. All of this data points to risk factors located outside of chromosomal hot spots, such as viral infection, diet, and gut microbiota. Viruses in T1D Initial interest in the link between T1D and viruses began with studies on rubella. Researchers noticed that infection with rubella was often subsequently followed by T1D onset (Ginsberg Fellner, 1984). The theory here is that viral infection can either mediate a direct cytolytic effect on pancreatic cells (Yoon, 1991) or induce immunity due to the homology between viral structures and beta cell antigen. The autopsies of newborn infants that died of Coxsackie virus infection revealed virus positive islets, while control pancreata did not (Foulis, 1990; Ylipaasto, 2004). In addition, enterovirus RNA was detected in 57% of individuals that developed T1D within 6 months co mpared to 31% of normal controls (Lonnrot, 2000). The exact role of viruses, however, is still controversial as others have observed no link with T1D development (Graves, 2003; Fuchtenbusch, 2001). Dietary Factors One of the biggest focal points in dietary arose when it was discovered that humans produced antibodies against bovine insulin (Vaarala, 1999). Bovine insulin is very homologous to human insulin as they differ at only 3 amino acid residues. Furthermore, studies have demonstrated that children that


38 ended breastfeeding at an earlier time point and began drinking CM were at an increased risk for T1D (Gerstein, 1994; Norris 1996). The elimination of CM related proteins in the first 6 8 months of life led to a 40 60% reduction in T1D associated to permeability early on in life. As the infant ages, the gut becomes more restricted, tightening junction between epithelial cell s (Vaarala, 1999). Therefore, dietary antigens Other Environmental Factors Aside from the items listed above, a few other environmental factors have been considered for their influen ce in T1D. Vitamin D is among the factors considered to have a negative correlation with T1D onset. A Finnish study observed that infant Vitamin D supplementation was associated with decreased incidence of T1D (EURODIAB Substudy 2 Study Group, 1999). Moreo ver, T1D onset occurs at a higher rate in winter months than summer months (Knip, 2005). As Vitamin D production is directly associated with exposure to sunlight, this may explain the seasonal difference in diabetes onset and the geographical differences m entioned above. In order to explain several of the environmental factors detailed above, several theories have been developed. The hygiene hypothesis states that regular, normal infections in childhood may induce tolerance later on in life. As more develo ped countries become cleaner and cleaner, there has been an observed spike in T1D disease incidence (Bach, 2001; Pundziute Lycka 2000; Viskari, 2000). A study by EURODIAB Substudy 2 demonstrated that regular preschool attendance and infectious disease expo sure was negatively correlated with T1D. The accelerator or beta cell stress/overload hypothesis has also emerged as a prominent explanation of trends in


39 diabetes incidence. Essentially, the accelerator hypothesis claims that obesity and excessive weight g ain in children leads to an increased demand for insulin and insulin resistance. Recent studies have shown that active beta cells are more susceptible to damage from cytokines than resting cells (Wilkin, 2001). Interestingly, world trends have shown that c ountries that exhibit an increase in Type 2 Diabetes (caused by insulin resistance and excessive weight gain) also show a parallel increase in T1D incidence (Onkamo, 1999; Akerblom, 1985;). Environmental factors are gaining credence as a major player in T1 D development. In addition to diet and virus infection, bacteria are being indicated as major influencers of the host, modulating immune responses and possibly altering T1D onset. Possible Treatments for T1D Over the last few decades, the NOD model has pro vided great insight into T1D development. However, there are over 200 successful treatments in NOD mice that prevent or delay onset of T1D (Anderson and Bluestone, 2005). A large majority of these treatments have proven unsuccessful in human translational studies. To date, there are several ongoing human trials investigating possible successful interventions and preventions of T1D. Insulin has been a center of focus, as the NOD mouse has shown resistance to T1D if exposed to oral insulin (Atkinson, 1990). T he Diabetes Prevention Trial Type 1 (DPT 1) has attempted to determine whether injected or oral insulin would be effective. While neither treatment entirely prevented onset a decrease in insulin autoantibodies and a delay in diabetes onset was observed in those who received oral insulin (Kupila, 2003). Further studies are being conducted to determine whether the dosage of insulin plays a role in the treatment (Harrison, 2010). GAD65, another marker the body generates autoantibodies against, is also being t ested for


40 treatment use. The NOD mouse saw prevention of T1D with GAD65 vaccination (Tisch, 1994). In humans, a 20 g dose of GAD65 maintained levels of C peptide (a protein that promotes the proper formation of insulin chains). This promising data has led to phase III trials in human patients recently diagnosed with T1D. In addition to the prevention strategies listed above, several studies are being conducted on intervention methods. The immunosuppression of CD3 cells seems like a plausible treatment, as the inhibition of autoreactive T cells would be beneficial. Anti CD3 antibodies have been used to target those T cells for destruction. In addition, anti thymocyte globulin (made by inoculating horses or rabbits with human thymocytes) treatment generates a polyclonal anti human T cell antibody preparation (Feng, 2008). Following the short term deletion of these T cells, Tregs may be selectively expanded, stopping any T effector cells that resurface. This treatment requires 6 to 14 day intravenous infusions, which may be a deterrent to patient sign up. There are also studies focused on stopping B cells, the producers of autoantibodies. Anti CD20 antibodies like Rituximab have been shown to be effective in limiting the signaling interaction with T cells. In ad dition to being potent in the NOD model (Xiu, 2008; Fiorina, 2008), Rituximab is a standard part of treatments in several human diseases, like rheumatoid arthritis and non the results of their two year t ime point in Rituximab treatments in T1D patients. There (Mycofenolate Mofetil (MMF)) and the depletion of activated T cells (Daclizumab (DZB)). To this point, however, neither drug or combination of drugs has been capable of affecting C peptide levels beneficially (Gottlieb, 2010).


41 Several NOD mouse studies have demonstrated that the transfer of mature or tolerogenic DCs into young, prediabetic mice was an effective method of preventing onset of T1D ( Feili Hariri 1999). However, the human rejection of foreign MHCs could prove to be a difficult barrier to overcome. As such, mesenchymal stem cells (MSC) have gained momentum as a possible avenue of treatment. MSCs generate little to no immunogenicity from host patients. In addition, MSCs are thought to suppress inflammation in localized areas. While MSCs do not live for extensive amounts of time in the human body, this could be beneficial as there are concerns about creating an im mortal cell line for in vivo their ability to avoid stimulating the immune system. MSCs have also been shown to cell repair and enhance the production of insulin (Fiorina, 2009; Madec, 2009). In order to create a proper treatment for T1D, the agents must be considered for efficacy and safety. As research progresses in this field, products are n ot only being selected simply because they were advantageous in NOD models. Agents are being selected because they have also shown beneficial effects in other autoimmune models like lupus or multiple sclerosis. In addition, these treatments must have limit ed side effects. In light of this, probiotics may be a new line of treatment for autoimmune disease like T1D. Many probiotic treatments use bacteria that are generally regarded as safe (GRAS) and have shown an enhanced ability to modulate the immune respon se. Modulation of the Immune Response by Bacteria As over 100 trillion microbiota inhabit the human digestive tract (Hooper, 2001), it is important to understand how they can influence the immune response. Gut bacteria can aid the host by absorbing otherwi se indigestible nutrients and can even limit the


42 ability of pathogenic bacteria to adhere to the epithelial cells of the intestine. This is done through the production of antimicrobial compounds, outcompeting pathogenic bacteria for adhesion sites, and mod ulating the immune system (Borchers, 2009). Typically, dendritic cells sample antigen from the intestinal lumen by reaching through gaps in epithelial cells or through encounters in M cells DCs transfer these antigens to he Mesenteric Lymph Nodes, where they may activate T cells (Figure 1 1). DCs treated with the supernatant of epithelial cell bacteria cocultures preferentially drove a TH2 or Treg response (Rimoldi, 2005). It has been shown that the influence of gut bacter ia on the immune system extends beyond the localized area of the gut. Dobber et al. reported that germ free mice possessed lower levels of CD4+ T cells in the spleen. These CD4+ cells were also biased to produce IL 4. Therefore, mice lacking gut microbiota are biased to a TH2 phenotype. Commensal bacteria could fix this TH1/TH2 imbalance, as recolonization by B. fragilis restored CD4+ T cell numbers and induced expression of IFN Interestingly, this ability was associated with a single product made by B. fragilis : polysaccharide A (PSA) R ecolonization by B. fragilis lacking PSA did not reproduce these results (Mazmanian, 2005). Probiotics are defined as a class of bacteria capa ble of surviving the intestinal transit (acid and bile tolerant), adhering to the mucosal surface, and colonizing it for a temporary amount of time. In addition, these bacteria can produce antimicrobial substances, antagonizing pathogenic bacteria. Typical ly, probiotic bacteria are also associated with well documented and validated health benefits (Borchers, 2009). Lactobacillus (LB), along with Bifidobacteria, are considered probiotic bacteria. While


43 the ability to stimulate IL 12 is varied in LB, a majori ty of the species are able to induce high levels of IL 10 (Fink, 2007). Several LB strains have been noted as potent inducers of APC maturation, which may mediate tolerance through anergy or AICD (Borchers, 2009). Oral feedings of Lactobacillus casei preve nted T1D in NOD mice, mainly by inducing IL 2 and IL 10 (Matsuzaki, 1997) VSL#3, a probiotic mixture of LB and Bifidobacteria strains, was shown to limit the production of TH1 associated cytokines and induce production of IL 10 from human DCs (Jijon, 2004 ; Hart, 2004). It also prevented onset of T1D in NOD mice, again through the production of IL 10 (Calcinaro, 2005). LB is also known to improve epithelial cell integrity, limiting the ability of microbes to breach into direct contact with the immune system (Madsen, 2001). Upon LB stimulation, gut DCs have also been shown to be capable of inducing Tregs and IgA production from B cells, which binds up pathogens in the gut (Tezuka, 2007). LB treated DCs limited inflammation in the gut via a MyD88 (a major prot ein involved in the recognition of pathogen associated molecular patterns) and TLR2 pathway (Round, 2010). Furthermore, Valladares et al. demonstrated that feeding Lactobacillus johnsonii N6.2 (LjN6.2), isolated from diabetes resistant rats, to diabetes pr one rats delayed onset of T1D. This delay of T1D was correlated with a TH17 cell bias observable in both the mesenteric lymph nodes and spleen (Lau, 2011). Taken together, this data shows that bacteria have a profound ability to alter the host immune respo nse. In the context of T1D, the probiotic treatment of LjN6.2 may limit the skewing of diabetogenic T cells to a TH1 phenotype, preventing the initiation step of the disease. Gut Bacteria and T1D Vaarala et al. have suggested that a series of immunological signs align and contribute to the onset of T1D. The first sign is an altered gut bacteria composition. Gut


44 bacteria play a major role in this theory, and has been supported by research showing that rodents derived by Caesarian means or kept in germ free e nvironment develop T1D at an increased pace (Like, 1991). In addition, certain antibiotics decreased the occurrence rate of T1D in NOD mice (Brugman, 2006). Rakoff Nahoum et al. demonstrated that a low level of activation of MyD88 is beneficial for epithel ial cells, rather than detrimental. Activation of the MyD88 pathway by commensal bacteria creates products associated with protection, tissue repair, and angiogenesis (the development of new blood vessels). Among these products, 6, a mediator of epithelial cell protection (Tebbutt, 2002), are particularly interesting, as they are involved in TH17 generation. While inflammation may be helpful in small doses, a constant, exacerbated level of gut activation through TLRs c ould be dangerous. Wen et al. demonstrated that MyD88 / NOD mice do not develop T1D. However, if these knockouts are kept germ free, diabetes incidence returns, and is only ameliorated by the addition of bacteria to the digestive tract. Clearly, the inter action between immune cells and gut bacteria can maintain a healthy homeostasis. If environmental factors change the composition of gut bacteria, abnormal immune responses may disrupt the established balance of tolerance and inflammation. Gut leakiness is the second focal epithelial cells tight and packed together, sealing off the luminal contents from direct interaction and activation of the immune system. Studies have s hown that both rodents and humans display an increased permeability in the gut following recent diagnosis of T1D. Increased exposure to the gut in an uncontrolled manner may set off this autoimmune reaction. An altered intestinal immunity is the final fact or in this model. This


45 may include aberrant immune responses in the gut. For example, lamina propia (the area directly underlying the epithelial cell layer) cells of diabetic patients show elevated levels of MHC Class II molecules and integrin, which is necessary to home to the intestines (Savilahti, 1999;Westerholm Ormio, 2003). In addition, diabetic patients may show decreased levels of Tregs and an increased presence of autoimmune cells in the gut (Tiittanen, 2008). These autoimmune cells, in turn, may release cytokines that promote an inflammatory environment and recruit more immune cells to the affected area. The perfect storm follows the old friends hypothesis, where the consistent presence of normal commensal bacteria maintains a tolerant environ ment, possibly stimulating Tregs to produce anti inflammatory molecules like IL 10 and TGF 2005). As infiltrating lymphocytes in the islets express the gut homing receptor, integrin, it is likely that they circulate from the gut to the pancre as (Yang, 1997; Hanninen, 1996). In addition, DCs carrying intestinal antigen may travel to the pancreatic lymph nodes to stimulate T cells. Therefore, specific interactions or aberrations in the gut can likely influence the development of T1D in the perip hery.


46 Figure 1 1. Gut antigen sampling and presentation by dendritic cells Dendritic cells (DC) sample antigen through the epithelial cell layer in the intestines. DC will traffic to various lymph nodes displaying the broken down peptide on its major histocompatability complex T cells that possess a T cell receptor (TCR) specific for the peptide will become activated while the DC provides costimulation and a cytokine milieu.


47 Figure 1 2. T helper cell differentiation. Following activation by DCs, nave CD4+ T cells will differentiate into different T Helper classes depending on the cytokines presented to it. Each T Helper class is listed with its hallmark cytokines required for differentiation, its main transcription factor, and its function in th e immune system.


48 Figure 1 3. IL 17 signaling. The IL 17R consists of IL 17RA and IL 17RC. Upon ligation, ACT1 is recruited to the SEF/IL 17R (SEFIR) domain. ACT1 recruits TNFR associated factor 6 (TRAF6). TRAF6 leads to the activation of Nuclear Facto r B (NF B). Act1 also activates extracellular signal regulated kinase (ERK), which phosphorylates CCAAT/enhancer binding protein When it is phosphorylated, its ability to promote transcription decreases. This is one of the few inhibitory activities IL 17 is known for.


49 CHAPTER 2 MATERIALS AND METHOD S Characterization of a TH17 Bias Induced by LjN6.2 Animals C 57BL/6 mice (The Jackson Laboratory Bar Harbor, ME) were maintained in specific pathogen free conditions at the Association for Assessment and A ccreditation for Laboratory Animal Care accredited University of Florida under the supervision of the Institutional Animal Care and Use Committee in strict accordance to approved protocols. Bone M arrow D erived D endritic C ell P reparation Bone marrow was re moved from the femur and tibia bones of NOD mice and washed. Progenitor cells were subsequently incubated in RPMI 1640 supplemented with 10% FBS, 1% antibiotic/antimycotic, and GM CSF (20 ng/m L ) in 24 well plates (1 10 6 cells/well). Old medium was remove d and replaced with 1 ml fresh complete RPMI 1640 medium containing 20 ng/m L GM CSF every 2 d. On day 8, aggregates were dislodged and transferred with complete RPMI 1640 medium into 100 mm petri dishes at a maximum of 1 10 7 cells/dish. At 24 and 48 h ti me points following transfer, nonadherent, nonproliferating, maturing bone marrow derived DCs (BMDCs) were collected from the dish and stored in a sterile flask. 70% of the BMDC preparation was identified as expressing CD11c through flow cytometry. Prior t o footpad injection into NOD mice, some BMDCs were incubated with LjN6.2 for 12 h, followed by extensive washing.


50 D endritic C ell V accinations Nine week old NOD mice received three weekly hind paw injections of either PBS or LjN6.2 pulsed BMDCs (5 10 5 ). Mice receiving the injections were sacrificed on week 13 of life, followed by isolation of pancreas, spleen, and various lymph nodes. T L ymphocyte P urification Lymph node and splenocytes from C57BL/6 mice were stained with biotin conjugated Abs to mouse B 220, CD11c, CD11b, and NK1.1 for 10 min at 4C, followed by streptavidin conjugated MACS beads followed by passage over a MACS LS column Bergisch Gladbach, Germany) ( Guay, 2007 ). The negatively selected fraction contained CD3 + lymphocytes (>95% pure as confirmed by FACS), and the positively selected fraction served as APCs. Of the APC fraction, the populations typically co nsist of 72% B cells (B220+), 14% macrophages (CD11b+), and 14 % dendritic cells (CD11c+). The percentage of NK cells has not been determined by our group. Proliferation A ssays Lymphocyte proliferation assays were performed, as previously described ( Larkin III, 2007 ), with modifications. Total splenocytes (4 10 5 ) or 2 10 5 MAC S purified T L anti CD3 ( clone 17A2; eBioscience San Diego, CA), 2 10 5 L anti CD28 (clone 37.51; eBioscience ) in the presence of LjN6.2 at various concentrations, as indicated. Cell cultures were incubated in supplemented RP MI 1640 (10 040 CV; Cellgro Manassas, VA) containing 10% FBS (10082 147; Life Technologies Carlsbad, CA) and 1% antibiotic/antimycotic (30 004 CI; Cellgro) in 96 well round bottom plates. After 72 h of incubation, cultures were pulsed with 0.5 mCi [ 3 H] thymidine (GE Healthcare Arlington Heights, IL) per well and


51 harvested 16 18 h later. [ 3 H] Thymidine incorporation was measured using a Beckman LS3801 Liquid Scintillation System In V itro C ytokine S ecretion A nalysis Leukocytes from C57BL/6 mouse sple en and lymph nodes were isolated and L anti L anti CD28 (BD Biosciences San Diego, CA), and LjN6.2 or LrTD1 at various concentrations. To inhibit IL 6 signaling, l euk ocytes were also coincubated with anti CD3, APCs, and LjN6.2 in the presence o r absence of an IL 6 signal neutralizing Ab mixture containing the following: 0.2 mg/m L rabbit polyclonal to IL 6 (Abcam L anti mouse IL L anti mouse CD126 (554459; BD Biosciences) for 48 h. At L supernatant was removed from each well and replenished with fresh medium, as previously described ( Lerman, 2004 ). (555138; BD Biosciences ) and IL 6 (5 55240) ELISA kits were obtained from BD Biosciences Capture mAb (555068) and detection mAb (555067) for IL 17A were also obtained from BD Biosciences Cytokine standard for IL 17 was purchased from eBioscience (14 8171 80). Role of TH17 Cells in an Autoimmune Diabetes Setting Animals NOR/LtJ and NOD/ShiLtJ (The Jackson Laboratory Bay Harbor, ME) were maintained in specific pathogen free c onditions at the Association for Assessment and Accreditation for Laboratory Animal Care (AAALAC) accredited University of Florida under the supervision of Institutional Animal Care and Use Committee (IACUC). Blood glucose levels of NOD/ShiLtJ mice were mo nitored using a blood glucose monitoring kit.


52 NOD mice were defined as diabetic following repeated blood glucose levels over 250 mg/d L for two consecutive days. NOD mice were euthanized following diabetes onset. Axillary lymph nodes, mesenteric lymph nodes pancreatic lymph nodes, and spleens were extracted from each mouse for in vitro or ex vivo analysis. Flow Cytometry Single cell suspensions of pooled l ymph nodes (axillary, inguinal, brachial, mesenteric, and superficial cervical), and spleen were staine d with the following mAbs for flow cytometric analysis: anti CD4 Pacific Blue (RM4 5;), anti MHC I FitC (KH95), anti CD11c PE (N418; eBioscience San Diego, CA), anti B220 APC (RA3 6B2, eBioscience ), anti CD11b A700 (M1/70), anti CD11b FitC (M1/70, eBios cience ), anti CD11c FitC (N418, eBioscience ), anti CD86 A700 (GL1), anti CD80 PE (16 10A1), DEC 205 APC (205yekta) and anti MHC II FC (39 10 8,) mAb. All flow cytometry antibodies were purchased from BD PharMingen unless otherwise stated. 50,000 100,000 live events were collected on a LSRII (BD PharMingen ) and analyzed using FlowJo software (Tree Star, San Carlos, CA). The absolute numbers of cells recovered from various organs was determined by multiplying the total number of cells isolated from vario us tissues by the percentage of total cells bearing a lineage specific marker denoted by flow cytometry. Pancreas RNA Isolation/Histology Pancreas was harvested from NOD and NOR mice and snap frozen in OCT (Fisher 14 373 65, Pittsburgh, PA) embedding med ium in a dewer of liquid nitrogen and 2 methylbutane (Fisher O3551 4). Blocks were sectioned on a Leica CM 1950 cryostat at a thickness of 40 microns. RNA was then isolated using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems KIT0204, Car lsbad, CA) and


53 protocol. Purity of RNA was confirmed using a Nanodrop ND 1000 Spectrophotometer 5 micron sections were cut and stained for H&Es. Photos were taken at 20x magnification using the Leica DM 2500 Microscope equipped with an Optronics color camera and MagnaFire software (Optronics Goleta, CA). RNA Isolation and RT qPCR. Total RNA was extracted from the lymph nodes and spleens of NOD or NOR mice using the SV Total RNA Isolation System (Promega Corp., Madison, WI, USA), according to the concentrations and purity of the total RNA were determined using a SmartSpec Plus Spectrophotometer ( BioRad Hercules, CA, USA ). First strand cDNA synthesis was performed using ImProm II Re verse Transcription System (Promega Corp., Madison, WI, USA) or iScript RT Supermix for RT qPCR (BioRad 170 8841) Absolute QPCR SYBR Green Mix (ABgene Epsom Surrey, UK) or iQ SYBR Green Supermix Sample (BioRad, 170 8880S) and gene specific prim ers (Table 1) at 200nM were used to amplify relative amounts of cDNA on a PTC 200 Peltier Thermal Cycler with a CHROMO 4 Continuous Fluorescence Detector ( BioRad ). A mplification was performed as previously described (Lau, JI 2011). The fold change in ex pression was T method (i.e. using the equation 2 ) using BioRad software. Bacterial Enumeration and Cell Lysis E. coli, L. brevis, L. johnsonii N6.2, and L. reuteri TD1 strains of bacteria were all provided courtesy of Dr. Graciela Lorca. Briefly, bacterial cells were lysed by treatment with 0.1 mm glass beads in a bead beater for 3 minutes. Cells were then centrifuged at 100,000 g for 15 minutes to separate the membrane (pellet) and cytoplasm


54 (supernatant) fractions. Co ncentration of bacteria was determined by performing ten fold serial dilutions, plating 0.1 mL of the sample on MRS plates. Plates were incubated for 48 hours at 37 C under anaerobic conditions. Statistical Calculations Statistically significant differences were d etermined using Graph Pad Prism software using an unpaired, two tailed student t test. Significance and statistics for studies involving diabetes inciden ce were determined using the Gehan Breslow Wilcoxon test.


55 CHAPTER 3 RESULTS Characterization of a LjN6.2 Mediated TH17 Bias LjN6.2 Induces Apoptosis at High Concentrations Valladares et al. demonstrated that feeding diabetes prone rats with LjN6.2 was cap able of mitigating T1D onset. While the beneficial effects of probiotics have frequently been demonstrated, they have not always been explained mechanistically. And, w hile there have been studies on the effects of LjN6.2 on onset of T1D in diabetes prone r ats (Valladares, 2010), the same has not been done in NOD mice. It is particularly important to use the NOD model as it shares several of the genetic defects found in T1D susceptible humans (see Idd genes listed above). Therefore, it is vital to understand how LjN6.2 can affect the onset of T1D, beginning with its in vitro effects on mouse immune cells. Several studies have stated that certain strains of Lactobacillus are capable of suppressing cytokine production by immune cells. In accordance, we initiall 6 production with increasing concentrations of LjN6.2. In addition, there was a severe reduction in proliferation in splenocytes treated with higher concentrations of LjN6.2. We next wanted to determine whether the reduced cytokine profile was due to active suppression by LjN6.2 or whether it was causing cell death. Leukocytes were incubated with LjN6.2, L actobacillus brevis E. coli (a gram negative control bacteria), or antiCD3 control. At the 48 hour time point, the cells were stained for Annexin V, an early marker of apoptosis. As seen in Figure 3 1, LjN6.2 induced a higher level of Annexin V expression than any other sample set. In addition, an increasing concentration of LjN6.2 corresponded to increased expression of An nexin V in both CD4+ and CD8+ T cells. As the killing of all immune cells provides little


56 insight into mechanism and serves as a detriment to the host, future experiments scaled back to LjN6.2 concentrations that yielded immune responses and reflected phy siological concentrations but did not cause overwhelming cell death. T Cells Require TCR Stimulation t o Create a TH17 Response to LjN6.2 Oral feedings of LjN6.2 were capable of preventing onset of T1D in diabetes prone rats. Lau and Benitez et al. demons trated that these rats had elevated levels of several TH17 related factors in the localized region of the mesenteric lymph nodes. Additionally, this effect was not restricted to the gut, as splenocytes also displayed this increase. Therefore, we characteri zed what factors are required for generating a TH17 bias. Studies have shown that T cells are capable of directly reacting to bacterial agonists through the use of Toll Like Receptors, which recognize common patterns expressed by bacteria and other microbi ota (McAleer, 2010). Therefore, we incubated splenocytes with increasing concentrations of LjN6.2 in the absence of antiCD3, which stimulates T cells through the T cell receptor, mimicking the act of antigen presentation by APCs (Leo, 1987). As seen in Fig ure 3 2, minimal proliferation was recorded in response to LjN6.2 without antiCD3 stimulation As APCs are the main producers of IL 6 and would be unaffected by a lack of antiCD3 (Diehl, 2002) IL 6 is generated in increasing amounts as higher doses of LjN 6.2 are added (until the CFU/mL crosses a concentration threshold and induces cell death) However, despite the presence of the TH17 differentiation factor, IL 6, T cells did not respond by producing IL 17 in the absence of antiCD3. And, at higher concentr ations of LjN6.2, no proliferation or cytokine production was seen, confirming that these doses likely killed all cultured cells, as indicated in Figure 3 1.


57 To test whether T cells require TCR stimulation to respond to LjN6.2, we cultured splenocytes and lymphocytes with antiCD3 (4 g/ml). The cells were also incubated with either LjN6.2 or Lactobacillus reuteri (LrTD1), the strain that increased T1D incidence in diabetes prone rats. Over the course of 48 hours, cells treated with antiCD3 and LjN6.2 respon ded with marked increases in IL 6 beginning as early at 12 hours of incubation. By 48 hours, LjN6.2 treated cells generated over 3 times as much IL 6 as antiCD3 controls (Figure 3 3A). Following IL 6 production, IL 17 protein is induced at 36 hours. By 48 hours, the IL 17 output by LjN6.2 stimulated cells is nearly 4 times the amount made in antiCD3 alone controls. While LrTD1 was capable of creating a small boost in IL 17, it is considerably less than its Lactobacillus counterpart, LjN6.2. In addition, we demonstrated that this IL 17 production was dependent on IL 6 (Serada, 2008). An antibody cocktail against IL 6 caused a 30% decrease in IL 17 production in LjN6.2 stimulated cells. APCs are Required to Create a LjN6.2 Mediated TH17 Bias. IL 6 is an import ant factor for TH17 differentiation. While APCs are generally the main producers of IL 6, T cells have also shown to be capable of making IL 6 (Sofi, 2009). In order to determine whether properly stimulated T cells alone could generate a TH17 bias, we sepa rated T cells from APCs on a MACS separation column. T cells cultured alone were incubated with antiCD3 and antiCD28, as proper costimulation in the absence of APCs is required for a T cell response (Clark, 1987). T cells incubated with APCs were given ant iCD3 as described before. Both sample sets were co cultured with LjN6.2 in increasing concentrations. As seen in Figure 3 4, both T cells alone and T cells with APCs were capable of proliferating at equal rates, as shown by trititated thymidine incorporati


58 However, T cells were unable to produce a strong IL 6 response in the absence of APCs. The presence of APCs allowed for a dose dependent increase in IL 6 production. In addition, without APCs, ve ry low levels of IL 17 were recorded. This data shows that in order to create a favorable TH17 environment, APCs are required for the generation of IL 6, which causes robust IL 17 production by T cells. BMDCs are Capable of Promoting a Long T erm TH17 Bias Dendritic cells are the main samplers of gut lumen and potent activators of T cells (Leser, 2009). In addition, several studies have reported that BMDC transfers into NOD mice were capable of preventing onset of T1D. Therefore, we examined whether LjN6.2 c ould mediate a TH17 bias using DCs as the main APC. BMDC precursors were isolated from bone marrow and matured. BMDCs were co incubated with pure, CD3+ T cell populations and a dose of LjN6.2 where indicated (Figure 3 5). As we have previously shown, the l ack of TCR stimulation did not yield IL 17 production, while the DCs were still able to produce IL 6. With the addition of LjN6.2, BMDCs were provided higher levels of IL 6 and were sufficient to provide a pro TH17 environment to allow for increased IL 17 production. As our treatment of BMDCs was sufficient to create a TH17 bias, we transferred LjN6.2 pulsed BMDCs (LJ BMDC) into 9 week old NOD recipient mice. A set of mice were sacrificed at 13 weeks of age, when all mice were still confirmed euglycemic an d non diabetic. mRNA was isolated from the spleens of the sacrificed mice and tested for TH17 factors. Mice receiving LJ BMDCs exhibited increased message for both IL 6 and IL 17 compared to control treated mice (Figure 3 6). TH17 cells have been reported as notoriously fickle in plasticity. They have been shown to convert into TH1 cells following transfer into NOD.SCID mice (Bending, 2009).


59 In order to determine the stability of the TH17 environment induced by LJ BMDCs, we observed long term LJ BMDC recip ient mice. NOD mice were allowed to live until they were determined diabetic by blood glucose levels. Mice were sacrificed at 20 weeks of age if they were still euglycemic at the end of our time course. Splenocytes were plated in vitro and supernatants wer e analyzed at 48 hours for cytokine production by ELISA. PBS control spleno cytes produced no IL 6 or IL 17 without antiCD3 stimulation. Interestingly, splenocytes of LJ BMDC mice produced large amounts of IL 6 and IL 17 even without TCR stimulation, in con trast to our in vitro work shown above. And, while PBS control mice do produce IL 6 and IL 17 with the addition of antiCD3, LJ BMDC splenocytes produce significantly higher levels of both cytokines (Figure 3 7) We also investigated whether LJ BMDC could p revent onset of T1D. While LJ BMDC slightly delayed onset of T1D, there was no difference in prevention of T1D in the two treatments by 20 weeks of age (Figure 3 8). The Role of TH17 Cells in an Autoimmune Diabetic Setting NOD Mice Display a TH17 Deficienc y Compared to NOR Mice While we have previously established a role for TH17 cells in the BBDP rat (Lau, 2011) their role in the NOD mouse is still being investigated. The NOD mouse is the preferred method of T1D study, as it allows for easy genetic manipu lation and avails itself to a large range of reagents. The NOR mouse serves as the non diabetic counterpart to NOD mice. Originally derived from NOD mice backcrossed with C57BL/6 mice, the NOR mouse still shares several defects associated with the NOD mous e, i.e., the prototypical diabetogenic MHC II Ag7 and defective Treg populations (Serreze, 1994) Yet despite these deficiencies, the NOR mouse does not develop T1D and serves as a control for the NOD mouse. To begin, we investigated the natural capability of NOD


60 lymphocytes to create a TH17 bias. We sacrificed prediabetic NOD mice and compared them to NOR mice. Lymph node cells were plated in vitro with antiCD3. At 48 hours, the supernatants were analyzed for cytokine secretion. There were no significant di and NOR lymphocytes (Figure 3 9 ). However, NOD lymphocytes produced no IL 6 and lower amounts of IL 17 compared to NOR mice. While NOD mice are competent in regards to TH1 cytokine production, they lack the sa me ability to produce TH17 factors. In addition to examining the peripheral lymph nodes, we focused on the autoreactive site of the pancreas. Pancreas from 11 week old age matched mice were snap frozen in liquid nitrogen and Optimal Cutting Temperature (O CT) liquid. This method allows for reliable RNA isolation. In addition, we cut thin sections for hematoxylin and eosin staining, providing a snapshot of possible infiltrating leukocytes. In agreement with published data, Figure 3 10 shows that NOD mice dis play massive insulitis, with leukocytes encroaching upon islets. While NOR mice also display infiltration, they do not exhibit insulitis. qPCR confirmed that NOD mice tend to display elevated levels of CD3+ cells compared to NOR counterparts. To restrict o ur qPCR to T cell messages, we used CD3 as a reference gene and examined the pancreas for several TH17 relevant messages (Fi gure 3 11 different, while IL 17 began to trend toward significance. Similar to what was observed in the peripheral lymph nodes, NOD mice display lower levels of IL 17. They also show lower levels of IL 6 and importantly, ROR cells. Despite possessing less T cells, the NOR pancreas expresses significantly higher levels of TH17 associated cytokines and transcription factors.


61 NOD Lymph Nodes Contain Lower Quantity of APCs Compared to NOR We next determined whether the lack of IL 17 production in the lymph node was due to a lack of T cells in the NOD mouse. Peripheral lymph nodes (axillary, mesenteric, cervical) from the NOD mouse were pooled and compared to NOR lymph node cells with f low cy tometry. As seen in Figure 3 12 A, there were no statistical differences between either CD4+ or CD8+ populations in the two strains of mice. We then decided to focus on APCs, and measured their percentages by staining for CD11b+ (macrophages), CD11c+ (DCs), and B220+ (B cells). Figure 3 12B shows that the NOR lymph nodes possess 1.6 times as many APCs as the NOD mouse. The NOD lymph nodes display an imbalance in the T cell to APC ratio, which may affect its ability to create TH17 cells. LjN6.2 Increase s NOD APC Numbers and Maturation Previous work has shown that NOD APCs suffer from several defects. BMDCs reportedly generate lower yields following harvesting from bone marrow. In addition, they can develop macrophage like qualities. NOD APCs are also les s adept at activating T cells through antigen presentation. In addition, even upon LPS stimulation, NOD APCs do not mature properly (as indicated by upregulation of MHC markers and costimulatory molecules) (Strid, 2001). With all of the deficiencies listed in NOD APCs, we sought to determine whether treatment with LjN6.2 could alleviate some of the issues. Splenocytes were incubated with antiCD3 and a concentration of either LjN6.2 or LrTD1 where indicated. Flow cytometry was performed, staining the cells f or MHC markers, costimulatory molecul es and other maturation markers (DEC 205). Treatment of APCs with LjN6.2 increased CD11b+ expression, increasing from 11.4% to 17.4% at the hi ghest concentration (Figure 3 13 ). A similar trend was observed in CD11c+


62 exp ression, increasing from 12.7% to 18.9%. Our data indicates LjN6.2 treatment may be capable of restoring the lack of APCs found in the NOD lymph nodes. With the observed increase in APC numbers, we next investigated whether LjN6.2 treatment could restore A PC antigen presenta tion and maturation. Figure 3 14 demonstrates that LjN6.2 treatment increased MHC class I and II expression on both macrophage and dendritic cell types. In both cell types, the percentage of MHC class I and II expressing cells were nearl y doubled. LrTD1, which increased diabetes onset in a rat model, was incapable of generating the same type of reaction, as the levels of MHC I and II remained relatively unchanged. As we observed an increase in MHC II, we next examined DEC 205. DEC 205 is dendritic cell surface receptor associated with the upregulation of MHC II, cell maturation, and receptor mediated endocytosis (Inaba, 1995). As we increased the concentration of LjN6.2, we noticed a corresponding drop in the expression of D EC 205 (Figure 3 15 ). Overall, there was an 18% drop in the amount of cells expressing DEC 205. Once again, this effect was not seen with LrTD1 treatment of APCs. DEC 205 was likely being internalized following stimulation with LjN6.2, transferring antigen internally and promoting the upregulation of MHC II. LjN6.2 Triggers BMDC Immunity Through a Surface Antigen As we have seen several immunogenic responses to LjN6.2, we next attempted to determine what antigen was stimulating APCs. LjN6.2 was lysed through the use of a bead beater and glass beads. The membrane and cytoplasm fractions were then separated by using an ultracentrifuge at 100,000g for 15 min. The pellet was considered the membrane fraction, while the supernatant was considered the cytoplasm fraction. We then treated NOD BMDCs with either whole bacteria cells, the membrane fraction,


63 or the cytoplasm fraction. Following 48 hours of incubation, supernatant from the culture was analyzed for IL 6 protein by ELISA. As seen in Figure 3 16A untreated BMDCs did not r espond by making any IL 6. Interestingly, the cytoplasm fraction also elicited very low levels of IL 6 from BMDCs. Only the membrane fraction of LjN6.2 was capable of mediating a high level of IL 6 production comparable to the unlysed bacter ial fraction. I n addition, we confirmed that the response elicited by the membrane fraction was not because a high concentration of bacteria survived the lysis process. As seen in Fig. 3 16B, membrane fractions of LjN6.2 showed no CFU/mL on the 10 6 dilution on MRS media, whereas whole LjN6.2 had a count of 23. Our data indicates a surface antigen likely triggers the observed immune responses and changes we have listed above. Given all the data we have obtained concerning the interaction between LjN6.2 and immune cells, a preliminary model has been created (Fig. 3 17) and will be discussed later on.


64 Table 3 1. Primers used and/or discussed in this study. Primer Sequence Annealing Temperature ( C) Actin F: 5' CCT TCC TTC TTG GGT ATG CA 3 55 R : 5' GGA GGA GCA ATG A TC TTG AT 3' 55 Actin (Pancreas Only) F: 5' CCA CAG CAC TGT AGG GTT TA 3' R: 5' ATT GTC TTT CTT CTG CCG TTC TC 3 55 55 CD3 F: 5' GAC CTG ACA GCA GTA GCC AT 3' R: 5' CTC CTT GTT TTG CCC TGT GG 3' 55 55 IFN F: 5' AAC TAT TTT AAC TCA AGT GGC AT 3' R: 5' AGG TGT GAT TCA ATG ACG 3' 55 55 IL 6 F: 5' GGA AAT GAG AAA AGA GTT GTG C 3' R: 5' CTC CAG AAG ACC AGA GGA AAT 3' 57 57 IL 23p19 F: 5' GCT CTC TCG GAA TCT CT 3' R: 5' AAG CAG AAC TGG CTG TTG T 3' 55 55 IL 23R F: 5' CAG AAA ATT GGA AGT TGG GAT ATG TT 3' R: 5' CAG AAA ATT GGA AGT TGG GAT ATG TT 3' 55 55 IL 17A F: 5' ACT CTC CAC CGC AAT GA 3' R: 5' CTC TTC AGG ACC AGG AT 3' 55 55 ROR t F: 5' ACA GCC ACT GCA TTC CCA GTT T 3 R: 5' TCT CGG AAG GAC TTG CAG ACA T 3' 63 63


65 Figure 3 1 Lactobacillus johnsonii induces apoptosis at high end concentrations Splenocytes and lymphocytes were incubated at a concentration of 4 x 10 5 cells/well for 48 hours with 4 g/m L antiCD3 and an indi cated dose of bacteria where indicated. Cells were then stained with CD4+, CD8+, and Annexin V antibodies for FLOW cytometry.


66 Figure 3 2. LjN6.2 mediation of a TH17 bias requires proper TCR stimulation Graphs show (A) proliferation, (B) IL 6 producti on, (C) or IL 17A production by leukocytes (4 x 10 5 L ) or indicated concentrations of LjN6.2 for 48 hours. Supernatants were removed at 48 hours for ELISAs, while tritiated thymidine was administered at 72 hours an d incubated for 16 18 hours. Data is representative of 3 separate experiments


67 Figure 3 3. LjN6.2 boost s production of TH17 related cytokines in a time dependent manner. Leukocytes were incubated in the presence of anti L ), and/or LjN6.2, L rTD1 (7.5 10 3 CFU/mL) for the indicated amounts of time. Graphs show A) IL 6 or B) IL 17 production mediated by the presence of LjN6.2 or LrTD1 over the indicated time periods. C ) Splenocytes were incubated at 4 10 5 cells/well for 48 hours with the ind icated concentration of LjN6.2 in the presence or absence of an anti IL6 antibody cocktail. Supernatants were removed at 48 hours for ELISAs. Data is representative of 3 separate experiments. *=p<0.05


68 Figure 3 4. LjN6.2 mediated TH17 bias requires th e presence of APCs Leukocytes were sorted on a MACS column. T cells only samples were incubated alone (4 x 10 5 L L antiCD28. T cell and APC samples contained 2 10 5 cells each of T cells and APCs in the presence or absence of anti L ). A) T ritiated thymidine was administered at 72 hours and incubated for 16 18 hours to analyze proliferation Supernatants were removed at 48 hours for B) IFN C) IL 6, or D) IL 17 analysis through ELISA. Data is representative of 3 i ndependent experiments.


69 Figure 3 5 B MDCs are sufficient to drive a LjN6.2 mediated TH17 bias Graph shows A) IL 6 and B) IL 17 production (as measured by ELISA) mediated by the incubation of NOD derived BMDCs and T cells together with antiCD3 (4 g/m L ) and LjN6.2 (7.5 10 3 CFU/mL ) where indicated for 48h. Data is representative of 3 separate experiments.


70 Figure 3 6. LjN6.2 pulsed BMDCs mediate a TH17 bias in recipient NOD mice 9 week old NOD mice received 3 weekly injections of 5 10 5 LjN6.2 pulsed BMDCs or PBS controls. At 13 weeks of age, the mice were sacrificed and the spleens were obtained for RNA isolation. Graph shows IL 6 and IL 17 message in the spleens of NOD mice receiving LjN6.2 DC vaccination compared with PBS treated control mice N =3. *=p<0.05


71 Figure 3 7. LjN6.2 pulsed BMDCs can mediate a longterm TH17 bias in recipient NOD mice 11 week old NOD mice received 3 weekly injections of 5 10 5 LjN6.2 pulsed BMDCs or PBS control. Mice were sacrificed upon two consecutive days of observed hyperglycemia or allowed to progress to 20 weeks of age. Upon sacrifice, 4 10 5 splenocytes were plated in the presence or absence of antiCD3 (4 g/m L ). Graphs show IL 6 and IL 17 ELISA data for supernatants obtained at 48 hours. 13 control mice and 9 LjN6.2 pulsed BMDC mice were used. *=p<0.05, ***=p<0.005


72 Figure 3 8. Transfer of LjN6.2 pulsed BMDC does not prevent onset of T1D in NOD mice. NOD mice were injected weekly at 9 weeks of age for a total of three treatments with either PB S or BMDC pulsed with LjN6.2. Mice possessing blood glucose levels over 250 mg/dL for two consecutive days were considered diabetic. p = 0.8905. Figure was provided courtesy of Erin Collins.


73 Figure 3 9 NOD lymphocytes display a reduced IL 6 and IL 17A production Lymph nodes were isolated from NOD and NOR mice 4 12 weeks old and were plated at a concentration of 4 x 10 5 cells/well in the presence of antiCD3 (4 g/m L ) and incubated at 37 C for 72 hours. A) Graph showing proliferation after a 16 18 hour incubation with tritiated thymidine following the 72 hour mark Supernatants were removed for ELISA anal ysis of B) IFN C) IL 6, and D) IL 17 at 48 hours and replenished with fresh media. 6 mice were used per species. ***=p < 0.005


74 Figure 3 10 NOD Pancreas display insulitis and higher infiltrating levels of T cells Pancreas was isolated from NOD and NOR mice and snap frozen in OCT medium using 2 methylbutane and liquid nitrogen. Sect ions were isolated with a cryostat machine. A) H&E stains performed on 5 micron thick pancreas sections isolated from NOD or NOR mice. Photos are at 20x magnification. Arrow indicates location of an islet. B) Graph showing CD3 qPCR results for 40 micron se ctions of pancreas relative to actin. A total of 6 mice per category were examined.


75 Figure 3 11 NOD Pancreas display reduced levels of TH17 related factors Pancreas was isolated from NOD and NOR mice and snap frozen in OCT medium using 2 methylbutan e and liquid nitrogen. 40 m sections were isolated with a cryostat machine. Graphs show qPCR results for A) IFN B) IL 17A, C) ROR t, and D) IL 6 expression. Results are relative to CD3. A total of 6 mice per category were examined. *=p <0.05


76 Figure 3 1 2 NOD lymph nodes display reduced levels of APCs Cells isolated from Axillary, Mesentery, and Cervical Lymp h Nodes were examined ex vivo using flow cytometry A) T cells were sta ined for CD8 and CD4 markers. B) APCs stained for CD3, B220, CD11b, and CD11c. Absolute cell number counts for CD11b+, CD11c+, and B220+ populations were obtained by gating first for th e CD3 population. **=p< 0.01. Data is averaged for 3 mice per set.


77 Figure 3 1 3 LjN6.2 increases CD11b+ and CD11c+ expression Splenocytes were harvested from NOD mice and plated at a concentration of 4 x 10 5 cells/well with 4 g/mL antiCD3 and a dos e of bacteria where indicated (CFU/mL). Following incubation at 37C for 48 hours, samples were analyzed through flow cytometry. Graphs show changes in CD11b or CD11c expression. Numbers represent percentage of the indicated cell population. Data is repres entative of 3 mice per set.


78 Figure 3 1 4 LjN6.2 enhances CD11b and CD11c expression of MHC I and II markers Splenocytes were harvested from NOD mice and plated at a concentration of 4 x 10 5 cells/well with 4 g/mL antiCD3 and a dose of bacteria wher e indicated (CFU/mL). Following incubation at 37C for 48 hours, samples were analyzed through flow cytometry. Graphs showing percentages of cells stained for CD11b, CD11c, MHC I, and MHC II for flow cytometry. Data is averaged on 3 mice per set. The p val ues for these figures are not statistically significant with the current sample size.


79 Figure 3 1 5 CD11c+ cells reduce expression of DEC205 in the presence of LjN6.2 Splenocytes were harvested from NOD mice and plated at a concentration of 4 x 10 5 ce lls/well with 4 g/mL antiCD3 and a dose of bacteria where indicated (CFU/mL). Following incubation at 37C for 48 hours, samples were analyzed through flow cytometry. A) Graphs showing DEC 205 expression by CD11c+ cells. Numbers represent percentage of CD 11c+ cells expressing DEC 205. B) Chart displaying percentage of CD11c+ cells positive for DEC 205.


80 Figure 3 1 6 LjN6.2 influences APCs via a membrane bound antigen. 4 x 10 5 NOD BMDC were plated per well and treated with whole 2.5 x 10 4 CFU/mL LjN6.2 or the fractions of an equivalent amount of cells Membrane and Cytoplasm fractions were created by lysing cells with glass beads a nd a Bead Beater machine The sample was then centrifuged for 15 min at 100,000 x g. The pellet was considered the membrane f raction, while the supernatant was the cytoplasm fraction. Supernatants were removed at 48 hours and A) analyzed for IL 6 production through ELISA. B) Serial dilutions were done and plated on MRS media and incubated anaerobically. P lates shown are for a 10 6 dilution to determine CFU/mL for whole (left) versus membrane fractions (right) of LjN6.2. 3 mice were used in this experiment


81 Figure 3 1 7 A proposed mechanism for the action of LjN6.2 in a diabetes model. In the NOD model, APCs are defective with respects to maturation and proper antigen presentation. The immature state of DCs may lead to a default TH1 bias. However, LjN6.2 may be capable of inducing maturation of DCs, altering the cytokine milieu, and preventing the onset of a TH1 phenotype and T 1D.


82 CHAPTER 4 DISCUSSION While genetic factors in T1D are being explored, the role of environmental factors has trailed behind. More and more, factors like diet and habitat are being discussed as viable options influencing the onset of T1D. Recently, the use of probiotics has yielded interesting results in animal models, preventing disease onset in both rats and mice. And, while the promise of therapeutic bacteria is encouraging, the mechanism behind its effects is poorly understood. Animal models have al lowed for detailed analysis for the differences between diabetes prone and resistant rodents. Roesch et al. demonstrated that different types of bacteria resided in T1D prone and resistant animals. The stool of the latter contained elevated levels of Lacto bacillus and Bacteroides, two genera typically made up of bacteria associated with providing beneficial health effects for its hosts. Furthermore, Valladares et al. demonstrated that feeding Lactobacillus johnsonii N6.2 from diabetes resistant rats to diab etes prone (DP) rats could delay onset of the disease. This trend was also common to NOD mice, as Lactobacillus casei Shirota strain feedings also prevented onset of the disease (Matsuzaki, 1997). In order to understand how these probiotics treatments medi ated its effects in T1D models, we conducted extensive research into the direct effects of LjN6.2 on immune cells, both in vitro and in vivo Our lab has showed that the DP rats treated with LjN6.2 that did not develop T1D displayed elevated TH17 factors locally in the mesenteric lymph nodes and in more distant sites like the spleen (Lau, 2011) Previous work has shown that gut bacteria can be potent inducers of a TH17 phenotype originating in the gut (Atarashi, 2010). And, as other work has suggested a po tentially beneficial role for TH17 cells in T1D, we sought


83 to investigate how LjN6.2 mediates this TH17 phenotype. Initial in vitro work required we determine the proper concentration of LjN6.2 that elicited an immune response but did not induce apoptosis in our mouse splenocytes. dogma typically states that T cells must receive proper activation through its TCR in order to properly differentiate (Nakayama, 2010) In accord ance with this, we demonstrated LjN6.2 was capable of causing an IL 6 response in the absence of TCR stimulation (typically associated with APCs), but not IL 17. With that knowledge, antiCD3 was used to stimulate our splenocytes and we observed that LjN6.2 was particularly adept at inducing IL 6 and IL 17 production in comparison with antiCD3 alone controls or LrTD1 treated splenocytes. Furthermore, we observed a 33% reduction in IL 17 production when we used an antibody cocktail against IL 6, confirming th e contributions of this TH17 inducing cytokine. While we would have predicted a larger decrease in IL 17 production with this cocktail treatment, it is possible that other cytokines may serve in a redundant capacity to provide for TH17 differentiation, as detailed above. Future experiments could use additional antibodies against TH17 related cytokines, like IL 23 and TGF the LjN6.2 mediated bias. We also demonstrated that APCs are the main providers of IL 6 for TH17 differ entiation in our LjN6.2 in vitro assays, as T cells alone generate minimal levels of IL 6. In turn, we have shown that APCs are required in the presence of TCR stimulation and LjN6.2 to promote TH17 development. Given the importance of APCs in our assay an d the antigen sampling role of DCs in the gut, we showed that DCs alone are capable


84 of creating a TH17 bias. Because several papers have reported that DC transfers are capable of preventing T1D onset (Feili Hariri, 1999; Clare Salzler, 1992 ), we tested the ability of our LjN6.2 pulsed DCs to mediate an immune response in recipient NOD mice. Interestingly, we have demonstrated that NOD mice receiving LjN6.2 pulsed DCs displayed an elevated TH17 response. This response was seen longterm, even in mice sacrific ed 11 weeks later. This effect is particularly important, as several papers stated that their attempt to transfer TH17 cells into NOD.SCID mice resulted in their near immediate conversion to TH1 cells. A longterm TH17 bias may prevent conversion into diabe togenic TH1 cells. Although we were able to observe this effect, we were unable to create a change in the onset of T1D between our sample sets. It is likely that our DC transfers occurred too late in the diabetes timeline. Reports indicate that typically t here cell apoptosis in the pancreas in NOD mice, first at 2 3 weeks of age, and again at 9 10 weeks of age (Turley, 2003). Labs that have reported successful BMDC treatments that delayed or stopped onset of T1D were performed in the time window between apoptosis events. Even though our treatment mediated a changed immune response, it occurred toward the end of the time window, and may have not been sufficient to reverse the compounding autoimmune damage. The knowledge of this treatment is very important, as it may only be effective in the initiation stages of this disease and not during the effector phase. These experiments will likely be conducted again, using earlier time points in BMDC treatments. In addition to the data listed above, w e demonstrated a role for TH17s in the autoimmune setting of the NOD mouse. Compared to NOR mice, prediabetic NOD mice display lower levels of TH17 related factors within the mesenteric lymph node and


85 directly in the pancreas. Additionally, NOD lymph nodes lack the ability to create a TH17 bias upon TCR stimulation. As this data came mostly from 11 week old mice, it would be interesting to monitor TH17 levels over the course of the disease. Preliminary data shows that NOD mice have a peak in lymph node IL 1 7 production at 8 weeks of age and subsequently drops off 4 weeks later. The use of anti IL 17 antibodies could elaborate on the effects of TH17 cells during the progression of T1D. In order to understand how LjN6.2 and its TH17 promoting effects would fu nction in an autoimmune setting, we investigated the effects LjN6.2 had on NOD APCs. Here, we showed that LjN6.2 treatment could increase APC numbers, which is particularly relevant because NOD lymph nodes hold lower percentages of APCs overall. Furthermor e, LjN6.2 induced upregulation of both MHC class markers and costimulatory molecules, suggesting the cells undergo maturation. This maturation is important because NOD APCs have several difficulties associated with proper maturation. Proper maturation of A PCs was listed as one of the main hypothesis as to how BMDC transfers prevented T1D onset in NOD.SCID models. Mature APCs allows for antigen induced cell death in improperly activated T cells, as a stronger signal, provided by costimulatory molecules, is r equired to cause cell death instead of activation. Along with AICD, cross antigen tolerance may also play a role in the restoration of APC function. We observed a decreased expression of surface DEC 205 on dendritic cells, a marker associated with endocyto sis and the process of cross antigen presentation. Cross antigen presentation involves the uptake of exogenous antigen and its presentation on MHC Class I. This theory states that self antigen may be presented to CD8+ T cells and induce cross tolerance thr ough apoptosis. The decrease in DEC 205 may seem contradictory at first,


86 but it is likely that DEC 205 is internalized during endocytosis. It would be interesting to observe intracellular staining through flow cytometry for DEC 205 or use immunofluorescenc e to monitor internally trafficking DEC 205. Finally, we have determined that LjN6.2 is likely stimulating APCs through a surface expressed antigen. This was determined by lysing LjN6.2 cells and separating membrane and cytoplasm fractions via ultra centr ifugation. Cytoplasmic fractions were unable to stimulate IL 6 production from BMDCs, while membrane fractions were capable of inducing robust IL 6. We have also shown that membrane stimulation of BMDCs was not due to a high concentration of LjN6.2 survivi ng the lysing process. Ardissone and Triplett et al. are currently examining differences in gene expression between LjN6.2 and LrTD1 and initial results are promising, demonstrating that LjN6.2 expresses higher levels of pili genes. otion of TH17 cells over TH1 cells may seem like exchanging one type of inflammation for another, other immune pathways must be considered. TH17 cells may recruit inflammatory cells during the effector phase of the disease, but TH1 cells have been indicate d as the initiators of T1D. Manirarora et al. (2011) demonstrated that certain Lactobacillus strains promote IL 10 production from DCs instead of IL 12. This was correlated with a decreased incidence of disease onset and again, points to the causative role of TH1s in T1D. As previously stated, CFA treatment of NOD mice prevented onset of T1D. This observation correlated with an increase in TH17 factors (Nikoopour, 2010). Manirarora et al. (2008) also stated that CFA treatment of APCs increased their ability to promote Regulatory T cells. They believe this was a function of increasing expression of B7 1, or CD80. Interestingly, we


87 have also shown that LjN6.2 treatment of APCs induced CD80 upregulation (data not shown). Fig. 3 17 demonstrates a preliminary mod el of what we believe could be occurring with LjN6.2 and APCs. Without any treatment, NOD APCs suffer from several of the APC defects listed above, including poor maturation. Due to these problems, NOD APCs naturally default to promoting a TH1 phenotype. W ith LjN6.2 treatments, NOD APCs upregulate MHC and costimulatory molecules, while decreasing DEC 205, indicating a shift from antigen uptake to antigen presentation. This may also change the cytokine production by APCs, preventing T cells from shifting to dangerous TH1 cells. This model still requires further research to confirm the downregulation of TH1 related factors (IL 12, T bet) upon LjN6.2 treatment. In addition, we have yet to determine whether LjN6.2 induces an antigen specific response or whether the changes observed are broad and non specific. While we still have a few issues to address with this model, we believe this could be a strong representation of how LjN6.2 influences the host immune response. While Treg numbers have been shown not to decl ine during the course of disease in NOD mice, their function becomes impaired (Tritt, 2008). This loss of function was correlated with a lack of Foxp3, the Treg transcription factor. Manirarora demonstrated that CFA treatment in NOD mice increased Foxp3 ex pression in Tregs. Treg development in the gut remains an unexplored, yet important part of our LjN6.2 treatments. It is possible that proper presentation of LjN6.2 to DCs allows them to not only promote a TH17 environment, but enhance the function and num bers of Tregs. The Tregs could then tolerize the host to the gut microbiota, even if bacteria are promoting an autoimmune response.


88 Probiotics have encouraging potential as a therapeutic agent. Several publications state the benefits of oral probiotic trea tment. Probiotics also suggest that certain sets of gut microbiota may be correlated with a healthy or dysfunctional immune system. If a collection of microbiota can be associated with Type 1 Diabetes, this may lead to a screening process that analyzes ind ividuals at risk for the disease. Furthermore, probiotics may be prescribed to help the patient maintain a healthy gut environment. Probiotics are also relatively cheap (compared to some treatment options) and can be produced by the liters in short amounts of time. Of course, translational studies must be conducted to determine the efficacy of probiotics in T1D patients. And, even though most probiotic strains are Generally Regarded as Safe (GRAS), they should be studied for their longterm effects. It would also be helpful to understand the immune profile generated by the strains of bacteria to be used, as a panel of T Helper cell differentiation could determine which strains could be most effective. Our studies have focused on illuminating the mechanisms be hind probiotic effect and function in the autoimmune model of T1D. With each bacterial strain that becomes characterized immunologically, we gain more insight into the interplay between environment and host, providing groundwork for future clinical success


89 LIST OF REFERENCES Acha Orbea H., and McDevitt H.O. 1987. The first external domain of the nonobese diabetic mouse class II I A beta chain is unique. Proc. Natl. Acad. Sci USA 84 2435 39. Akerblom, H.K., and Reunanen, A. 1985. The epidemiology of ins ulin dependent diabetes mellitus (IDDM) in Finland and in northern Europe. Diabetes Care 8(Suppl 1), 10 16. Akerblom, H.K.,Virtanen, S.M., Hamalainen, A.M., Ilonen, J., Savilahti, E., Vaarala, O.,Reunanen, A., Teramo, K., and Knip, M. 1999. Emergence of di abetes associated autoantibodies in the nutritional prevention of IDDM (TRIGR) project. Diabetes 48(Suppl 1) A45. Anderson, M.S., and Bluestone, J.A. 2005. The NOD mouse: a model of immunedysregulation. Annu. Rev. Immunol. 23 447 485. Andoh, A., Zhang, Z., and Inatomi, O. et al. 2005. Interleukin 22, a member of the IL 1 0 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts. Gastroenterology 129 969. Arreaza, G., Salojin, K., Yang, W., Zhang, J., Gill, B., Mi, Q.S., Gao, J.X ., Meagher, C., Cameron, M., and Delovitch, T.L. 2003. Deficient activation and resistance to activation induced apoptosis of CD8+ T cells is associated with defective peripheral tolerance in nonobese diabetic mice. Clin. Immunol. 107 103 115. Asao, H., O kuyama, C., Kumaki, S., Ishii, N., Tsuchiya, S., Foster, D., and Sugamura, K. 2001. Cutting edge: the common gamma chain is an indispensable subunit of the IL 21 receptor complex. J. Immunol. 167 1 5. Ashton Rickardt, P.G., Bandeira, A., Delany, J.R., Kae r, L.V., Pircher, H B., Zinkernagel, R.M., and Tonegawa, S. 1994. Evidence for a differential avidity model of T cell selection in the thymus. Cell 76 651 663. Atarashi, K., Tanoue, T., and Honda, K. 2010. Induct ion of lamina propia TH17 cells by intestin al commensal bacteria. 28(50), 8036 8038. Atkinson, M.A. et al. 1990. Insulitis and diabetes in NOD mice reduced by prophylactic I nsulin therapy. Diabetes 39 933 937. Bach, J.F. 1994. Insulin dependent diabetes mellitus as an autoimmune disease. Endocr. R ev 15 516 542. Bach, J.F. 2001. Protective role of infections and vaccinations on autoimmune diseases. J. Autoimmun. 16 347 353.


90 Barnett, A.H., Eff, C., Leslie, R.D.G., and Pyke, D.A. 1981. Diabetes in identical twins. A study of 200 pairs. Diabetologia 20 87 93. Bending, D., H. De La Pena, M. Veldhoen, J. M. Phillips, C. Uyttenhove, B. Stockinger, and A. Cooke. 2009 Highly purified Th17 cells from BDC2.5NOD mice convert into Th1 like cells in NOD/SCID recipient mice. J. Clin. Invest. 119 565 572. Ben ding, D., Newland, S., Krejci, A., Phillips, J.M., Bray, S., and Cooke, A. 2011. Epigenetic changes at il12rb2 and tbx21 in relation to plasticity behavior of Th17 cells. J. Immunol. 186 3373 3382. Bettelli, E., Carrier, Y ., and Gao, W., et al. 2006. Reci procal developmental pathways for the generation of pathogenic effector Th17 and regulatory T cells. Nature 441(7090), 235 238. Boehm, B., Rosinger, S., Sauer, G., Manfras, B.J., Palesch, D., Schiekofer, S., Kalbacher, H., and Burster, T. 2009. Protease re sistant human GAD derived altered peptide ligands decrease TNF 17 production in peripheral blood cells from patients with type 1 diabetes mellitus. Mol. Immunol. 46 2576 2584. Borchers, A.T., Selmi, C., Meyers, F.J., Keen, C.L., and Gershwin, M.E 2008 Probiotics and Immunity. J. Gastroenterol. 44 26 46. Bour Jordan, H., Salomon, B.L., Thompson, H.L., Szot, G.L., Bernhard, M.R., and Bluestone, J.A. 2004. Costimulation controls diabetes by altering the balance of pathogenic and regulatory T cell s. J. Clin. Invest. 114 979 987. Bradshaw, E.M., Raddassi, K., Elyaman, W., Orban, T., Gottlieb, P.A., Kent, S.C., and Hafler, D.A. 2009 Monocytes from patients with type 1 diabetes spontaneously secrete proinflammatory cytokines inducing Th17 cells. J. Immunol. 183(7) 4432 4439. Brandt, K., Bulfone Paus, S., Jenckel, A., Foster, D.C., Paus, R. and Ruckert, R. 2003. Interleukin 21 inhibits dendritic cell mediated T cell activation and induction of contact hypersensitivity in vivo. J. Invest. Dermatol. 12 1 1379 1382. Brugman, S., Klatter, F.A., Visser, J.T., Wildeboer Veloo, A.C., Harmsen, H.J., Rozing, J., and Bos, N.A. 2006. Antibiotic treatment partially protects against type 1 diabetes prone rat. Is the gut flora involved in the development of type 1 diabetes? Diabetologia 49(9), 2105 2108. Brustle, A., Heink, S., Huber, M., Rosenplanter, C., Stadelmann, C., and Yu, P., et al. 2007. The development of inflammatory T(H) 17 cells requires interferon regulatory factor 4. Nat. Immunol. 8(9), 958 66. Bryces on, Y.T., Chiang, S.C., Darmanin, S., Fauriat, C., Schlums, H., Theorell, J., and Wood, S.M. 2011. Molecular mechanisms of natural killer cell activation. J. Innate Immun. 3(3) 216 226.


91 Calcinaro, F., Dionisi, S., Marinaro, M., Candeloro, P., Bonato, V., Marzotti, S., Cornell, R.B., Ferretti, E., Gulino, A., Grasso, F., De Simone, C., Di Mario, U., Falorni, A., Boirivant, M., Dotta, F. 2005. Oral probiotic administration induces interleukin 10 production and prevents spontaneous autoimmune diabetes in the non obese diabetic mouse. Diabetologia, 48(8), 1565 1575. Chatzigeorgiou, A., Harokopos, V., Mylona Karagianni, C., Tsouvalas, E., Aidinis, V., and Kamper, E.F. 2010 The pattern of inflammatory/anti inflammatory cytokines and chemokines in type 1 diabete s patients over time. Ann. Med. 42(6) 426 438. Chen, J., Reifsnyder, P.C., Scheuplein, F., Schott, W.H., Mileikovsky, M., Soodeen Karamath, S., Nagy, A., Dosch, M.H., Ellis, J., Koch Nolte, F., and Leiter, E.H. 2005. Agouti NOD: identification of a CBA de rived Idd locus on chromosome 7 and its use for chimera production with NOD embryonic stem cells. Mamm. Genome 16 775 783. Christianson, S.W., Shultz, L.D., and Leiter, E.H. 1993. Adoptive transfer of diabetes into immunodeficient NOD scid/scid mice: rela tive contributions of CD4+ and CD8+ T lymphocytes from diabetic versus prediabetic NOD.NON Thy 1 a donors. Diabetes 42 44 55. Clare Salzler, M.J., Brooks, J., Chai, A., Herle, K.V., and Anderson, C. 1992 Prevention of diabetes in nonobese diabetic mice by dendritic cell transfer. J. Clin. Invest. 90 741 7 48. Clark, E.A. and Draves, K.E. 1987. Activation of macaque T cells and B cells with agonistic monoclonal antibodies. Eur. J. Immunol. 17 1799 1805. Crome, S.Q., Wang, A.Y., and Levings, M.K. 2009. Tra nslational mini review on TH17 cells: function and regulation of human T helper 17 cells in health and disease. Clin. Exp. Immunol. 159 109 119. Cua, D.J., and Tato, C.M. 2010. Innate IL 17 producing cells: the sentinels of the immune system. Nat. Rev. 1 0 479 89. Dall, T. M., and Zhang, Y., et al. 2010. T he Economic Burden of Diabetes. Health Affairs 29 (2) 297 303. Decallonne, B., Van Etten, E., Giulietti, A., Casteels, K., Overbergh, L., Bouillon, R., and Mathieu, C. 2003. Defect in activation induced cell death in non obese diabetic (NOD) T lymphocytes. J. Autoimmun. 20 219 226. Diana, J., Gahzarian, L., Simoni, Y. and Lehuen, A. 2011. Innate immunity in type 1 diabetes. Discov. Med. 11(61) 513 520. Diehl, S. and Rincon, M. 2002. The two faces of IL 6 on TH1/TH2 differentiation. Mol. Immunol. 38 531 536.


92 Delovitch, T.L., and Singh, B. 1997. The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD Immunity 7 291 297. Dobber, R., Hertogh Huijbregts, A., Rozing J., Bottomly, K., and Nagelkerken, L. 1992. The involvement of the intestinal microflora in the expansion of CD4+ T cells with a naive phenotype in the periphery. Dev. Immunol. 2 141 50. Driver, J.P., Serreze, D.V., and Chen, Y.G. 2011. Mouse models for the study of autoimmune type 1 diabetes: a NOD to similarities and differences to human disease. Semin. Immunopathol. 33 67 87. Durant, L., Watford, W.T., Ramos, H.L., Laurence, A., Vahedi, G., and Wei, L., et al. 2010. Diverse targets of the transcripti on factor STAT3 contribute to T cell pathogenicity and homeostasis. Immunity 32(5), 605 615. Elyaman, W., Bradshaw E.M., Uyttenhove, C., Dardalhon, V., Awashti, A., Imitola, J., Oukka, M., van Snick, J., Renauld, J.C., K uchroo, V.K., and Khoury, S.J. 2009 IL 9 induces differentiation of TH17 cells and enhances function of FoxP3+ natural regulatory T cells. Proc. Natl. Acad. Sci. USA 106(31) 12885 12890. Emamaullee, J.A., Davis, J., Merani, S., Toso, C., Elliott, J.F., Thiesen, A., and Shapiro, A.M. 2009 Inhibition of Th17 cells regulates autoimmune diabetes in NOD mice. Diabetes 58(6), 1302 1311. Estella, E., McKenzie, M.D., Catterall, T., Sutton, V.R., Bird, P.I., Trapani, J.A., Kay, T.W., and Thomas, H.E. 2006. Granzyme B mediated death of pancreatic b eta cells requires the proapoptotic BH3 only molecule bid. Diabetes 55(8), 2212 2219. Esteban, L.M., Tsoutsman, T., Jordan, M.A., Roach, D., Poulton, L.D., Brooks, A., Naidenko, O.V., Sidobre, S., Godfrey, D.I., Baxter, A.G. 2003. Genetic control of NKT ce ll numbers maps to major diabetes and lupus loci. J. Immunol. 171, 2873 2878. EU RODIAB Substudy 2 Study Group. 1999. Vitamin D supplement in early childhood and risk of Type I (insulin dependent) diabetes mellitus. Diabetologia 42 51 54. EURODIAB Substudy 2 Study Group. 2000 Infections and vaccinations as risk factors for childhood type I (insulin dependent) diabetes mellitus: a multicentre case control investigation. Diabetologia 43 47 53. Falcone, M., and Sarvetnick, N. 1999. Cytokines that regulate au toimmune responses. Curr. Opin. Immunol. 11 670 676. Faveeuw, C., Gagnerault, M.C., and Lepault, F. 1995. Isolation of leukocytes infiltrating the islets of langerhans of diabetes prone mice for flow cytometric analysis. J. Immunol. Methods 187 163 169.


93 Feili Hariri, M ., Dong, X ., Alber, S.M ., Watkins, S.C ., Salter, R.D ., and Morel, P.A 1999 Immunotherapy of NOD mice with bone marrow derived dendritic cells. Diabetes 48(12) 2300 2308. Feng, X., Kajigaya, S., and Solomou, E.E., et al. 2008. Rabbit ATG but not horse ATG promotes expansion of functional CD41 CD25 high FOXP3+ regulatory T cells in vitro. Blood 111(7) 3675 3683. Fink, L.N., Zeuthen, L.H., Ferlazzo, G., and Frkir, H. 2007. Human antigen presenting cells respond differently to gut derived probiotic bacteria but mediate similar strain depe ndent NK and T cell activation. FEMS Immunol. Med. Microbiol. 51 535 546. Fiorina, P., Vergani, A., Dada, S., Jurewicz, M., Wong, M., Law, K., Wu, E., Tian, Z., Abdi, R., Guleria, I., Rodig, S., Dunussi Joannopoulo s, K., Bluestone, J., and Sayegh, M.H. 2008 Targeting CD22 reprograms B cells and reverses autoimmune diabetes. Diabetes 57 3013 3024. Fiorina, P., Jurewic z, M., and Augello, A., et al. 2009. Immunomodulatory function of bone marrow derived mesenchymal s tem cells in experimental autoimmune type 1 diabetes. J. Immunol. 183(2), 993 1004. Foulis, A.K., Farquharson, M.A., Cameron, S.O., McGill, M., Schnke, H., and Kandolff, R. 1990. A search for the presence of the enteroviral capsid protein VP1 in pancrease s of patients with type 1 (insulin dependent) diabetes and pancreases and hearts of infants who died of coxsackie viral myocarditis. Diabetologia 33 290 298. Fuchtenbusch, M., Irnstetter, A., Jager, G., and Ziegler, A.G. 2001 No evidence for an associati on of coxsackie virus infections during pregnancy and early childhood with development of islet autoantibodies in offspring of mothers or fathers with Type 1 diabetes. J.Autoimmun 17, 333 340. Gagnerault, M. C., Luan, J. J., and Lotton, C. et al. 2002. Pa ncreatic lymph nodes are required for priming of cell reactive T cells in NOD mice. J. Exp. Med. 196 369 377. Gale, E.A. 2002. The rise of childhood type 1 diabetes in the 20 th century. Diabetes 51 3353 3361. Gao, X., Ding, G., Wang, Z., Fu, H., Ni, Z. Ma, J., Song, S., Liu, F., and Fu, Z. 2010. Adjuvant treatment suppresses IL 17 production by T cell independent myeloid sources in nonobese diabetic mice. Mol. Immunol. 47(14), 2397 2404. Gaskins, H.R., Prochaza, M., Hamaguchi, K., S erreze, D.V., and Le iter, E.H. 1992. Beta cell expression of endogenous xenotropic retrovirus distinguishes diabetes susceptible NOD/Lt from resistant NON/Lt mice. J. Clin. Invest. 90, 2220 2227.


94 Geboes, L., Dumoutier, L., and Kelchtermans, H. et al. 2009. Proinflammatory rol e of the Th17 cytokine interleukin 22 in collagen induced arthritis in C57BL/6 mice. Arthritis Rheum. 60, 390. Gerstein, H.C. overview of the clinical literature. Diabetes Care 17 13 19. G horeschi, K., Laurence, A., Yang, X.P., Tato, C.M., McGeachy, M.J., Konkel, J.E., Ramos, H.L., Wei, L., Davidson, T.S., Bouladoux, N., Grainger, J.R., Chen, Q., Kanno, Y., Watford, W.T., Sun, H.W., Eberl, G., Shevach, E.M., Belkaid, Y., Cua, D.J., Chen, W. Generation of pathogenic T(H)17 cells in the absence of TGF signaling Nature 467(7318), 967 971. Ginsberg Fellner, F., Witt, M.E., Yagihashi, S., Dobersen, M.J., Taub, F., Fedun, B., McEvoy, R.C., Roman, S.H., Davies, T.F., Coo per, L.Z., Rubinstein, P., and Notkins, A.L. 1984. Congenital rubella syndrome as a model for Type 1 (insulin dependent) diabetes mellitus: increased prevalence of islet cell surface antibodies. Diabetologia 27, 87 89. Glocker, E. O., Kotlarz, D., and Bozt ug, K. et al. 2009. Inflammatory bowel disease and mutations affecting the interleukin 10 receptor. N. Engl. J. Med. 361, 2033. Gottlieb, P.A., Quinlan, S., and Krause Steinrauf, H., et al. 2010. Failure to preserve beta cell function with mycophenolate mo fetil and daclizumab combined therapy in patients with new onset type 1 diabetes. Diabetes Care 33(4), 826 832. Graves, P.M., Rotbart, H.A., Nix, W.A., Pallansch, M.A., Erlich, H.A., Norris, J.M., Hoffman, M., Eisenbarth, G.S., and Rewers, M. 2003. Prospec tive study of enteroviral infections and development of beta cell autoimmunity. Diabetes autoimmunity study in the young (DAISY). Diabetes Res. Clin. Pract. 59 51 61. Gregori, S., Giarratana, N., Smiroldo, S., and Adorini, L. 2003 Dynamics of pathogenic and suppressor T cells in autoimmune diabetes development. J. Immunol. 171 4040 4047. Guay, H.M., Larkin III, J., Picca, C.C., Panarey, L., and Caton, A.J. 2007. Spontaneous autoreactive memory B cell formation driven by a high frequency of autoreactive C D4+ T cells. J. Immunol. 178, 4793 4802. Hamilton Williams, E.E., Serreze, D.V., Charlton, B., Johnson, E.A., Marron, M.P., Mullbacher, A., and microglobulin as a diabetes susceptibility gene in NOD mice. Proc. Natl. Acad. Sci. USA 98 11533 11538.


95 Hamilton Williams, E.E., Martinez, X., Clark, J., Howlett, S., Hunter, K.M., Rainbow, D.B., Wen, L., Shlomchik, M.J., Katz, J.D., Beilhack, G.F., Wicker, L.S., and Sherman, L.A. 2009. Expression of diabetes associated genes by dendritic cells and CD4 T cells drives the loss of tolerance in nonobese diabetic mice. J. Immunol. 183, 1533 1541. Hanninen, A., Salmi, M., Simell, O., and Jalkanen, S. 1996. Mucosa associated (beta7 integrin high ) lymphocytes accumulate early in the pancreas of NOD mice and show aberrant recirculation behavior. Diabetes 45, 1173 1180. Harrison, L. INIT II study web site. 2010. Retrieved August 28, 2011. < http://www.diabetestrials.org/initii.html> Hart, A.L., Lammers, K., Brigidi, P., Vitali, B., Rizzello, F., and Gionchetti, P., et al. 2004. Modulation of human dendritic cell phenotype and function by probiotic bacteria. Gut 53 1602 1609. Herber, D., Brown, T.P., Liang, S., Young, D.A., Collins, M., and Dunussi Joannopoulos, K. 2007. IL 21 has a pathogenic role in a lupus prone mouse model and its blockade with IL 21R.Fc reduces disease progression. J. Immunol. 178, 3822 3830. Hinrichs, C.S., Spolski, R., Paulos, C.M., Gattinoni, L., Kerstann, K.W., Palmer, D.C., Klebanoff, C.A., Rosenberg, S.A., Leonard, W.J., and Restifo, N.P. 2008 IL 2 and IL 21 confer opposing differentiation programs to CD8+ T cells for adoptive immunother apy. Blood 111 5326 5333. Hoglund, P., Mintern, J., and Waltzinger, C. et al. 1999. Initiation of autoimmune diabetes by developmentally regulated presentation of islet cell antigens in the pancreatic lymph nodes. J. Exp. Med. 189 331 339. Hong, S., Wils on, M.T., Serizawa, I., Wu, L., Singh, N., Naidenko, O.V., Miura, T., Haba, T., Scherer, D.C., Wei, J., Kronenberg, M., Koezuka, Y., and Van Kaer, L. 2001. galactosylceramide prevents autoimmune diabetes in non obese diab etic mice. Nat. Med. 7 1052 1056. Honkanen, J., Nieminen, J.K., Gao, R., Luopajarvi, K., Solo, H.M., Ilonen, J., Knip, M., Otonkoski, T., and Vaarala, O. 2010. IL 17 immunity in human type 1 diabetes. J. Immunol. 185, 1959 1967. Hooper, L.V., and Gordon, J.I. 2001. Commensal host bacterial relationships in the gut. Science 292, 1115 1118. Hu, C.Y., Rodriguez Pinto, D., Du, W., Ahuja, A., Henegariu, O., Wong, F.S., Shlomchik, M.J., and Wen, L. 2007. Treatment with CD20 specific antibody prevents and reverse s autoimmune diabetes in mice. J. Clin. Invest. 117 3857 3867.


96 Inaba, K., Swiggard, W.J., Inaba, M., Meltzer, J., Mirza, A., Sasagawa, T., Nussenzweig, M.C., and Steinman, R.M. 1995. Tissue distribution of the DEC 205 protein that is detected by the mon oclonal antibody NLDC 145. I. Expression on dendritic cells and other subsets of mouse leukocytes. Cell Immunol. 163(1), 148 56. Ivanov, I.I., McKenzie, B.S., Zhou, L., Tadokoro, C.E., Lepelley, A., and Lafaille, J.J., et al. 2006. The orphan nuclear recep tor RORgammat directs the differentiation program of proinflammatory IL 17 + T helper cells. Cell 126(6), 1121 33. Jarpe, A.J., Winter, W.E., and Peck, A.B. 1990.The changing profile of mononuclear cell populations infiltrating the islets of Langerhans dur ing the progressive insulitis phase in prediabetic NOD mice. Allergy Immunol 9, 283 292. Jijon, H., Backer, J., Diaz, H., Yeung, H., Thiel, D., and McKaigney, C., et al. 2004. DNA from probiotic bacteria modulates murine and human epithelial and immune fu nction. Gastroenterology 126, 1358 1373. Juvenile Diabetes Research Foundation International. Type 1 Diabetes Facts. Dec. 2010. Retrieved 10 July 2010. < http://www.jdrf.org/index.cfm?page_id=102585 > Ki, S. H., Park, O. and Zheng, M. et al. 2010. Interleukin 22 treatment ameliorates alcoholic liver injury in a murine model of chronic binge ethanol feeding: role of signal transducer and activator of transcriptio n 3. Hepatology 52, 1291. Kikutani, H., and Makino, S. 1992. The murine autoimmune diabetes model: NOD and related strains. Adv. Immunol 51, 285 322. King, C., Ilic, A., K oelsch, K., and Sarvetnick, N. 2004. Homeostatic expansion of T cells during immune insufficiency generates autoimmunity. Cell 117 265 277. Knip, M., Veijola, R., Virtanen, S.M., Hyoty, H., Vaarala, O., and Akerblom, H.K. 2005. Environmental triggers and determinants of type 1 diabetes. Diabetes 54(Suppl 2) S125 136. Kom, T., et al. 200 7. IL 21 initiates an alternative pathway to induce proinflammatory TH17 cells. Nature 448, 484 487. Koopman, G., Reutelingsperger, C.P., Kuijten, G.A., Keehnen, R.M., Pals, S.T., and van Oers, M.H. 1994. Annexin V for flow cytometric detection of phosphat idylserine expression on B cells undergoing apoptosis. Blood 84(5), 1415 1420. Kumar, D., Gemayel, N., Deapen, D., Kapadia, D., Yamashita, P., Lee, M., Dwyer, J., Roy Burman, P., Bray, G., and Mack, T. 1993. North American twins with IDDM. Genetic, etiolog ical, and clinical significance of disease concordance according to age, zygosity, and the interval after diagnosis in first twin. Diabetes 42 1351 1363.


97 Kupila, A,. Sipila, J., an d Keskinen, P., et al. 2003. Intranasally administered insulin intended for prevention of type 1 diabetes a safety study in healthy adults. Diabetes Metab. Res. Rev. 19(5), 415 20. Larkin III, J., Picca, C.C., and Caton, A.J. 2007. Activation of CD4+CD25+ regulatory T cell suppressor function by analogs of the selecting peptide. Eur. J. Immunol. 37 139 146. Lau, K., Benitez, P., Ardissone, A., Wilson, T.D., Collins, E.L., Lorca, G., Li, N., Sankar, D., Wasserfall, C., Neu, J., Atkinson, M.A., and Larkin, J. 3 rd 2011. Inhibition of type 1 diabetes correlated to a Lactobacillus jo hnsonii N6.2 mediated Th17 bias. J. Immunol. 186(6), 3538 3546. Lee, Y.K., Turner, H., Maynard, C.L., Oliver, J.R., Chen, D., Elson, C.O., and Weaver, C.T. 2009. Late developmental plasticity in the T helper 17 lineage. Immunity 30 92 107. Lejeune, D., Du moutier, L., Constantinescu, S., Kruijer, W., Schuringa, J. J. and Renauld, J. C. 2002. Interleukin 22 (IL 22) activates the JAK/STAT, ERK, JNK, and p38 MAP kinase pathways in a rat hepatoma cell line. Pathways that are shared with and distinct from IL 10. J. Biol. Chem. 277 33676. Leo, O., Foo, M., Sachs, D.H., Samelson, L.E., and Bluestone, J.A. 1987. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc. Natl. Acad. Sci. USA 84, 1374 1378. Lerman, M.A., Larkin III, J., Cozz o, C., Jordan, M.S., and Caton, A.J. 2004. Activation of CD4+CD25+ regulatory T cell repertoire formation in response to varying expression of a neo self antigen. J. Immunol. 173 236 244. Leser, T.D. and Molbak, L. 2009. Better living through microbial ac tion: the benefits of the mammalian gastrointestinal microbiota on the host. Environ. Microbiol. 11 2194 2206. Lexberg, M.H., Taubner, A., Forster, A., Albrecht, I., Richter, A., Kamradt, T., Radbruch, A., and Chang, H.D. 2008. Th memory for interleukin 1 7 expression is stable in vivo. Eur. J. Immunol. 38, 2654 2664. Liang, S.C., Long, A.J., Bennett, F., Whitters, M.J., Karim, R., Collins, M., Goldman, S.J., Dunussi Joannopoulos, K., Williams, C.M., Wright, J.F., and Fouser, L.A. 17F/A Heterod imer Protein Is Produced by Mouse Th17 Cells and 179, 7791 7799. Lien, E., and Zipris, D. 2009. The role of toll like receptor pathways in the mechanism of type 1 diabetes. Current Molecular Medicine 9, 5 2 68.


98 Like, A.A., Guberski, D.L., and Butler, L. 1991. Influence of environmental viral agents on frequency and tempo of diabetes mellitus in BB/Wor rats. Diabetes 40, 259 262. Litherland, S.A., Grebe, K.M., Belkin, N.S., Paek, E., Elf, J., Atkinson, M., Morel, L., Clare Salzler, M.J., and McDuffie, M. 2005. Nonobese diabetic mouse congenic analysis reveals chromosome 11 locus contributing to diabetes susceptibility, macrophage STAT5 dysfunction, and granulocyte macrophage colony stimulating factor overpro duction. J. Immunol. 175, 4561 4565. Lonnrot, M., Korpela, K., Knip, M., Ilonen, J., Simell, O., Korhonen, S., Savola, K., Muona, P., Simell, T., Koskela, P., and Hyoty, H. 2000. Enterovirus infection as a risk factor for beta cell autoimmunity in a prospe ctively observed birth cohort: the Finnish Diabetes Prediction and Prevention Study. Diabetes 49, 1314 1318. Lund, B.T., Ashikian, N., Ta, H.Q., Chakryan, Y., Manoukian, K., Groshen, S., Gilmore, W., Cheema, G.S., Stohl, W., Burnett, M.E., Ko, D., Kachuck, N.J., and Weiner, L.P. 2004. Increased CXCL8 (IL 8) expression in Multiple Sclerosis. J. Neuroimmunol. 155(1 2), 161 171. Lund T., O'Reilly L., Hutchings P., Kanagawa O., Simpson E., Gravely R., Chandler P., Dyson J., Picard J.K., Edwards A., Kioussis D. and Cooke A. 1990. Prevention of insulin dependent diabetes mellitus in non obese diabetic mice by transgenes encoding modified I chain or normal I chain. Nature 345, 727 729. Lyons, P.A., Armitage, N., Argentina, F., Denny, P., Hill, N.J., Lord, C.J., Wilusz, M.B., Peterson, L.B., Wicker, L.S., and Todd, J.A. 2000. Congenic mapping of the type 1 diabetes locus, ldd3, to a 780 kb region of mouse chromosome 3: identification of a candidate segment of ancestral DNA by haplotype mapping. Genome Res. 10 446 453. Madec, A.M., Mallone, R., and Afonso, G., et al. 2009. Mesenchymal stem cells protect NOD mice from diabetes by inducing regulatory T cells. Diabetologia 52(7), 1391 1399. Madsen, K., Cornish, A., Soper, P., MacKainey, C., Jijon, H., and Yachi mec, C., et al. 2001. Probiotic bacteria enhance murine and human intestinal epithelial barrier function. Gastroenterology 121 580 591. Makino, S., Kunimoto, K., Muraoka, Y., Mizushima and Y., Katagiri, K., et al. 1980. Breeding of a non obese, diabetic strain of mice. Jikken Dobutsu 29 1 13. factor beta induces development of the T(H)17 lineage. Nature 441(7090), 231 234.


99 Manirarora, J.N., Kosiewicz, M.M. Parnell, S.A., and Alard, P. 2 008. APC Activation restores functional CD4+CD25+ regulatory T cells in NOD mice that can prevent diabetes development. PLoS One 3(11), e3739. Manirarora, J.N., Parnell, S.A., Hu, Y.H., Kosiewic z, M.M., and Alard, P. 2011. NOD dendritic cel ls stimulated with Lactobacilli preferentially produce IL 10 versus IL 12 and decrease diabetes incidence. Clin. Dev. Immunol 2011 e630187. Martin Orozco, N., Y. Chung, S. H. Chang, Y. H. Wang, and C. Dong. 2009. Th17 cells promote pancreatic inflammatio n but only induce diabetes efficiently in lymphopenic hosts after conversion into Th1 cells. Eur. J. Immunol. 39 216 224. Mathews, C. 2005. Utility of murine models for the study of spontaneous autoimmune type 1 diabetes. Pediatric Diabetes. 6, 165 177. M atsuda, J., Mallevaey, T., Scott Browne, J., and Gapin, L. 2008. CD1d restricted iNKT 358 368. Matsuzaki, T., Nagata, Y., Kado, S., Uchida, K., Kato, I., Hashimoto, S., and Yok okura, T. 1997. Prevention of onset in an insulin dependent diabetes mellitus model, NOD mice, by oral feeding of Lactobacillus casei. APMIS 105(8), 643 649. Mayo Clinic. Type 1 Diabetes Symptoms. May 2011. Retrieved 26 August 2011. < http://www.mayoclinic.com/health/type 1 diabetes/DS00329/ DSECTION=symptoms > Mazmanian, S.K., Liu, C.H., Tzianabos, A.O., and Kasper, D.L. 2005. An immunomodulatory molecule o f symbiotic bacteria directs maturation of the host immune system. Cell 122, 107 118. McAleer, J.P. and Vella, A .T. 2010 Educating CD4 T cells with vaccine adjuvants: lessons from lipopolysaccharide. Trends Immunol. 31(11), 429 435. McGuire, H.M., Vogelza ng, A., Hill, N., Flodstrom Tullberg, M., Sprent, J., and King, C. 2009. Loss of parity between IL 2 and IL 21 in the NOD Idd3 locus. Proc. Natl. Acad. Sci. USA 106, 19438 19443. Mellanby, R.J., Thomas, D., Phillips, J.M., and Cooke, A. 2007. Diabetes in n on obese diabetic mice is not associated with quantitative changes in CD4+ CD25+ Foxp3+ regulatory T cells. Immunology 121, 15 28. Mehta, D.S., Wurster, A.L., Whitters, M.J., Young, D.A., Collins, M., and Grusby, M.J. 2003. IL 21 induces the apoptosis of r esting and activated primary B cells, J. Immunol. 170, 4111 4118.

PAGE 100

100 Miyazaki, A., Hanafusa, T., and Yamada, K. et al. 1985. Predominance of T lymphocytes in pancreatic islets and spleen of pre diabetic non obese diabetic (NOD) mice: a longitudinal study Cli n. Exp. Immunol. 60 622 630. Miyazaki T., Uno, M., Uehira, M., Kikutani, H., Kishimoto, T., Kimoto, M., Nishimoto, H., Miyazaki, J., and Yamamura, K. 1990. Direct evidence for the contribution of the unique I A nod to the development of insulitis in non ob ese diabetic mice. Nature 345, 722 724. Monteleone, G., Caruso, R., Fina, D., Peluso, I., Gioia, V., Stolfi, C., Fantini, M.C., Caprioli, F., Tersigni, R., Alessandroni, L., MacDonald, T.T., and Pallone, F. 2006 Control of matrix metalloproteinase producti on in human intestinal fibroblasts by interleukin 21. Gut 55 1774 1780. 23 mediates Toxoplasma gondii induced immunopathology in the gut via matrixmetallo proteinase 2 and IL 22 but independent of IL 17. J. Exp. Med. 206 3047. Nakayama, T., and Yamashita, M. 2010. The TCR mediated signaling pathways that control the direction of helper T cell differentiation. Semin. Immunol. 22(5), 303 309. Nikolic, B., Takeuchi, Y., Leykin, I., Fud aba, Y., Smith, R.N., and Sykes, M. 2004. Mixed hematopoietic chimerism allows cure of autoimmune diabetes through allogeneic tolerance and reversal of autoimmunity. Diabetes 53, 376 383. Nikoopour, E., Schwart, J.A., Huszarik, K., Sandrock, C., Kroughly, O., Lee Chan, E., and Singh, B. 2010. Th17 polarized cells from nonobese diabetic mice following mycobacterial adjuvant immunotherapy delay type 1 diabetes. J. Immunol. 184(9), 4779 4788. Noorchashm, H., Noorchashm, N., Kern, J., Rostami, S.Y., Barker, C.F ., and Naji, A. 1997. B cells are required for the initiation of insulitis and sialitis in nonobese diabetic mice. Diabetes 46, 941 946. Norris, J.M., Beaty, B., Klingensmith, G., Yu, L., Hoffman, M., Chase, H.P., Erlich, H.A., Hamman, R.F., Ei senbarth, G. S., and Rewers, M. 1996 Lack of association cell autoimmunity. Diabetes Autoimmunity Study in the Young (DAISY). Jama. 276 609 614. Nurieva, R., Yang, X.O., Martinez, G., Zhang, Y., Panopoulos, A.D., Ma, L., Schluns, K., Tian, Q., Watowich, S.S., Jetten, A.M., and Dong, C. 2007. Essential autocrine regulation by IL 21 in the generation of inflammatory T cells. Nature 448(7152) 480 483. Onkamo, P., Vaananen, S., K arvonen, M., and Tuomilehto, J. 1999. W orldwide increase in incidence of Type I diabetes the analysis of the data on published incidence trends. Diabetologia 42 1395 1403.

PAGE 101

101 Oppmann, B., Lesley, R., Blom, B., Timans, J.C., Xu, Y., Hunte, B., Vega, F., Yu, N., Wang, J., and Singh, K., et al. 2000 Novel p19 protein engages IL 12p40 to form a cytokine, IL 23, with biological activities similar as well as distinct from IL 12. Immunity 13 715 725. Ouyang, W., Kolls, J., and Zheng, Y. 2008. The biological functions of T helper 17 cell effector cytoki nes in inflammation. Immunity 28, 454 467. Ozaki, K., Kikly, K., Michalovich, D., Young, P.R., and Leonard, W.J. 2000 Cloning of a type I cytokine receptor most related to the IL 2 receptor beta chain. Proc. Natl. Acad. Sci. USA 97 11439 11444. Pakala, S V., Kurrer, M. O. and Katz, J. D. 1997. T helper 2 (Th2) T cells induce acute pancreatitis and diabetes in immune compromised nonobese diabetic (NOD) mice. J. Exp. Med. 186 299 306. Parrish Novak, J., et al. 2000 volved in NK cell expansion and regulation of lymphocyte function. Nature 408 57 63. Pickert, G., Neufe rt, C., and Leppkes, M. et al. 2009. STAT3 links IL 22 signaling in intestinal epithelial cells to mucosal wound healing. J. Exp. Med. 206, 1465. Pillai A.B., George, T.I., Dutt, S., and Strober, S. 2009 Host natural killer T cells induce an interleukin 4 dependent expansion of donor CD4+CD25+Foxp3+ T regulatory cells that protects against graft versus host disease. Blood 113, 4458 4467. Pundziute Lycka A., Ur bonaite, B., and Dahlquist, G. 2000. Infections and risk of Type I (insulin dependent) diabetes mellitus in Lithuanian children. Diabetologia 43, 1229 1334. Rabinovitch, P.S., Torres, R.M., and Engel, D. 1986. Si m ultaneous cell cycle analysis and t wo color surface immunofluorescence using 7 amino actinomycin D and single laser excitation: applications to study of cell activation and the cell cycle of murine Ly 1 B cells. J. Immunol. 136(8), 2769 2775. Radaeva, S., Sun, R., Pan, H. N., Hong, F. and G ao, B. 2004. Interleukin 22 (IL 22) plays a protective role in T cell mediated murine hepatitis: IL 22 is a survival factor for hepatocytes via STAT3 activation. Hepatology 39, 1332. Reis e Sousa, C. 2006. Dendritic cells in a mature age. Nat. Rev. Immunol 6, 476 483. Ren, X., Hu, B. and Colletti, L. M. 2010. IL 22 is involved in liver regeneration after hepatectomy. Am. J. Physiol. Gastrointest. Liver Physiol. 298, G74. Rimoldi, M., Chieppa, M., Larghi, P., Vulcano, M., Allavena, P., and Rescigno, M. 200 5. Monocyte derived dendritic cells activated by bacteria or by bacteria stimulated epithelial cells are functionally different Blood 106 2818 2826.

PAGE 102

102 Ringrose, L., Ehret, H., and Paro R. 2004. Distinct contributions of histone H3 lysine 9 and 27 methylat ion to locus specific stability of polycomb complexes. Mol. Cell 16, 641 653. Roesch, L.F., Lorca, G.L., Casella, G., Giongo, A., Naranjo, A., Pionzio, A.M., Li N., Mai, V., Wasserfall, C.H., Schatz, D., Atkinson, M.A., Neu, J., and Triplett, E.W. 2009. Cu lture independent identification of gut bacteria correlated with the onset of diabetes in a rat model. ISME J. 3(5), 536 548. Rook, G.A., and Brunet, L.R. 2005. Microbes, immunoregu lation, and the gut. Gut 54, 317 320. Round, J.L., O'Connell, R.M., and Ma zmanian, S.K. 2010. Coordination of tolerogenic immune responses by the commensal microbiota. J. Autoimmunity 34, 220 225. Ruckert, R., Bulfone Paus, S., and Brandt, K. 2008. Interleukin 21 stimulates antigen uptake, protease activity, survival and inducti on of CD4+ T cell proliferation by murine macrophages. Clin. Exp. Immunol. 151 487 495. Ruddy, M. J. et al. 2004. Functional cooperation between interleukin 17 and tumor members. J. Bi ol. Chem 279 2559 2567. Ryu, S., Kodama, S., Ryu, K., Schoenfeld, D., and Faustman, D. 2001. Reversal of cell function. J. Clin. Invest. 108(1), 63 72. Salomon, B., Lenschow, D.J., Rhee, L., Ashourian, N., Singh, B., Sharpe, A., and Bluestone, J.A. 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12 431 440. Santos Rosa, H., R. Schneider, B. E. B ernstein, N. Karabetsou, A. Morillon, C. Weise, S. L. Schreiber, J. Mellor, and T. Kouzarides. 2003. Methylation of histone H3 K4 mediates association of the Isw1p ATPase with chromatin. Mol. Cell 12, 1325 1332. Savilahti, E., Ormala T., Saukkonen, T., San dini Pohjavuori, U., Kantele, J.M., Arato, A., Ilonen, J., Akerblom, H.K. 1999. Jejuna of patients with insulin dependent diabetes mellitus (IDDM) show signs of immune activation. Clin. Exp. Immunol 116 70 77. Schweitzer, A.N., and Sharpe, A.H. 1998. St udies using antigen presenting cells lacking expression of both B7 1 (CD80) and B7 2 (CD86) show distinct requirements for B7 molecules during priming versus restimulation of Th2 but not Th1 cytokine production. J. Immunol. 161 2762 71.

PAGE 103

103 Sebzda, E., Wallac e, V.A., Mayer, J., Yeung, R.S .M., Mak, T., and Ohashi, P.S. 1994. Positive and negative thymocyte selection induced by different concentrations of a single peptide. Science 263 1615 1618. Serada, S., Fujimoto, M., Mihara, M., Koike, N., Ohsugi, Y., Nomur a, S., Yoshida, H., Nishikawa, T., Terabe, F., and Ohkawara, T. et al. 2008. IL 6 blockade inhibits the induction of myelin antigen specific TH17 cells and TH1 cells in experimental autoimmune encephalomyelitis. Proc. Natl. Acad. Sci. USA 105, 9041 9046. S underlying abnormal macrophage development and maturation in NOD/Lt mice: defective regulation of cytokine receptors and protein kinase C. Proc. Natl. Acad. Sci. USA 90, 9 625 9629. Serreze, D.V., Gaskins, H.R., and Leiter, E.H. 1993. Defects in the differentiation and function of antigen presenting cells in NOD/Lt mice. J. Immunol. 150 2534 2543. Serreze, D.V., Prochazka, M., Reifsnyder, P.C., Bridgett, M.M., and Leiter, E .H. 1994. Use of recombinant congenic and congenic strains of NOD mice to identify a new insulin dependent diabetes resistance gene. JEM 180, 1553 1558. Serreze, D.V., Fleming, S.A., Chapman, H.D., Richard, S.D., Leiter, E.H., and Tisch, R.M. 1998. B lymph ocytes are critical antigen presenting cells for the initiation of T cell mediated autoimmune diabetes in nonobese diabetic mice. J. Immunol. 161, 3912 3918. Sharif, S., Arreaza, G.A., Zucker, P., Mi, Q S., Sondhi, J., Naidenko, O.V., Kronenberg, M., Koezu ka, Y., Delovitch, T.L., Gombert, J M., Leite de Moraes, M., Gouarin, C., Zhu, R., Hameg, A., Nakayama, T., Taniguchi, M., Lepault, F., Lehuen, A., Bach, J galactosylceramide treatment pr events the onset and recurrance of autoimmune type 1 diabetes. Nat. Med. 7 1057 1062. Shultz, L.D., Schweitzer, P.A., Christianson, S.W., Gott, B., Schweitzer, I.B., Tennent, B., McKenna, S., Mobraaten, L., Rajan, T.V., and Greiner, D.L. et al. 1995. Mul tiple defects in innate and adaptive immunologic function in NOD/LtSz scid mice. J. Immunol. 154 180 191. Siegemund, S., Schutze, N., and Schulz, S. et al. 2009. Differential IL 23 requirement for IL 22 and IL 17A production during innate immunity against Salmonella enterica serovar. Enteritidis. Int. Immunol. 21 555. Silveira, P.A., Chapman, H.D., Stolp, J., Johnson, E., Cox, S.L., Hunter, K., Wicker, L.S., and Serreze, D.V. 2006. Genes within the Idd5 and Idd9/11 diabetes susceptibility loci affect the pathogenic activity of B cells in nonobese diabetic mice. J. Immunol. 177, 7033 7041.

PAGE 104

104 Silverberg, M. S., Cho, J. H., Rioux, and J. D. et al. 2009. Ulcerative colitis risk loci on chromosomes 1p36 and 12q15 found by genome wide association study. Nat. Genet 41, 216. Slattery, R.M., Kjer Nielsen, L., Allison, J., Charlton, B., Mandel, and Miller, T. JFAP 1990. Prevention of diabetes in non obese diabetic I A k transgenic mice. Nature 345, 724 726. Sofi, M.H., Li, W., Kaplan, M.H., and Chang, C.H. 2009. Elevat ed IL 6 expression in CD4 T cells via PKCtheta and NF kappaB induces TH2 cytokine production. Mol. Immunol. 46, 1443 1450. Spolski, R., Kashyap, M., Robinson, C., Yu, Z., and Leonard, W. 2008. IL 21 signaling is critical for the development of type 1 diabe tes in the NOD mouse. PNAS 105(37), 14028 14033. Spolski, R., Kim, H.P., Zhu, W., Levy, D.E., and Leonard, W.J. 2009. IL 21 mediates suppressive effects via its induction of IL 10. J. Immunol. 182, 2859 2867. Strengell, M., Lehtonen, A., Matikainen, S., a nd Julkunen, I. 2006. IL 21 enhances SOCS gene expression and inhibits LPS induced cytokine production in human monocyte derived dendritic cells. J. Leukoc. Biol. 79 1279 1285. Strid, J., Lopes, L., Marcinkiewicz, J., Petrovska, L., Nowak, B., Chain, B.M. and Lund, T. 2001. A defect in bone marrow derived dendritic cell maturation in the non obese diabetic mouse. Clin. Exp. Immunol. 123(3), 375 381. Suarez Pinton, W., Rajotte, R.V., Mosm ann, T.R., and Rabinovitch, A. 1996. Both CD4+ and CD8+ T cells in s yngeneic islet grafts in NOD mice produce interferon gamma during beta cell destruction. Diabetes 45(10), 1350 1357. Sugimoto, K., Ogawa, A., and Mizoguchi, E. et al. 2008. IL 22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis. J Clin. Invest. 118, 534. Sujino, T., Kanai, T., Ono, Y., Mikami, Y., Hayashi, A., Doi, T., Matsuoka, K., Hisamatsu, T., Takaishi, H., Ogata, H., Yoshimura, A., Littman, D.R., Hibi, T. 2011. Regulatory T cells suppress development of colitis, blocking diff erentiation of T Helper 17 into alternative T Helper 1 cells. Gastroenterology Jun 7 (Epub ahead of print). Takatori, H. et al. 2009. Lymphoid tissue inducer like cells are an innate source of IL 17 and IL 22. J. Exp. Med 206 35 41. Tartar, D.M., VanMor ian, A.M., Wan, X., Guloglu, F.B., Jain, R., Haymaker, C.L., Ellis, J.S., Hoeman, C.M., Cascio, J.A., Dhakal, M., Oukka, M., and Zaghouani, H. 2010. Foxp3+ RORgammat+ T helper intermediates display suppressive function against autoimmune diabetes. J. Immun ol. 184(7), 3377 3385.

PAGE 105

105 Tiittanen, M., Westerholm Ormio, M., Verkasalo, M., Savilahti, E., and Vaarala, O. 2008. Infiltration of Foxp3 expressing cells in jejunal mucosa in celiac disease but not in type 1 diabetes. Clin. Exp. Immunol 152, 498 507. Tebbu tt, N.C., Giraud, A.S., Inglese, M., Jenkins, B., Waring, P., Clay, F.J., Malki, S., Alderman, B.M., Grail, D., and Hollande, F., et al. 2002. Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT mediated trefoil gene activation in gp130 mutant mice. Nat Med. 8, 1089 1097. Tisch, R., Yang, X.D., and Liblau, R.S., et al. 1994. Administering glutamic acid decarboxylase to NOD mice prevents diabetes. J. Autoimmun. 7(6), 845 850. Todd, J.A., Acha Orbea, H., Bell, J.I., Chao, N., Fronek, Z., Ja cob, C.O., McDermott, M., Sinha, A.A., Timmerman, L., Steinman, L., and McDevitt, H.O. 1988. A molecular basis for MHC class II associated autoimmunity. Science 240, 1003 1009. Tritt, M., Sgouroudis, E., d'Hennezel, E., Albanese, A., and Piccirillo, C.A. 20 08. Functional waning of naturally occurring CD4+ regulatory T cells contributes to the onset of autoimmune diabetes. Diabetes 57, 113 123. death triggers priming o f self reactive T cells by dendritic cells in a type 1 diabetes model. JEM 198(10), 1527. Ucker, D.S., Meyers, J., and Obermiller, P.S. 1992 Activation driven T cell death. II. Quantitative differences alone distinguish stimuli triggering nontransformed T cell proliferation or death. J. Immunol. 149 1583 1592. Ueda, H., et al. 2003. Association of the T cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 423, 506 511. Vaarala, O., Knip, M., Paronen, J., Hamalainen, A.M., Muona, P., Vaatainen, M., Ilonen, J., Simell, O., and Akerbl om, H.K. primary immunization to insulin in infants at ge netic risk for type 1 diabetes. Diabetes 48, 1389 1394. Diabetes 57 255 5 2562. Valladares, R., Sankar, D., Li, N., Williams, E., Lai, K.K., Abdelgeliel, A.S., Gonzalez, C.F., Wasserfall, C.H., Larkin, J., Schatz, D., Atkinson, M.A., Triplett, E.W., Neu, J., and Lorca, G.L. 2010. Lactobacillus johnsonii N6.2 mitigates the deve lopment of type 1 diabetes in BB DP rats. PLoS One 5(5), e10507. Veldhoen, M., Hocking, R.J., Atkins, C.J., Locksley, R.M., and Stockinger, B. 2006. TGF beta in the context of an inflammatory cytokine milieu supports differentiation of IL 17 producing T ce lls. Immunity 24(2), 179 189.

PAGE 106

106 Veldhoen, M., Hirota, K., Westendorf, A.M., Buer, J., Dumoutier, L., and Renauld, J.C., et al. 2008. The aryl hydrocarbon receptor links TH17 cell mediated autoimmunity to environmental toxins. Nature 453(7191), 106 109. Viska ri, H.R., Koskela, P., Lonnrot, M., Luonuansuu, S., Reunanen, A., Baer, M., and Hyoty, H. 2000. Can enterovirus infections explain the increasing incidence of type 1 diabetes? Diabetes Care 23 414 416. Walker, L.S., and Abbas, A.K. 2002. The enemy within: keeping self reactive T cells at bay in the periphery. Nat. Rev. Immunol. 2 11 19. Wang, B., Gonzalez, A., Hoglund, P., Katz, J.D., and Benoist, C., et al. 1998. Interleukin 4 deficiency does not exacerbate disease in NOD mice. Diabetes 47, 1207 1211. W ang, J., Cho, S., Ueno, A., Cheng, L., Xu, B.Y., Desrosiers, M.D., Shi, Y., and Yang, Y. 2008. Ligand dependent induction of noninflammatory dendritic cells by anergic invariant NKT cells minimizes autoimmune inflammation. J. Immunol. 181 2438 2445. Wen, L., Ley, R.E., Volchkov, P.Y., Stranges, P.B., Avanesyan, L., Stonebraker, A.C., Wong, F.S., Szot, G.L., Bluestone, J.A., Gordon, J.I., and Chervonsky, A.V. 2008. Innate immunity and intestinal microbiota in the development of Type 1 diabetes. Nature 455(7 216), 1109 1113. Westerholm Ormio, M., Vaarala, O., Pihkala, P., Ilonen, J., Savilahti, E. 2003. Immunologic activity in the small intestinal mucosa of pediatric patients with type 1 diabetes. Diabetes 52, 2287 2295. Wilkin, T.J. 2001. The accelerator hypo thesis: weight gain as the missing link between Type I and Type II diabetes. Diabetologia 44 914 922. Wilson, S.S., and Deluca, D. 1997. NOD fetal thymus organ culture an in vitro model for the development of T cells involved in IDDM. J. Autoimmun. 10, 461 472. Wolk, K., Witte, E., and Wallace, E. et al. 2006. IL 22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis. Eur. J. Immunol. 36, 1309. Wol k, K., Haugen, H. S., and Xu, W. et al. 2009 IL 22 and IL 20 are key mediators of the epidermal alterations in psoriasis while IL 17 and IFN gamma are not. J. Mol. Med. 87 523. Xiu, Y., Wong, C .P., and Bouaziz, J.D., et al. 2008. B lymphocyte depletion b y CD20 monoclonal antibody prevents diabetes in nonobese diabetic mice despite isotype specific differences in Fc gamma R effector functions. J. Immunol. 180(5), 2863 2875.

PAGE 107

1 07 Yamanouchi, J., Rainbow, D., Serra, P., Howlett, S., Hunter, K., Garner, V.E., Gonz alez Munoz, A., Clark, J., Veijola, R., Cubbon, R., Chen, S.L., Rosa, R., Cumiskey, A.M., Serreze, D.V., Gregory, S., Rogers, J., Lyons, P.A., Healy, B., Smink, L.J., Todd, J.A., Peterson, L.B., Wicker, L.S., and Santamaria, P. 2007. Interleukin 2 gene var iation impairs regulatory T cell function and causes autoimmunity. Nat. Genet. 39, 329 337. Yang, X.D., Sytwu, H.K., McDevitt, H.O., and Michie, S.A. 1997. Involvement of beta 7 integrin and mucosal addressin cell adhesion molecule 1 (MAdCAM 1) in the deve Diabetes 46, 1542 1547. Yang, X.O., Pappu, B.P., Nurieva, R., Akimzhanov, A., Kang, H.S., Chung, Y., Ma, L., Shah, B., Panopoulos, A.D., Schluns, K.S., Watowich, S.S., Tian, Q., Jetten, A.M., and Dong, C. 2008. T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma. Immunity 28(1), 29 39. Yang Hau, V., Lee, W.H., Ortiz, S., Lee, M.H., Qin, H.J., and Liu, C.P. 2009. All trans retinoic acid inhibits type 1 diabetes by T regulatory (Treg) dependent suppression of interferon producing T cells without affecting TH17 cells. Diabetes 58, 146 155. Ylipaasto, P., Klingel, K., Lindberg, A.M., Otonkoski, T., Kandolf, R., Hovi, T., and Roivainen, M. 2004. Enterovirus infection in human pancreatic islet cells, islet tropism in vivo and receptor involvement in cultured islet beta cells. Diabetologia 47 225 239. Yoon, J.W. 1991. Role of viruses in the pathogenesis of IDDM. Ann. Med. 23, 437 445. You, S., Belghith, M., Cobbold, S., Alyanakian, M.A., Gouarin, C., Barriot, S., Garcia, C., Waldmann, H., Bach, J.F., Chatenoud, and L. 2005. Autoimmune diabetes onset results from qualitative rather than quantitative age dependent changes in pathogenic T cells. Diabetes 54, 1415 1422. Young D.A., et al. 2007. Blockade of the interleukin 21/interleukin 21 receptor pathway Ameliorates disease in animal models of rheumatoid arthritis. Arthritis Rheum. 56, 1152 1163. Zenewicz, L. A., Yancopoulos, G. D., Valenzuela, D. M., Murphy, A. J., S tevens, S. and Flavell, R. A. 2008. Innate and adaptive interleukin 22 protects mice from inflammatory bowel disease. Immunity 29 947. cell death promotes priming of diabetogenic CD8 T lymphocytes. J. Immunol. 168 1466 1472. Zheng, Y., Valdez, P. A., and Danilenko, D. M. et al. 2008. Interleukin 22 mediates early host defense against attaching and effacing bacterial pathogens. Nat. Med. 14, 282.

PAGE 108

108 Zhou, L., Ivanov, I.I., Spolski, R., Min, R., Shenderov, K., Egawa, T., Levy, D.E., Leonard, W.J., Littman, D.R. 2007 IL 6 programs T(H) 17 cell differentiation by promoting sequential engagement of the IL 21 and IL 23 pathways. Nat. Im munol. 8, 967 974.

PAGE 109

109 BIOGRAPHICAL SKETCH Kenneth Kit Lau was born in Manhattan, New York in 1985 and moved with his family to Miami, Florida a few years later. Following high school, he attended the University of Florida for his undergraduate studies, comp leting a Bachelor of Science degree in m icrobiology and c ell s cience in the spring of 2007. Inspired by research topics revolving around benchside to bedside translational studies, he received his Ph.D. from the University of Florida in the fall of 2011, f ocusing on immunology and Type 1 Diabetes.