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Impact of Myeloid Derived Suppressor Cells during Polymicrobial Sepsis

Permanent Link: http://ufdc.ufl.edu/UFE0021985/00001

Material Information

Title: Impact of Myeloid Derived Suppressor Cells during Polymicrobial Sepsis
Physical Description: 1 online resource (151 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: inflammation, myd88, myeloid, sepsis
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Sepsis is defined as bacterial infection accompanied by an overwhelming systemic inflammatory response that results in both innate and adaptive immune system dysfunction. Specifically, polymicrobial sepsis alters the adaptive immune response, and induces T-cell suppression and TH2 immune polarization. In this dissertation, we identified a population of GR-1+CD11b+ cells whose numbers dramatically increase and remain elevated in the spleen, lymph nodes and bone marrow during polymicrobial sepsis. Phenotypically, these cells are heterogeneous, immature, predominantly myeloid progenitors that express IL-10, TNF?, MCP-1 and a number of other cytokines and chemokines. Splenic GR-1+ cells effectively suppress antigen-specific CD8+ T-cell interferon-? production, but only modestly suppress antigen-specific and nonspecific CD4+ T-cell proliferation. GR-1+ cell depletion in vivo prevents both the sepsis-induced augmentation of TH2-dependent and depression of TH1-dependent antibody production. Signaling through MyD88 and the SDF-1 pathways, but not TLR4, TRIF, the IFN?/? receptor, CCR2 receptor, IL-10, IL-4 or the M-CSF pathways are required for complete GR-1+CD11b+ expansion. GR-1+CD11b+ cells contribute to sepsis-induced T-cell suppression and preferential TH2 polarization.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Ramphal, Reuben.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021985:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021985/00001

Material Information

Title: Impact of Myeloid Derived Suppressor Cells during Polymicrobial Sepsis
Physical Description: 1 online resource (151 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: inflammation, myd88, myeloid, sepsis
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Sepsis is defined as bacterial infection accompanied by an overwhelming systemic inflammatory response that results in both innate and adaptive immune system dysfunction. Specifically, polymicrobial sepsis alters the adaptive immune response, and induces T-cell suppression and TH2 immune polarization. In this dissertation, we identified a population of GR-1+CD11b+ cells whose numbers dramatically increase and remain elevated in the spleen, lymph nodes and bone marrow during polymicrobial sepsis. Phenotypically, these cells are heterogeneous, immature, predominantly myeloid progenitors that express IL-10, TNF?, MCP-1 and a number of other cytokines and chemokines. Splenic GR-1+ cells effectively suppress antigen-specific CD8+ T-cell interferon-? production, but only modestly suppress antigen-specific and nonspecific CD4+ T-cell proliferation. GR-1+ cell depletion in vivo prevents both the sepsis-induced augmentation of TH2-dependent and depression of TH1-dependent antibody production. Signaling through MyD88 and the SDF-1 pathways, but not TLR4, TRIF, the IFN?/? receptor, CCR2 receptor, IL-10, IL-4 or the M-CSF pathways are required for complete GR-1+CD11b+ expansion. GR-1+CD11b+ cells contribute to sepsis-induced T-cell suppression and preferential TH2 polarization.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Ramphal, Reuben.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021985:00001


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IMPACT OF MYELOID DERIVED SUPPRESSOR CELLS DURING POLYMICROBIAL SEPSIS By MATTHEW J. DELANO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Matthew J. Delano 2

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To Nicole Dobija for her indefatigab le spirit and unending, selfless support. 3

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ACKNOWLEDGMENTS In this brief and finite space I can only begin to adequately express my gratitude, thanks, and appreciation to the multitude of individuals who have unselfi shly sacrificed their time and efforts to contribute to my sc ientific growth. I would like to first thank the Department of Surgery for allowing me the flexibility to ta ke a significant amount of time away from a demanding clinical schedule and pursue my resear ch interests. Specifi cally, I would like to express my sincere thanks to Dr. William Cance for his foresight into the future of academic surgery and the surgeon researcher Dr. Kevin Behrns also deserv es recognition for his practical prioritization of the residency programs efforts to train clinical practitioners, research scientist, and sometimes both, all in rapid sequence. My Committee members, Dr. Lyle Moldawer Dr. Michael Clare-Salzler, Dr.Westley Reeves, Dr. Reuben Ramphal, and Dr. Willaim Ca nce, all deserve my highest gratitude and thanks for their mentorship and guidance through out the dissertation process. The Committees guidance has provided me with the necessary structure and foundation paramount to my evolution as a scientist surgeon. The members of the Reeves lab deserve my gratitude for their assistance in method development and experiment al ideas. The numerous discussions with Pui Lee regarding the phenomenon of myeloid cells in various infl ammation models were invaluable to my work. Jason Weinstein deserves speci al recognition for his as sistance with numerous methodologies and for laying the foundation and build ing the data trail for the B cell story in sepsis. I owe many thanks to Kindra Kelly-Scumpia formerly of the Reeves lab and now in the Moldawer lab. Her collaborative and organizationa l skills during large experiments were crucial to my success. Dr. Clare-Salzler is commended for his conti nued support scientifica lly, academically, and personally; and for his willingness to act as a sounding board for scientific ideas and career 4

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planning. Dr. Ramphals support, guidance and sobe ring view of practitione r/researcher reality do and will serve as a platform for my future endeavors. I would like to thanks the individuals at Schereing Plough Biopharma (formerly DNAX) for a ll of their guidance and support namely: Dr. Drake LaFace, Dr. Paul Heyworth and Dr. Joe Philips. Dr. Dominique Coco also deserves special thanks for his interpretation of the histol ogical sections. Dr. Al fred Ayala and lab at Brown deserve kudos for their assistance in seve ral studies that they made happen in quick fashion with in demand limited mouse recourses. I have intentionally saved the specific individuals for last who have made an astounding contribution to my success as a researcher and individual person. The fi rst would be Dr. Kerri OMalley, who taught me how to balance research with life and was always available on short notice to lend a hand and advice in a pinch durin g large experiments. Next is Dr. Jim Wynn who, after spending three years of his career in the same office as me, should be given a Good Samaritan Award. Jims input advice and data interpretation were paramount to my success. I wish that I had more space to dedicate to thank Dr. Philip O. Scumpia who made numerous unsolicited contributions to my work and freely gave of himself and his own time to assist me. The data and knowledge that I genera ted, and the directions that I have chosen to navigate, would not have been made possible w ithout Phils assistance and companionship. To him I am indebted and owe a treme ndous amount or thanks and praise. Although this whole process was daunting to me at times it was not to Dr. Lyle (Linc) Moldawer, and that was what served me most effectively. Many people speak of individuals who dedicate their lifes work to scientific investigation; I only know one person and that are Linc Moldawer. There will be no other person in my career who will have as positive an impact on me as this man. From his unwavering compa ssion to his genuine concern for our scientific 5

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growth, there is no possible way to summarize th e positive and significant contribution that he has made to my maturation as a pers on and as a scientist. Thank you. This work was supported in part by grants R37GM-40586 and R01GM-62041 awarded to Dr. Lyle Moldawer by the National Institute of General Medical Sciences. U.S.P.H.S. I was supported by a training grant awarded to the Depart ment of Surgery in burns and trauma research (T32GM-08431), also awarded by the National In stitute of General Medical Sciences. I would also like to than k my immediate family, Arthur and Rhonda Delano for encouraging me to pursue medicine and science and for creating a family foundation for me to build and grow. My brother Dr. Ben Delano also deserves credit for listening to me fret about bungled experiments late at night. Finally none of this could have been made po ssible without the loving support of my wife and life companion, Dr. Nicole Dobija. 6

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES .........................................................................................................................10LIST OF FIGURES .......................................................................................................................11 ABSTRACT....14 CHAPTER 1 INTRODUCTION ................................................................................................................ ..15Sepsis Significance .................................................................................................................15Sepsis, the Cecal Ligation and Puncture (CLP) Model, and Immune Dysfunction ...............16Myeloid Derived Suppressor Cells (MDSCs) and Disease ....................................................18Myeloid Derived Suppressor Cells Function During Inflammation .....................................19Hypothesis ..............................................................................................................................20Specific Aim 1 .................................................................................................................21Specific Aim 2 .................................................................................................................21Specific Aim 3 .................................................................................................................212 EXPANSION OF AN IMMATURE GR-1+CD11B+ POPULATION INDUCES TCELL SUPPRESSION AND TH2 POLARIZATION IN SEPSIS .........................................23Specific Aim 1 ........................................................................................................................23Introduction .................................................................................................................. ...........23Results .....................................................................................................................................24GR-1+CD11b+ Cells Accumulate in the Spleen after Sepsis ...........................................24GR-1+CD11b+ Cells are Phenotypically, Heterogenous Cells ........................................26Myeloid Derived Suppressor Cells A ccumulate in Secondary Lymphoid Organs After Sepsis. ....................................................................................................27Myeloid Derived Suppressor Cells are Capable of Inflammatory Mediator Production ....................................................................................................28Myeloid Derived Suppressor Cells Affect Adaptive Immune Responses In Sepsis .......29MyD88 Signaling Pathways Are Required for GR-1+CD11b+ Cells to Accumulate in the Spleen After Polymicrobial Sepsis. ...................................................................31Discussion .................................................................................................................... ...........32Materials and Methods ...........................................................................................................38Mice .................................................................................................................................38Cecal Ligation and Puncture ...........................................................................................39Flow Cytometry ...............................................................................................................39Cell Purification ...............................................................................................................40Ex Vivo Stimulation and Cytokine Production ...............................................................40Ex Vivo Differentiation and Colony Formation ..............................................................40 7

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Antigen Specific CD8+ T-Cell IFNProduction ............................................................40Enzyme-Linked Immunospot Assay (ELISpot) ..............................................................41Antigen Specific and Nonspecific CD4+ T-Cell proliferation .........................................41Immunization with NP-KLH and Humoral Immune Responses .....................................42Morphologic and Histologic Analysis .............................................................................42Statistics .................................................................................................................... .......433 INTERLEUKIN-4, INTERLEUKIN-10 AND NITRIC OXIDE SIGNALING DO NOT CONTRIBUTE TO THE MYELOID DERI VED SUPPRESSOR CELL EXPANSION OR SUPPRESSOR FUNCTION DURI NG POLYMICROBIAL SEPSIS ............................62Specific Aim 2 ........................................................................................................................62Introduction .................................................................................................................. ...........62Results .....................................................................................................................................64MDSCs are Capable of Immune Mo dulatory Cytokine Production ...............................64MDSCs Inhibit Antigen Specific CD8+ T-Cell IFNProduction Independent of Interleukin-4, Interleukin-10, and iNOS Production ...................................................64MDSCs Inhibit Antigen Specific CD8+ T-Cell Cytotoxicity ..........................................65Discussion .................................................................................................................... ...........66Materials and Methods ...........................................................................................................68Mice .................................................................................................................................68Cecal Ligation and Puncture ...........................................................................................68Flow Cytometry ...............................................................................................................68Cell Purification ...............................................................................................................69Ex Vivo Stimulation and Cytokine Production ...............................................................69Antigen Specific CD8+ T-Cell IFNProduction ............................................................69Enzyme-Linked Immunospot Assay (ELISpot) ..............................................................69CD8+ T Cell-Cytotoxicity ................................................................................................70Statistics .................................................................................................................... .......704 MYELOID DERIVED SUPPRESSOR CELL EXPANSION IS DEPENDENT ON CXCL12 MEDIATED COMMON MYELO ID PROGENITOR EXPANSION DURING SEPSIS ...................................................................................................................76Specific Aim 3 ........................................................................................................................76Introduction .................................................................................................................. ...........76Results .....................................................................................................................................79Hematopoietic Stem Cell Proliferatio n and Differentiation do not Dependent on MyD88 or TRIF Signaling In Vivo .............................................................................79MDSC Expansion During Sepsis Occurs Independently of the CCR2 Signaling Pathway. ......................................................................................................81M-CSF Receptor Signaling Modestly Inhibi ts Immature Myeloid Cell Expansion .......83MDSC Expansion During Sepsis Occu rs Independently of Neutrophil Elastase Activity. .........................................................................................................84CXCL12 is Required for Complete MDSC Expansion During Sepsis. ..........................85 Discussion .................................................................................................................... ...........87 8

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Materials and Methods ...........................................................................................................91Mice .................................................................................................................................91Inhibitors .................................................................................................................... ......92Cecal Ligation and Puncture ...........................................................................................92Flow Cytometry ...............................................................................................................93Ex Vivo Stimulation and Cytokine Production ...............................................................93Statistics .................................................................................................................... .......945 MYELOID-DERIVED SUPPRESSOR CELLS AND THEIR CONTRIBUTION TO POST-INJURY AND SEPSIS IMMUNE SUPPRESSION .................................................106Introduction .................................................................................................................. .........106Sepsis and Immune Dysfunction ..........................................................................................106Myeloid Derived Suppressor Cells Play a Role in Injury, Sepsis and Trauma ....................107MDSCs and Sepsis-Induced Immune Suppression ..............................................................119Myeloid Derived Suppresor Cells and Future Directions .....................................................126LIST OF REFERENCES .............................................................................................................136BIOGRAPHICAL SKETCH .......................................................................................................151 9

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LIST OF TABLES Table page 5-1 MDSC Phenotypes in Various Inflammation Models. ....................................................1305-2 MDSCs Proliferate When Cu ltured With Growth Factors. ............................................1315-3 MDSC Fold Change Increases in Various Models of Inflammation. ..............................132 10

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LIST OF FIGURES Figure page 2-1 Bacteremia and blood leukocyte responses to sepsis produced by a cecal ligation and puncture..............................................................................................................................442-2 Differential white blood cell c ounts in response to sepsis.. ...............................................452-3 Selected plasma cytokine concentrati ons in septic and sham-treated mice.. .....................462-4 Sepsis induced splenomegaly. ...........................................................................................472-5 Appearance of GR-1+CD11b+ cells in spleens from septic and sham mice. ..................482-6 Ter119 Staining of GR-1+CD11b+ splenocytes harvested from sham and septic mice.. ...492-7 MHC Class II expression in GR-1+CD11b+ splenocytes harvested from sham and septic mice.. .......................................................................................................................502-8 Flow cytometric analysis of GR-1+ splenocytes cultured with GM-CSF, G-CSF or erythropoietin (EPO) ex vivo. ..........................................................................................512-9 Bone marrow and lymph node GR-1+CD11b+ cells from septic and sham mice.. ............522-10 Hematoxylin and eosin stained spleens from septic mice ten days after CLP. ..................532-11 Effect of ex vivo lipopolysaccharide s timulation on cytokine expression in GR-1+ splenocytes obtained from septic mice.. ............................................................................542-12 Immunoglobulin production following NP-K LH immunization in sham, septic and septic mice depleted of GR-1+ cells.. .................................................................................552-13 Effect of GR-1 an tibody depletion on CD11b+GR-1+ populations in the spleen and lymph nodes. ......................................................................................................................562-14 Immunoglobulin production following NP-K LH immunization in sham, and septic mice.. ........................................................................................................................ ..........572-15 Effect of GR-1+ cells from septic mice on antigen specific CD4+ T-cell proliferation and CD8+ T-cell IFN responses.. .....................................................................................582-16 Effects of lipopolysaccharide (LPS) and tr ansgenic mice on expansion of the splenic GR-1+CD11b+ population ten days after CLP.. .................................................................592-17 Requirement for MyD88-/signaling in the expansion of GR-1+CD11b+ splenocytes in response to prolonged sepsis..........................................................................................60 11

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2-18 Immunoglobulin production following NP-K LH immunization in sham, and septic mice 12 weeks after cecal ligation and puncture.. .............................................................613-1 Effect of ex vivo lipopolysaccharide stimulation on cytokine expression in GR-1+ splenocytes obtained from septic mice.. ............................................................................713-2 Flow cytometry analysis of IL-10 and IL-4 expression in response to ex vivo lipopolysaccharide stimulation. .........................................................................................713-3 GR-1+CD11b+ myeloid cell expansion is not depe ndent on the presence of IL-4 or IL-10 production. ...............................................................................................................733-4 Effect of GR-1+ cells from septic mice on antigen specific CD8+ T-cell IFN responses.. ................................................................................................................... .......743-5 Effect of GR-1+ cells from septic mice on antigen specific CD8+ T-cell cytotoxic function. ..................................................................................................................... ........754-1 The absence of MyD88 and TRIF si gnaling pathways does not impact HSC expansion during sepsis.. ...................................................................................................954-2 MyD88 and TRIF signaling do not impact bone marrow progenitor expansion during sepsis. ....................................................................................................................... ..........964-3 MyD88 and TRIF signaling do not effect immature myeloid cell expansion in the bone marrow during sepsis. ...............................................................................................974-4 Splenic and bone marrow MDSC expansion is not dependent on CCR2 receptor signaling pathway.. ............................................................................................................984-5 CCR2-/mice exhibit fewer GR-1intermediate cells. ................................................................994-6 Splenic and bone marrow expansion of progenitor cells is not dependent on CCR2 receptor signaling pathway.. ............................................................................................1004-7 Splenic MDSC expansion is not dependent on cfms receptor signaling. .......................1014-8 Splenic and bone marrow expansion of immature myeloid and bone marrow progenitor cells is not dependent on neutrophil elastase activity. ...................................1024-9 Splenic expansion of myeloid derive d suppressor cells is dependent on CXCL12 signaling.. ................................................................................................................... ......103410 CXCL12 inhibited the splenic accumula tion of common myeloid progenitors during sepsis. ....................................................................................................................... ........1044-11 Bone marrow HSC and CMP expansion does not depend on CXCL12 during sepsis. ...105 12

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5-1 Panels A and B represent a cytospin preparation of GR-1+ cells isolated from the spleen of a 10 day septic mice .........................................................................................1295-2 Gemcitibine, a nucleoside analog that inhibits rapidly proliferating cells. ......................1335-3 H&E preparations of mouse spleens 10 days after either sham or sepsis treatment. .......1345-4 Proposed model by which MDSCs can impact both components of acquired and innate immunity. .............................................................................................................. 135 13

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14 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IMPACT OF MYELOID DERIVED SUPPRE SSOR CELLS DURING POLYMICROBIAL SEPSIS By Matthew J. Delano May 2008 Chair: Reuben Ramphal Major: Medical Sciences-Im munology and Microbiology Sepsis is defined as bacterial infectio n accompanied by an overwhelming systemic inflammatory response that resu lts in both innate and adaptive immune system dysfunction. Specifically, polymicrobial sepsis alters the adaptive immune response, and induces T-cell suppression and TH2 immune polarization. In this disser tation, we identified a population of GR1+CD11b+ cells whose numbers dramatically increase and remain elevated in the spleen, lymph nodes and bone marrow during polymicrobial sepsis. Phenotypically, these cells are heterogeneous, immature, predominantly my eloid progenitors that express IL-10, TNF MCP-1 and a number of other cytokines and chemokines. Splenic GR-1+ cells effectively suppress antigen-specific CD8+ T-cell interferonproduction, but only modestly suppress antigenspecific and nonspecific CD4+ T-cell proliferation. GR-1+ cell depletion in vivo prevents both the sepsis-induced augmentation of TH2-dependent and depression of TH1-dependent antibody production. Signaling through MyD88 and the SDF-1 pathways, but not TLR4, TRIF, the IFN / receptor, CCR2 receptor, IL-10, IL-4 or th e M-CSF pathways are required for complete GR-1+CD11b+ expansion. GR-1+CD11b+ cells contribute to seps is-induced T-cell suppression and preferential TH2 polarization.

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CHAPTER 1 INTRODUCTION Sepsis Significance Sepsis occurs when an overwhelming microbial infection leads to a systemic inflammatory response, manifesting clinically as fever, leukocytosis, increased car diac output, and reduced peripheral vascular resistance, leading to multi-system organ failure and death. Despite progress in antibiotic administration, ve ntilator management, and flui d resuscitation over the past 20 years, sepsis remains the leading cause of deat h in the intensive care with over 750,000 cases and 210,000 deaths annually in the United States ( 1, 2). Recently, significant advancements in understanding sepsis pathophysiology have occurr ed with a better appreciation of inflammation science and the innate immune system (3, 4). Un fortunately, our scientific understanding has had little impact on the mortality rate from severe sepsis (1, 2), with the sepsis incidence compounded by the ever increasing elderly U.S. population. A multitude of proposed approaches, including antitumor necrosis factor(TNF) therapies, corticosteroids, antibodies against endotoxin, inhibitors of prostaglandins, bradykini ns, PAF, and interleukin (IL)-1 receptor antagonist, have all failed during c linical trials (5). There are on ly partially efficacious sepsisrelated therapies currently availabl e, including activated protein C (6), replacement steroids for sepsis-associated adrenal insuffi ciency (7), and insulin thera py for blood glucose maintenance (8). However, as monotherapy or in combinati on, these approaches still only modestly improve sepsis survival (5). Furthermore most labo ratory work investigating these early antiinflammatory therapies were based on intravenously or intraperitoneally administered bacteria or endotoxin, or pretreatment prophylactic approaches which did not replicate the septic patients pathophysiology or the disease process adequately (9, 10) For the aforementioned reasons, many investigators have focused recent attention on experimental models of sepsis that modulate 15

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cellular apoptosis and effector cell populations of both the inna te and adaptive immune systems during sepsis to produce in animal models what is observe d in human sepsis. Sepsis, the Cecal Ligation and Puncture (CLP) Model, and Immune Dysfunction For years, investigators have observed that severe seps is produces a state immune suppression illustrated by a loss of delayed type hypersensitivity (11), an inability to eradicate primary infections (12), a predisposition to develop secondary nosocomial infections(12, 13), and a failure to respond to skin testing with specific antigens from microbes to which they were already exposed and had tested positive (11, 14). Furthermore, anim al models of sepsis indicate that immune dysfunction is crucial to the pathogenesis of seps is with a plethora of immune responses intertwining both the innate and adap tive immune systems (15-17). Loss of MHC II expression (18), defects in antigen presentati on (19), apoptosis induc ed depletion of CD4+ T cells (20), dendritic cell apopt osis (21), dendritic cell exha ustion of paralysis (22, 23), suppression of T-cell proliferative responses (24-27), re duced inflammatory and TH1 cytokine production by monocytes and tissue macropha ges (28), all contri bute to immunologic compromise during sepsis and culminate in a shift from the proinflammatory TH1 to the antiinflammatory TH2 immune profile (29-32). Moreover, recent attention has focused on the TH1 to TH2 immune profile shift as an explanati on for post-sepsis immune suppression (33-35); however, the underlying mechanisms that orches trate the shift in imm une polarization during sepsis are still unknown. With the failure of therapies in human sepsis, further research has resulted in a better understanding of the immune dysfunction during sepsis and the development of more appropriate murine models to better mimic human sepsis. Many of these findings can be recapitulated using the murine cecal ligation and puncture model (C LP) of sepsis (21, 36-39). Although the CLP model is valuable and replicates most of the ma nifestations of human sepsis, several limitations 16

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to this approach remain. First, it is difficult to hemodynamically monito r mice. Second, although acute fluid resuscitation is provided, actual fluid requirements in itially and chronically are not easily measurable or administratable. Furtherm ore, the pathophysiologic changes that occur in human sepsis occur in a more abbreviated tim e frame compared to human sepsis. Lastly, antibiotic administration varies among investigators and is not tailored to target the specific infective microbe, but is usually a broad sp ectrum antibiotic administered one time (39). Although there are some inherent differences in human sepsis and experimental murine sepsis from a CLP model, the similarities are profound and generally characterized by two distinct immune phases that occur either concomita ntly or sequentially (40). The early phase to a CLP is characterized by a hyperdynamic state with elevated cardiac output, tissue perfusion, and decreased vascular resistance. The hallmark of the early phase is inflammation mediated by neutrophils, macrophages, dendritic cells and monocytes, stimulated by microbes and/or their toxins. The second phase is characterized by a hypodynamic response beginning between 12-24 hrs after CLP and includes d ecreased macrovascular and micr ovascular blood flow, decreased cardiac function and output, and increased or gan injury and dysfunction. Along with the physiologic and inflammatory alterations observe d during the first 24 hours of sepsis,there is concomitant adaptive immune system dysfunction,where the adaptive immune system exhibits defective antigen presentation, decreased major histocompatibility complex type II (MHC II) expression, loss of phagocytic function, decreased TH1 CD4+ lymphocyte cytokine production and decreased TH1 proliferative response to exogenous mitogens (41). However, in the face of this TH1 immune dampening, there is a concomitant expansion of TH2 CD4+ lymphocyte cytokine production, expansi on in regulatory T cell populati ons (42, 43), immature myeloid populations, and an increased susceptibility to s econdary infections (3, 4). Insights into the 17

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specific immune modulatory cell populations have in promo ting sepsis induced immune suppression will help us understand sepsis syndromes. Myeloid Derived Suppressor Cells (MDSCs) and Disease Currently little is known about the origins and function of the MDSC population. The cancer literature describes them as a heteroge nous, immature population of the myeloid lineage derived from stem cells and bone marrow progenito rs. This population has been referred to as: natural suppressor cells(44), myeloid de rived suppressor cells( 45), early myeloid cells(46), and inhibitory macrophages(47) in the past deca des and now the population goes by the name of myeloid derived suppressor cells. Phenotypically, these cells exhibit a high expression of cell surface markers CD11b and GR-1. However, other cells of myeloid lineage can express low levels of these receptors su ch as macrophages and neutrophils. Other cell surface markers including CD115, CD31, CD34, c-K it, and F4/80 may also be displayed. Recent studies suggest that the accumulati on of MDSCs in bone marrow, spleen and lymph nodes is a conserved response to an array of disparate insults (48-50) which may explain the altered immune reactivity associated with these insults. Bront e and colleagues have postulated that MDSCs play an important role inhibiting T cell activati on during the resolution phase of an inflammatory insult or an immune response (47, 48, 51). Several cytokines, such as GM-CSF, CSF-1, IL-6, IL-10 and VEGF have been shown to regulate the expansion of MDSC populations (48, 52). Because of their relative immature and undiffe rentiated phenotype, there is considerable functional variability among thes e cells with a suppressive ph enotype elicited upon exposure to TH2 cytokines (IL-4, IL-10 and TGF ) often increased in sepsis (47, 53). On the other hand exposure of MDSCs to TH1 cytokines (TNF ) stimulates differentiation along macrophage pathways, and enhances T cell cytotoxic respon ses. For example when MDSCs obtained from 18

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tumor bearing mice were administered into healt hy nave animals, they differentiated into normal dendritic cells and macrophages, but when administered into othe r tumor bearing animals, they maintained their suppressor cell phenotype (54). Most of the information on the MDSC popula tions stems from the oncology literature, using human and murine tumors that result in MDSC expansion (49, 55-57). In mouse tumor models, immune suppression to growing tumors could be ameliorated by GR-1+CD11b+ cell depletion with all trans retinoic acid restoring T-cell respons es (58, 59). MDSCs can inhibit T cell activation through cell-cell co ntact or immediate juxtapositi on. The mechanisms of MDSC T-lymphocyte inhibition are not yet fully unders tood; however, they appear to depend on Larginine metabolism to decrease T lymphocyte re sponsiveness to subsequent antigen stimulation (48, 49). Furthermore induction of iNOS with NO release an d peroxynitrites formation only accounts for some of the T cell unresponsiveness in tumor models (60-62). Over production of arginase I by MDSCs can result in local argi nine starvation that can inhibit T-lymphocyte proliferation (63, 64). In addition GR-1+ cells also secrete IL-4 and IL-10 (65), reactive oxygen species, and TGF all of which can have im munosuppressive properties. Myeloid Derived Suppressor Cells Function During Inflammation The role that MDSCs play in acute inflammation has undergone little investigation. Many of the proinflammatory cytokines, factors and mediators that ar e produced during acute inflammation could play a role on the development of MDSCs and their immune modulatory phenotype. G-CSF, GM-CSF, CSF-1, IL-4, IL-6, a nd IL-10 are just a few of the mediators; however, few investigators have studied th e MDSC populations during sepsis. Holda and colleagues demonstrated that small injections of lipopolysaccharide markedly enhanced what he termed natural suppressor cell activity (66). Murphey, Sherw ood and colleagues reported a two19

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fold increase in natural suppressor cells in m ouse spleens five days after CLP (67). Ochoa and colleagues reported a seven fold increase in the GR-1+CD11b+ cell population in the spleens of mice 12 hours following operative trauma (68), and demonstrated that the MDSCs suppress Tcell proliferation to anti-CD 3, anti-CD28 stimulation through depletion of the amino acid, Larginine. These recent studies explain some of the earlier findings that arginase activity is increased in myeloid cells after trauma (69-72), and its increase is modulated by TH2 cytokines often increased in trauma (73). Hypothesis Although the role of MDSCs in tumor-indu ced immune suppression is evolving, the contribution of GR-1+CD11b+ MDSCs to the development of im mune suppression in sepsis is currently unknown. We propose that expansion of the MDSC populations in the secondary lymphoid organs during sepsis contributes to th e adaptive immune system defects observed in sepsis, and also serves as a ta rget for therapeutic in tervention. Preliminary research shows that MDSCs serve disparate immune modulatory role s in acute infection and tumor-associated inflammation. MDSCs simultaneously promote antigen specific T effector cell tolerance, while concomitantly modulating B cell antibody produc tion culminating in a shift from a TH1 to TH2 type immune profile. Therefor e, our overarching hypothesis is that MDSCs play simultaneous and competing roles in sepsis where the host utilizes their suppressive potential to dampen the magnitude of the inflammatory response facilitating an an ti-inflammatory TH2 immune polarization; however, accumulation or expansio n of these populations may also lead to a compensatory anti-inflammatory immune suppre ssion and increase the hos t susceptibility to secondary infections. 20

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Specific Aim 1 To characterize the biological impact of MDSCs during a prolonged model of polymicrobial sepsis and whether the expans ion of MDSC populati ons in the secondary lymphoid organs is dependent upon a sustained micr obial infection as seen in sepsis. The goal was to determine whether a polymicrobial infection and an accompanying systemic inflammatory response provide the proper milieu to facilitate the expansion of the MDSC population in the spleen, lymph nodes, a nd bone marrow. Mice underwent a prolonged infectious sepsis stim ulus (sublethal CLP, LD20) and the MDSC numbers and activity were determined in a time course fashion at various intervals up to 16 week s. MDSC numbers (GR1+CD11b+) were determined and phe notypically characterized by co-staining with various myeloid markers and analyzed using fl ow cytometry and histologic sectioning. Specific Aim 2 The goal of this aim was to determine the effect of MDSC expansion on the adaptive immune system in a prolonged model of polymicr obial sepsis. The suppr essor cell function was evaluated by cocu lturing the MDSCs in vivo with T-cell receptor specific CD8+ lymphocytes and measuring the T lymphocyte specific responses to antigen specific prolif erative signals. Since the MDSCs must be in physical proximity with other cells to impart their immunosuppressive properties (56, 74, 75), and are known to secrete IL-4, IL-10 (65) and nitric oxide (NO) (49), we tested whether these mediators contribute dir ectly to the MDSC suppression in sepsis by generating MDSCs from IL-4, IL-10 and iNOS (NOS2) null animals. Specific Aim 3 The goal in this aim was to confirm the si gnaling pathways that are required for the expansion of the MDSC populations in polymicrobial sepsis. Our preliminary data suggests that MyD88 null animals fail to undergo peripheral expa nsion of their MDSC populations in sepsis; 21

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22 however, the role that MyD88 si gnaling is playing is unclear. We demonstrate that MyD88 null mice experience the same fluctuations in bone marrow stem cells and myeloid progenitors as wild-type mice at both 1 and 7 days after seps is distinguishing the MyD88 induced signaling defects in the bone marrow from any effects on peripheral MDSC expansion. Since the absence of MyD88 signaling only delaye d, and not prohibited, the ultimate expansion of the splenic MDSC population, we attempted to investigate ot her signaling pathways that are involved in myeloid cells proliferation and differentiation during chronic sepsis. We found that signaling through MyD88 and the SDF-1 pathways, but not TLR4, TRIF, IFN / receptor, CCR2 receptor, IL-10, IL-4 or the M-CSF pathways are required for complete GR-1+CD11b+ expansion.

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CHAPTER 2 EXPANSION OF AN IMMATURE GR-1+CD11B+ POPULATION INDUCES T-CELL SUPPRESSION AND TH2 POLARIZATION IN SEPSIS Specific Aim 1 As discussed earlier, we set out in Specific Aim 1 to character ize the biological impact of MDSCs during a model of prolonged polymicrobial sepsis and whether the expansion of MDSC populations in the secondary lymphoid organs is dependent upon a sustained microbial infection as seen in sepsis. The goal was to determ ine whether a polymicrobial infection and an accompanying systemic inflammatory response provide the proper milieu to facilitate the expansion of the MDSC population in the sp leen, lymph nodes, and bone marrow. The proceeding experimental design was developed to achieve the goals set forth in Specific Aim 1. Introduction Sepsis is the systemic inflammatory response to severe microbial infection. It is well recognized that patients with sepsis are often immune suppressed, as il lustrated by failure to eradicate their primary infections a predisposition to develop secondary nosocomial infections, and an attenuated delayed type hypersensitivity response (4, 76). Animals with polymicrobial sepsis also exhibit widespread dysfunction in both antigen-presenting cells and T-lymphocytes. Reduced CD4+ T-cell numbers due to apoptosis-induced depletion (20, 77) and a suppression of T-cell proliferative responses (43) have been shown to contribute to sepsis-associated morbidity. Moreover, interest has focused on the shift from a TH1 to a TH2 profile as contributing to sepsisassociated immune dysfunction (78, 79). The role that suppressor cell populations play in polymicrobi al sepsis is unknown. The present report examined the ro le of myeloid derived suppresso r cells in sepsis, and their contribution to the sepsis-induced defects in acquired immunity. Myel oid derived suppressor cells with suppressor functions have been previo usly observed in the sple ens and tumors of mice 23

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with transplantable tumors (48, 80) and in models of chronic inflammation (50). In tumorbearing mice, these cells contribute to the tu mor-associated antigen specific T-cell dysfunction and tolerance (54, 59, 74, 80-82). Splenic GR-1+CD11b+ cells may also play an instrumental role in priming of B ce ll antibody production (65). However, there has been only modest explorat ion of these cell populations in sepsis or other acute inflammatory processe s. Here, we observed that an ongoing septic process induces a dramatic expansion of the GR-1+CD11b+ population in bone marrow, spleen and lymph nodes. The splenic infiltration with th ese cells was associated with splenic enlargement and lymphoid follicle disruption. The GR-1+CD11b+ cells contained immature progenitors and expressed IL10, TNF and other cytokines and chemokines. Furt hermore, using a depleting antibody, we demonstrated that expansion of GR-1+ cells in vivo contributed to the induced TH2 polarization of antibody responses following sepsis. These cells were also capable of causing CD8+ T-cell tolerance, as demonstrated by the suppr ession of antigen specific interferon(IFN) production by CD8+ T-lymphocytes in non-septic immunized mice. Finally, we observed that signaling through MyD88, but not TLR4, TRIF, or the IFN / receptor, was required for the early and full expansion of this cell population, highlight ing the importance of inflammation and TLR signaling other than that induced by microbial endotoxin in the regulati on of myeloid derived suppressor cells in sepsis. Results GR-1+CD11b+ Cells Accumulate in th e Spleen after Sepsis To examine the long-term effects of polymic robial sepsis on the expansion of the GR1+CD11b+ populations, the studies were conducted in a murine model of polymicrobial sepsis (generalized peritonitis) induced by ligation of the cecum and a double enterotomy created with a 27 gauge needle. Mortality in this model was approximately 10-20%, and occurred 24

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predominantly in the first 96 hours; thereafter, surviving mice developed abscesses surrounding the devitalized cecum. As shown in Figure 2-1, th e presence of sepsis was confirmed for at least ten days by a transient bacteremia (lasting 24 ho urs) and prolonged bacterial contamination of the peritoneal cavity. The animals exhibite d a significant early leukopenia, followed by a profound granulocytosis, Figure 2-2. Plasma cytokine concentrations over the first ten days were consistent with an early exaggerated system ic inflammatory response, and by sustained elevations in the plasma IL-6, KC and MIP-1 concentrations, Figure 2-3. Interestingly, surviving septic mice develope d a dramatic splenomegaly with the spleen mass increasing by 300% 10 days afte r initiation of polymicrobial sepsis, Figure 2-4, panel A. The dramatic increase in spleen mass suggested an expansion of one or more cell populations within the spleen. Periodic histological analys is of the spleens of mi ce over 10 days of sepsis, Figure 2-4 panel B, demonstrated that the apparent splenomegaly was associated with extramedullary hematopoeisis and marked e xpansion of immature myelomonocytic cells, including forms with ringed nuclei in the periarte riolar sheaths and subc apsular space, with the focal involution of lymphoid follicles. Analysis of lymphoid and nonlymphoid cell populations did not reveal an increase in the number of cells expressing CD3, B220, or CD11c. However, a dramatic accumulation of cells expressing GR-1 and CD11b occurred. As shown in Figure 2-5, splenocytes harvested from septic mice at various intervals after cecal ligation a nd puncture, or sham procedures, exhibited a striking increase in the percentage and absolute number of GR1+CD11b+ cells (Panels A, and B). The dramatic increases in the percentage and absolute numbers of GR-1+CD11b+ cells did not occur until at least three days after the induction of sepsis, and the percentages continued to incr ease to a plateau, until about 7-10 days. The numbers and proportion of these cells remained elev ated even out to 12 weeks in surviving mice. 25

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By ten days, the absolute numbers of these GR-1+CD11b+ cells in the spleen had increased 50fold. GR-1+CD11b+ Cells are Phenotypica lly, Heterogenous Cells GR-1+CD11b+ cells represent a heterogenous populati on of cells encompassing mature and immature myeloid forms. To further characterize these GR-1+CD11b+ cells, splenocytes were also stained for CD31, a marker of immature my eloid development that is lost with more terminal cell differentiation (83), and Ter119, a ma rker of erythroid lineag e, as well as F4/80, a marker for myeloid lineage development. As shown in Figure 2-5, panel C, approximately 40% of the GR-1+CD11b+ cells were also CD31 positive, and the numbers of GR-1+CD11b+CD31+ cells were increased nearly 70-fold during sepsis. Similar results were obtained examining the GR-1+CD11b+F4/80+ triple positive cells in the spleen and bone marrow ( data not shown ), suggesting that the GR-1+ cells contained a subpopulation of developing myeloid cells. In contrast, only 6% of the GR-1+CD11b+ positive cells were Ter119+, suggesting that only a small proportion of these cells may still possess the ability to differentiate into cells of erythroid lineage, Figure 2-6. Interestingly, ho wever, less than 3% of these GR-1+CD11b+ cells were also MHC II+, Figure 2-7. As shown in Figure 2-8, pa nel D, the enriched GR-1+ cell population from the spleens of septic mice contained large numbers of phenotypical ly heterogeneous cells. Many of these cells had characteristic circular or 'ringed-shaped' nuclei. Gauging on their nuc lear size, complexity, and cytoplasmic granularity as described previously in the literature, most of these cells were determined to be immature myeloid forms (18). These cells were identified on the cell-sorted cytospin preparations. To examine whether the GR-1+CD11b+ population were an immature proliferating precursor population sensitive to myeloid growth factors, the GR-1+ enriched splenocytes from 26

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septic mice were cultured ex vivo on 24 well-plat es with GM-CSF. In the absence of GM-CSF, these cells rapidly died. In c ontrast, seven days of culture w ith GM-CSF led to approximately 17% of these cells differen tiating into conventional CD11chighMHCIIhigh dendritic cells and approximately 22% differentiating into F4/80high macrophages (Figure 2-8 Panels A, B and C) and all of these cells demons trated increased MHCII expression over freshly isolated Gr1+CD11b+ cells. In addition, culturing these GR-1+ cells from septic mice in soft methylcellulose with either GM-CSF or G-CSF, but not erythropoietin, for seven days led to a significant increase in the numbers of colonies fo rmed (Figure 2-8 Panel E). Interestingly, the GR-1+ cells from sham-treated mice did not co ntain any significant number of progenitors capable of forming colonies in response to these growth factors. The failure of GR-1+ cells from septic mice to generate a significant number of colonies in re sponse to erythropoietin, and the low Ter119+ staining, suggest that during sepsis these expanded numbers of GR-1+CD11b+ cells represent a mixed population of immature, prolif erating, progenitors committed predominantly to a myeloid, and not an erythroid, pathway. Myeloid derived suppressor cells Accumulate in Secondary Ly mphoid Organs after Sepsis. We also looked for these GR-1+CD11b+ cells in other secondary lymphoid and reticuloendothelial organs. For these cross-sectio nal analyses, we selected intervals after cecal ligation and puncture or a sham procedure when th e changes in the spleen were maximal. No significant increases in GR-1+CD11b+ cells were seen in ei ther the liver or lung ( data not shown). In peripheral lymph nodes, however, marked increases in the percentage and numbers of these GR-1+CD11b+ cells were evident at 10-14 days afte r sepsis, and were still increased at 12 weeks (Figure 2-9, Panel B and C). Numbers remained significantly elevated in mesenteric lymph nodes in direct proximity to the cecal liga tion and puncture at 12 weeks, whereas numbers declined somewhat, but still remained significantly elevated, in more distal inguinal and axillary 27

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lymph nodes at 12 weeks. Furthermore, in the bone marrow (Figure 2-9, Panel A), the numbers of these GR-1+CD11b+ cells doubled within th ree days, and by seven days after sepsis, accounted for nearly 90% of th e cells in the bone marrow. Histological confirmation of thes e cells in the spleens of mice with severe sepsis is shown in Figure 2-10 panels A, B, and C. Over the co urse of 10 days, there was progressive expansion of the red pulp by extramedulla ry hematopoiesis and marked e xpansion of the periarteriolar sheaths (Figure 2-10, Panel B) and subcapsu lar space (Figure 2-10, Panel C) by immature mononuclear cells and myelomonocytic cells with ringed nuclei. These cells were also found in small clusters within the interfollicular areas. The myeloid derived suppressor cells with ringed nuclei in all of these loca tions were uniformly CD11b+ in the spleens of mice that underwent cecal ligation and puncture (Figure 2-10 Panel D, E, and G). In contrast, CD11b showed only scattered reactivity in the sham treated mice, mainly highlighting mature granulocytes within the interfollicular areas (Figure 2-10, Panel E). The increasing numbers and overall percentage of immature myelomonocytic cells w ith ringed nuclei correlated w ith the time progression after sepsis and was associated with cuffing in the pe rivascular/periarteriolar sheaths and subcapsular spaces. Finally, there was focal involution of the lymphoid follicles that paralleled the expansion of the red pulp. No immature my elomonocytic cells with ringed nucle i were identified within the follicular areas. Myeloid derived suppressor cells are Capable of Inflammatory Mediator Production Since myeloid derived suppressor cells obtaine d from tumor bearing hosts are known to be immunomodulatory (48, 80), we next examined wh ether cells obtained from septic mice could produce immunosuppressive and inflammatory medi ators, including IL-10, a cytokine generally regarded as a requisite for T-cell suppression and TH2 polarization during sepsis. When GR-1+ enriched splenocytes from septic mice were cultured ex vivo they produced low levels of several 28

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inflammatory mediators, including IL-10, TNF, RANTES (CCL5) and MIP-1 (CCL4). When stimulated ex vivo with bacter ial lipopolysaccharide, the GR-1+ cells from septic mice produced significantly greater amounts (>5 fo ld) of IL-10 than similar GR-1+ cells from sham-treated animals (Figure 2-11). They also produced increased quantities of TNF, RANTES and MIP1 but did not produce increas ed quantities of other TH2 cytokines IL-4 or IL-13. Unstimulated or lipopolysaccharide stimulated GR-1+ splenocytes also did not produce measurable quantities of GM-CSF, IL-12p40, IL-12p70, IL-2, IL3, IL-5, IL-9, IL-17, VEGF or IFN( data not shown). In addition, GR-1+ splenocytes obtained from septic mice also produced TNF (218 67 vs 4 1 pgs/ml) and IL-10 (165 43 pgs/ml) in response to stimulation with 5 g/ml flagellin (TLR5 agonist), albeit in lesser quantities than with LPS stimulation. Myeloid Derived Suppressor Cells Affect Ad aptive Immune Responses In Sepsis To examine whether these cells could affect adaptive immune func tion, sham-treated and septic mice were immunized with NP-KLH using the adjuvant alum when GR-1+ cells reached a maximal proportion in the spleen (ten days fo llowing cecal ligation and puncture), and the NP specific serum immunoglobulin re sponse determined ten days later. The serum immunoglobulin response to NP-KLH with alum is a T-cell dependent, B-cell re sponse, and the immunoglobulin isotypes can be used to assess polar ization of the T-cell response ( 19). Polymicrobial sepsis was not associated with any significan t changes in total IgM or IgG responses (Figure 2-12, Panel A and B); however, when the total IgG response was dissected into its isot ypic components, the serum IgG2a response was significantly decreased while the IgG1 response increased in the septic mice, consistent with a shift from a TH1 to a TH2 T-cell response (Figure 2-12 Panel C and D). When the septic mice were treated with a GR -1 depleting antibody, producing a greater than 80% reduction in total GR-1+CD11b+ splenocytes (Figure 2-13), th e sepsis-induced increase in IgG1 and the decrease in IgG2a responses were abolished, demonstrating the involvement of the 29

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GR-1+ cells in this polarization. As expected by the T-cell depe ndent nature of this antibody response, depletion of CD4+ cells significantly attenuated the IgM responses and completely prevented the IgG class switching in the septic animals (Figure 2-14, Panels A, B, C, and D). To further confirm the in vivo role that thes e cells play in suppressing an antigenic T-cell response, the effect of GR-1+ cells on the CD8+ T-cell IFNresponse by splenocytes from OT-I TCR-transgenic mice (C57BL/6-Tg(TCR TCR )1100mjb) immunized with OVA-derived peptide (H-2Kb restricted, aa 257, SIINFEKL) was examined (Figure 2-15 Panel A). GR1+ cells were obtained from either ten day septic or sham-treated mice, and were infused into C57BL/6 mice that had previously received CD8+ T-cells from OT-1 TCR-transgenic mice, and simultaneously immunized with OVA-derived specific peptide. Ten days later, the spleens from these animals were removed, and IFNresponses to ex vivo stimulation with OVA-derived specific peptide were examined. IFNproduction was markedly reduced when the animals were administered GR-1+ splenocytes from septic animals when compared to sham-treated mice, confirming that GR-1+ splenocytes from these septic mice could suppress a CD8+ T-cell IFNresponse. To determine whether GR-1+ cells could directly suppre ss an antigen specific or nonspecific CD4+ T cell proliferative response, D011.10 OVA-TCR transgenic mice were made septic and at 10 days, CD4+ splenocytes from septic mice were cultured with irradiated GR-1+ containing antigen presenting cells from the spleen s of 10 day septic, sham-treated, and control mice, and were incubated with either OVA pe ptide, bovine serum albumin or on CD3/CD28 coated plates. As shown in Figure 2-15, Panel B, culturing CD4+ cells with irradiated GR-1+ cells from septic mice only modestly, but still significantly, reduced both the antigen-specific (OVA) and non-specific (CD3/CD2 8) proliferative responses. 30

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MyD88 Signaling Pathways Are Required for GR-1+CD11b+ Cells to Accumulate in the Spleen after Polymicrobial Sepsis. Polymicrobial sepsis produced by cecal liga tion and puncture releases a large number of microbial products that are recognized by the inna te immune system, in large part through tolllike receptor signaling pathways. To examine the cell signaling pathways required to elicit the expansion of these immunomodulatory GR-1+CD11b+ cells, wild-type C57BL/6 mice were first injected with a sublethal dose (5 mg/kg BW) of the TLR4 agonist, bacterial lipopolysaccharide. The numbers of these GR-1+CD11b+ splenocytes were examined at daily intervals after lipopolysaccharide injection. As shown in Figure 2-16, panel A, the administration of bacterial lipopolysaccharide produced a more rapid, but transient increase in the percentage and absolute numbers of GR-1+CD11b+ splenocytes suggesting the likely pathway involves TLR4 signaling. The expansion of GR-1+CD11b+ splenocytes by lipopolysacchari de occurred by 24 hours, reached a peak by five days, and declined thereafter until it reached a near-baseline level by day 10. Interestingly, the increased numbers of splenic GR-1+CD11b+ cells were very modest compared to mice undergoing a cecal ligation and puncture, and even the sham procedure produced some modest increase in GR-1+CD11b+ cell numbers. To determine whether TLR4 signaling is a requirement for the expansion of these cell populations in our model of polymicrobial seps is, cecal ligation and puncture was performed in C3H/HeJ mice that have a spontaneous mutation in TLR4, and the results were compared to C3H/OuJ mice. As shown in Figure 2-16, Panel B, TLR4 mutant C3H/HeJ mice had a comparable increase in splenic GR-1+CD11b+ cell numbers as in the C3H/OuJ controls at seven days, suggesting that intact TLR4 signaling is not required for th e early expansion of these GR1+CD11b+ cells in sepsis. Thus, although lipopolys accharide signaling via TLR4 can induce an 31

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expansion of this GR-1+CD11b+ cell population, TLR4 signali ng is not required during polymicrobial sepsis, and there are redundant signaling pathways. Such findings are not completely unexpected since polymicrobial sepsis is generally associated with the release of large numbers of different mi crobial products that can signal simultaneously through a number of different TLR receptors. Since TLR signaling occurs through MyD88and TRIF-dependent pathways, and may involve the secretion of type I interferons, cecal ligation and puncture was also performed in MyD88-/-, TRIF-/and IFN / R-/mice. The increased expansion of GR-1+CD11b+ cells after cecal ligation and puncture at seven days was markedly attenuated in only the MyD88-/(B6x129) mice and not in either the TRIF-/or IFN / R-/mice, highlighting the requirement of My D88 signaling for the early expansion of this cell population during sepsis (Figure 2-16, Panel C, D and E). To confirm that the requirement for MyD88 signaling was not dependent upon the background of the animals, and was sustained duri ng prolonged sepsis, the studies were repeated in MyD88-/animals backcrossed onto a C57BL/6 b ackground. As shown in Figure 2-17, after seven days of sepsis, there wa s again no expansion of the GR-1+CD11b+ population in the MyD88-/(B6) mice. By 14 days, there was some expansion of the GR-1+CD11b+ cell populations, but it was still markedly attenuated wh en compared to the wild-type B6 controls. Discussion T-cell dysfunction is a common response to polymic robial sepsis (4), ultimately leading to increased susceptibility to ongoing and opportu nistic infections, and poor outcome. Recent attention has focused on the shift from a more proinflammatory TH1 to a more anti-inflammatory TH2 immune profile as an explanation for pos t-sepsis immune suppression; however, the underlying mechanisms that orchestrate this T-cell suppression and TH2 polarization during sepsis are still unknown. 32

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Although there has been some speculation that endogenous and inducible T-regulatory cells may contribute to the T-cell suppression and TH2 polarization in sepsis (42, 84), more recent studies have refuted some of those cl aims (85, 86). Several years ago, Murphey and colleagues observed an increased number of macrophage-like cells in the spleens of mice surviving a cecal ligation and puncture (67), alt hough they did not explore their suppressor cell function. More recently, Makarenkova and coll eagues observed increased numbers of myeloid derived suppressor cells in the spleens of mice within 12 hour s of a traumatic injury, and identified them as a source of arginase I activity (87). Using a model of polymicrobial sepsis that produces only limited early mortality but sustained inflammation as determined by elev ated plasma cytokine concentrations and neutrophilia, we observed a pr ofound splenomegaly that persis ted for weeks in surviving animals. On closer examination, it became evid ent that associated with this ongoing septic process, there was marked disintegration of th e follicular regions, increased extramedullary hematopoiesis, and replacement of the splenic cellularity with large numbers of myeloid derived suppressor cells (Figure 2-5). Hotchkiss and others, including ourselves, had previously shown that in similar models of polymicrobial se psis, there is a rapid apoptotic loss of CD4+ T-cells and dendritic cells in the first 24 hours of sepsis, a nd these cellular losses contribute to the adverse outcome (17, 21, 88). Although there has been co nsiderable explorati on into the role of extramedullary hematopoiesis in chronic infect ious and inflammatory processes, none have explored or even described this massive expa nsion of an immature myeloid cell population (GR1+CD11b+CD31+) in the bone marrow, spleen and lymph nodes of mice with ongoing septic processes. We have clearly shown that by ten days after cecal ligation and puncture, almost 40% of the spleen and 90% of the bone marro w cellularity repres ent immature GR-1+CD11b+ cells. 33

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Interestingly, the administration of near lethal doses of bacterial lipopolysaccharide could also produce some expansion of this immature myeloi d cell population. Howeve r, the increases were transient and modest compared to that seen in the septic an imals, suggesting that an ongoing inflammatory process may be required for co mplete manifestation of the response (50). The phenotype of these cells, the kinetics of their expansion, a nd their anatomical location in the spleen argue agai nst these cells being functionally or phenotypically similar to those reported previously by Makarenkova and coll eagues (68) immediately after trauma, or as merely components of extramedullary hemat opoiesis. Makarenkova et al. observed a rapid influx of GR1+CD11b+ cells into the spleens of mice 12-24 hours after traumatic injury. However these cells, which produced large quantities of arginase I, were located in the mantle surrounding the lymphocyte rich follicles, and very fe w ring cells were detect ed. In contrast, we saw a transient decline in GR1+CD11b+ splenocytes in the first 24 hours of sepsis, and only saw expansion of our splenocyte populat ion after three to five days of sepsis. Furthermore, although both populations are clearly hete rogenous, our cells contained hi gher proportions of immature precursors that were concentrated in perivascular/periarteriolar and subcapsular regions of the spleen, exhibited less MHC class II expression, and made copious amounts of IL-10 when stimulated ex vivo. These cells obtained from the spleens of septic animals contained predominantly precursors committed to a myeloid and not erythroid lineage. Less than 6% of the GR-1+CD11b+ cells recovered from the spleens of septic mice were Ter119+ positive, and when cultured in soft methylcellulose with erythropo ietin, had only a minimal capacity for colony or burst formation (Figure 2-6). In contrast, almost 40% of the cells differentiated into macrophages or dendritic cells when cultured with GM-CSF (Figure 2-8 panel C), and large 34

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numbers were capable of forming colonies when in cubated in soft methylcellulose with either GCSF or GM-CSF (Figure 2-8 panel E). This dramatic expansion of the GR-1+CD11b+ response to sepsis in the spleen is very similar to the response previously observed in mice with actively grow ing tumors. Similar immature myeloid cell populations have been sh own to accumulate in th e spleens, infiltrating into tumors in several animal tumor models (54, 55, 74, 81, 82, 89), and also in the blood of some patients with cancer (90). In mice with transplantable tumors, these myeloid-derived suppressor cells, as they are now being termed ( 91), have also been shown to inhibit antigenspecific and nonspecific T-cell func tions via several different mech anisms, including arginase I, nitric oxide, reactive oxygen species, and TGF Given the abilities of my eloid derived suppressor cells to facilitate immune suppression in murine cancer models, as well as to suppress antigen-specific T cell responses and to influence B cell antibody production, the expa nsion of these immature myeloi d populations in sepsis may similarly orchestrate the TH1 to TH2 immune polarization that is known to occur in sepsis. Challenging mice with T-cell depe ndent antigens, such as NP-KLH, offers the opportunity to explore in vivo the shift in an tibody class switching from IgG2a to IgG1 production, which is dependent upon cytokines including IFN and IL-4, and reflects this predilection towards a TH2 versus a TH1 CD4+ T-cell response (65, 92). Ten days after sepsis, immunization with NP-KLH led to an increase in the IgG1 production at the expense of IgG2a, consistent with a preferential TH2 response. In contrast, partial depletion of the GR-1+ cells in vivo blocked the characteristic increase in the IgG1 secretion while also preventing the fall in the IgG2a responses, demonstrating a contributory role for these GR-1+ cells in this shift from a TH1 to TH2 response. 35

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Consistent with these findings was the observation that GR-1+ cells from septic mice significantly attenuated the IFNresponse by CD8+ T-cells to specific antigenic stimulation. These results are very similar to the suppression of cytotoxi c T-cells to antigen-specific stimulation by GR-1+ cells obtained from tumor-bearing hos ts (81). Surprising, however, were the only modest reductions in antigen-specific CD4+ T-cell proliferation that we saw when GR1+ cells were administered to DO11.10 CD4+ OVA transgenic mice immunized with OVA peptide. The answer may simply be that these GR-1+ cells express very little MHC class II, although they retain relatively high levels of MHC class I expr ession (81). Therefore, in the DO11.10 CD4+ OVA transgenic mouse model, these GR-1+ cells were probably unable to present antigen via MHC class II, and ther efore, could not di rectly affect CD4+ T-cell proliferative responses. In contrast, they c ould present antigen in the context of class I expression and suppress CD8+ T-cell IFNresponses. Interestingly, the numbers of myeloid de rived suppressor cells decreased in mice in which the abscess had spontaneous ly resolved 12 weeks after seps is. Furthermore, when these same animals with abscess resolution were immunized with NP-KLH and alum, the antibody response more closely approximated baseline isot ype levels Figure 2-18. Similar decreases in the numbers of myeloid derived suppressor cells have been reported in tumor bearing animals upon resection of their primary tumor (93). The similarities in the appearance of these my eloid-derived suppressor cells in sepsis, and in animals with tumors or other chronic infla mmatory processes, suggest that there are common signals involved in the expansion of these cell po pulations. Polymicrobial sepsis produced by a ligation of the cecum and expression of fecal cont ents releases large quantities of microbial products into the peritoneum and systemic circ ulation. It is not at all surprising that 36

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polymicrobial sepsis produced a comparable expansion of the GR1+CD11b+ population in C3H/HeJ mice lacking a functional TLR4 recepto r, suggesting that although lipopolysaccharide contributes to the septic response in wild-type mice, signaling through TLR4 is not required during polymicrobial sepsis as signaling through other TLRs by pathogen associated molecular patterns likely also contributes to the expansion of these cell p opulations. Considering the fact that mice with defective TLR4 si gnaling also exhibit comparable mortality to wild-type mice after sublethal polymicrobial seps is (94), the findings suggest th at neither murine expansion of myeloid-derived suppressor cells nor survival after sepsis are entirely TLR4 dependent. Two major pathways activated by TLR signaling include the induction of both inflammatory mediators through MyD88 and type I interferon producti on. Since both MyD88 and TRIF pathways can lead to type I interf eron production, mice deficien t in the type I IFN receptor were used to ascertain the role of type I IFN in sepsis-induced myeloid cell accumulation. The increases in the GR1+CD11b+ population were unaffected in mice lacking TRIF or IFN / R signaling, indicating that TLR signaling to Type I interferons is not important in the induction of these cells. However, their expansion was completely prevented for the first seven days in septic mice lacking MyD88 signa ling, and was attenuated after 14 days. The studies were confirmed in MyD88-/mice on two backgrounds B6 and B6x129(F1)) to assure that these findings were not stra in dependent. This observation in particular suggests that the expansion of this cell population represents a fundamental component of inflammatory signaling in the host innate immune res ponse to TLR ligation by pathogens. The observation that even MyD88-/mice could expand their cell population to some extent after a cecal ligation and puncture reveals the fundamental nature of this response and the redundant signaling pathways involved in its invocation. In response to mi crobial products released during cecal ligation and 37

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puncture, activation of inflammatory signaling through MyD88, presumably through ligation of multiple TLR receptors, plays some still indete rminant role in the expansion of these cell populations. In summary, the results demonstrate for the first time that the numbers of GR-1+ CD11b+ cells increase dramatically in the spleen, ly mph nodes and bone marro w during polymicrobial sepsis, and remain elevated for up to 12 weeks. These cells are he terogenous, metabolically active, can secrete a number of cytokines, and are immature, but are predominantly committed to development along myeloid pathways. Signali ng through MyD88 is required for the full expansion of these cell populations, with an incomplete increase in the numbers of these cells in the spleen or bone marrow in the absence of MyD 88 signaling. In mice, these cells contribute to the T-cell suppression seen af ter sepsis by suppressing CD8+ T cell IFN production, and the repolarization from a TH1 to a TH2 type immune response exhibited by augmenting B cell antibody production towards IgG1 (TH2) and away from IgG2a (TH1). What remains unresolved is whether a comparable expansion of these immature myeloid populations also occurs in human sepsis, and contributes to the immune suppression and polariza tion that occurs. Further studies will be required to determine whether these find ings translate to a be tter understanding of the human innate and adaptive immune responses to severe sepsis. Materials and Methods Mice All experiments were approve d by the Institutional Animal Care and Use Committees at the University of Florida College of Medicine Rhode Island Hospital and Brown University or Schering-Plough Biopharma. Specific pathogen -free C57BL/6 mice, C3H/HeJ mice (TLR4 receptor mutation) and their control mice (C3H /HeOuJ), OT-1 TCR transgenic mice C57BL/6Tg(Tcr Tcr )1100Mjb/J, D011.10 OVA TCR transgenic mice (BALB/c-TgN(DO11.10)10Loh) 38

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and B6.129PF1/J mice were all purchased from Th e Jackson Laboratory (Bar Harbor, ME). IFNR/A129 mice on the 129S6/SvEv background (H-2b) and wildtype Sv129 mice were purchased from B & K Universal (Hull, East Yorkshire, UK). All mice were maintained at the University of Florida College of Medicine. MyD88-/mice on a B6x129(F1) background and TRIF-/mice were a kind gift of Dr. Shizuo Akira to Schering-Plough Biopharma, and were maintained at Schering-Plough Biopharma, Palo Alto, CA. MyD88-/mice on a B6 background were obtained from Dr. Shizuo Akira, and ma intained at Rhode Island Hospital and Brown University. Cecal Ligation and Puncture For induction of polymicrobial sepsis, mi ce underwent a cecal ligation and puncture or sham procedure as previously described (4, 32) to obtain a mortality of 10-20% by 10 days. At various intervals, bacterial counts from the blood and peritoneal wash (3 mls of phosphatebuffered saline) were determined by culturing aliquots on sheep RBC-agar plates. Total and differential white blood cell counts were also determined using an automated cell counter. Plasma cytokines were determined by Lumine x technology using reagents obtained from Upstate Cell Signaling Solutions (Beadlyte Mouse Multi-Cy tokine Detection System) (Temecula, CA). Flow Cytometry Spleens, lymph nodes and bone marrow cells were analyzed by flow cytometry as previously described (95). Antibodies included anti-GR1 (Ly6G and Ly6C (RB6-8C5)) conjugated to APC, anti-CD11b (Integrin aM, chain Mac-1a ch ain (M1/70)) conjugated to FITC, anti-MHC II (I-A/I-E (2G9)) conjugated to FITC, anti-F 4/80 Antigen (Pan Macrophage Marker (BM8)) conjugated to PE, Ter119 conjugated to FITC (c lone ter119) and anti-CD11c (N418) conjugated to APC, Fc-Block (CD16/CD32 Fc g III/II Re ceptor (2.4G2), and 7 amino actinomycin D 39

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(7AAD) F4/80 and CD11c specific antibodies we re purchased from eB ioscience all other antibodies were purchased from BD Pharmingen. Cell Purification All magnetic bead kits were obtained from Miltenyi Biotec. Er ythrocyte-depleted splenocytes and lymphocytes were isolated usin g either anti-GR-1 (Ly6G and Ly6C (RB6-8C5)) conjugated to APC followed by anti-APC MicroBeads for GR-1+ splenocytes. Anti-CD8a (Ly2) MicroBeads were used alone for OT-1 CD8+ splenocyte isolation. Ex vivo Stimulation and Cytokine Production Enriched GR-1+ cells were plated at 1 x 106 cells/well with RPMI 1640 supplemented with 10% fetal calf sera, 2 mM L-glutamine, 200 units /ml penicillin and 50 g/ml streptomycin, and stimulated with 10 g/ml of bacterial li popolysaccharide (E. coli 0111:B4). The culture supernatant was analyzed for cytokines. Ex vivo Differentiation and Colony Formation 1 x 106 GR-1+ splenocytes positively enriched an d cultured with RPMI 1640 medium supplemented with 10% fetal calf sera, 2 mM L-glutamine 200 units/ml penicillin and 50 g/ml streptomycin. Cells were stimulated for 7 days with 10 ng/ml of GM-CSF (R&D Systems). The cells were phenotyped by flow cytometric analyses. For the colony forming assays, 1 x 105 GR-1+ splenocytes from sham and septic mice were cultured in Methocult methylcellulose media (Stem Ce ll Vancouver, Canada) containing recombinant mouse G-CSF, GM-CSF, or erythropoe itin (R&D Systems; all at 10 ng/ml) for ten days. Colonies containing >30 cells were enumerated. Antigen Specific CD8+ T-Cell IFN Production 3 x 106 purified T-cells from OT-1 TCR transgen ic mice were injected i.v. into naive C57BL/6 recipient mice. Two days later, mice were injected i.v. with 5 x 106 GR-1+ cells 40

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obtained ten days after sepsis or sham procedure. Mice were immunized subcutaneously with the specific peptide (100 g of OVA-derived peptide SIINFEKL ) mixed with Incomplete Freund s Adjuvant. Ten days later, lymph node and spleen cells were isolated, and restimulated in vitro with specific (OVA-derived peptide SIINFEKL) or contro l peptide (RAHYNIVTF), and analyzed via IFNELISpot. Enzyme-Linked Immunospot Assay (ELISpot) Millipore MultiScreen HA plates were coated with and blocked with phosphate-bufferedsaline with 1% bovine serum albumin prior to pl ating. Spleen and lymph node cells were plated at 2.5 x 105 cells/well with HL-1 (Cam brex) supplemented with 2 mM L-glutamine, 200 units/ml penicillin and 50 g/ml streptomycin. Cells were stimulated with eith er 10 M OVA257-264 or bovine serum albumin for 48 hours. The cells we re treated with 1 g/m l biotinylated antimouse-IFN((XMG1.2) BD Bioscience), then a 1: 1000 dilution of stre pavidin-alkaline phosphatase conjugate and developed with 5-br omo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Pierce). The spots per well were manually counted. Antigen Specific and Nonspecific CD4+ T-cell proliferation The MHC class II-restricted OVA T-cell tran sgenic mouse strain DO11.10 was utilized to determine whether GR-1+ cells suppress antigen specific or nonspecific CD4+ T-cell proliferation. DO11.10 transgenic mice were made septic and immunized with 100 g OVA323 339 peptide (Genscript) in alum. At day 7, er ythrocyte-depleted splenocytes and lymph node cells underwent CD4+ T-cell purification by positive selec tion (>98% purity). The cells were irradiated with 3000 rads. 2 x 105 cells from sham-treated mice (containing <1x104 GR-1+ cells) or 2 x 105 cells from septic mice (containing 7.5 x 104 GR-1+ cells) were mixed with 2.5 x 104 CD4+ T cells. Cells were restimulated with 10 g/ml OVA323 peptide or anti-CD3 (1 g/ml) and anti-CD28 (1 g/ml) or 10 g/ml of bovine serum albumin for 48 hours. In the final 41

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16 hours of culture, 1 Ci 3H-thymidine (Amersham Biosciences,) was added. Proliferation was determined by the incorporation of 3H-thymidine in the cell coculture. Immunization with NP-KLH and Humoral Immune Responses An NP-KLH immunization model was used as described by Hurov and colleagues (96). When indicated, animals were depleted of either GR-1+ cells or CD4+ T-cells as described above. At day 10 post surgery, mice were immunized subcutaneously with 100 g of the T-celldependent antigen 4-hydroxy-3-nitrophenyl ace tylkeyhole limpet hemocyanin (NP-KLH) (Biosearch Tech) and alum. Serum titers of NP speci fic antibodies were determined by ELISA. Immulon 4HBX 96 well plates (Dynex Tech) we re coated with 1 mg of NP-bovine serum albumin (Biosearch Tech) per ml. NP-specifi c antibodies were bound to biotin-conjugated goat anti-mouse Ig isotype antibodies anti-IgM, anti-IgG1, anti-IgG2a, and anti-IgG3 (CalTag). Streptavidin-conjugated horseradish peroxidase was incorporated to detect the biotin-Ig with 2,2-azino-di(3-ethylbenzthiazolines ulfonate) (ABTS) substrate. Morphologic and Histologic Analysis For cytosipns 1 x 105 enriched GR-1+ splenocytes were stained using One Step II WrightGiemsa Stain Solution (Criterion Sciences). He matoxylin and eosin stai ning tissues were fixed in 10% neutral buffered formalin with 0.03% Eosin (Sigma Aldrich), paraffin embedded, sectioned (5 m) and mounted for staining. For immunohi stochemical staining, spleens were mounted in Tissue Tek O.C.T. compound (Sakura Finetek), and fl ash-frozen. 4 m cryosections were stained with a rat anti-mouse CD11b at a 1:50 dilution (BD Pharmingen), followed by a biotinylated rabbit anti-rat (Vector Laborator ies) secondary antibody, and visualized using Vulcan Fast Red (VFR) (Biocare Medical). Slides were counterst ained with hematoxylin (Vector Laboratories) followed by bluing with Tris-buffered saline. 42

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Statistics Continuous variables were first tested for nor mality and equality of variances. Differences among groups in flow cytometric analyses were evaluated by analysis of variance for multiple groups and Students t-test for two groups. Significance was designated at the 95% confidence level. 43

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Figure 2-1. Bacteremia and blood leukocyte respons es to sepsis produced by a cecal ligation and puncture. Mice underwent either a cecal ligation and puncture, or a sham procedure, as described in the Materials and Methods. At selected interval s, surviving animals were killed and blood and peritoneal bacter emia were determined. Blood bacteremia was only detected on the first day after ceca l ligation and puncture, whereas bacteria could be recovered from the peritoneal cavity for up to ten days. Blood and peritoneal washes were sterile from sham-treated animals (data not shown). Sepsis was associated with a profound leukopenia that lasted three days. Values represent the mean of three (sham) to five (septic) animals per group ( S.D.). Differences in the response were determined by Students t-test. 44

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Figure 22. Differential white blood cell counts in response to sepsis. Total differential leukocyte counts were performed at intervals in septic and sham-treated mice. Sepsis was associated with a transient pan cyt openia lasting approxim ately three days. Afterwards, there was a sustained neutrophilia that lasted approximately 7-10 days. Values represent the mean of three (sham) to five (septic) animals per group ( S.D.). Differences in the response were determined by Students t-test. 45

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Figure 2-3. Selected plasma cyt okine concentrations in septic and sham-treated mice. The plasma concentrations of 22 cytokines were determined after sepsis. The concentrations of 17 cytokines significantly changed in response to the cecal ligation and puncture (CLP), although only four are graphically presented. Concentrations peaked one-three days after sepsis, but remained significantly elevated for at least seven to ten days. Values represent the mean of three (sham) to five (septic) animals per group ( S.D.). Differences in the re sponse were determined by Students t-test. 46

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Figure 24. Sepsis induced splenomegaly. Spleen s were harvested from mice ten days after an LD10 cecal ligation and puncture (CLP). Panel A. Spleen mass increased nearly fivefold in ten days. Panel B. Hemotoxy lin and eosin stained spleen from sham mouse taken ten days after surgical pr ocedure (magnification 100 x). Panel C. Hematoxylin and eosin stained spleen fr om 10 day septic mouse (magnification 100 x). Splenomegaly and loss of follicular architecture is evident in spleens from septic animals 10 days after CLP. Values repres ent the mean and standard error of 5-10 animals per group. p<0.001 by Student s t-test. (Bar, 1000 m.) 47

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Figure 2-5. Appearance of GR-1+CD11b+ cells in spleens from septic and sham mice. Panel A. Flow cytometry histogram of vi able splenocytes gated on GR-1+ and CD11b+ staining. Panel B. Percentage and total numbers of GR-1+CD11b+ splenocytes recovered from mice at intervals after CLP a nd sham-treatment. Panel C. Percentage and total numbers of GR-1+CD11b+CD31+ splenocytes recovered from mice at intervals after CLP and sham-treatment. Sepsis induced by CLP produced 50-fold increases in numbers of GR-1+CD11b+ splenocytes. Values represent the mean and standard error of 5-10 animals per group. p<0.01 by analysis of variance and Student-Newman Keuls multiple range test. 48

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Figure 2-6. Ter119 Staining of GR-1+CD11b+ splenocytes harvested from sham and septic mice. Spleens were harvested from mice ten days after induction of sepsis by cecal ligation and puncture (CLP) and splenocytes were stained for GR-1, CD11b and Ter119. The percentage of Ter119+ cells increased in sepsis, but still remained less than 6% of the GR-1+CD11b+ population. Values represent the m ean of three to five animals per group ( S.D.). Differences in the res ponse were determined by Students t-test (p<0.05). 49

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Figure 2-7. MHC Class II expression in GR-1+CD11b+ splenocytes harvested from sham and septic mice. Spleens were harvested from mice ten days after th e induction of sepsis by cecal ligation and puncture (CLP) and sple nocytes were stained for GR-1, CD11b and MHC II. A GR-1-CD11bsplenocyte population (presumably B cells) were used to gate the MHCII+ population, and those gates were then applied to the GR1+CD11b+ populations from the sham and septic mice. Values represent the mean of three to five animals per group ( S.D.). Differences in the response were determined by Students t-test (p<0.05). 50

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Figure 2-8. Flow cytometric analysis of GR-1+ splenocytes cultured with GM-CSF, G-CSF or erythropoietin (EPO) ex vivo. Panel A. Flow cytometric analysis of enriched GR-1+ splenocytes obtained from septic mice 10 da ys after CLP. Panel B. Cells were cultured for 7 days with either nothing or GM-CSF and cell viability was determined by 7-AAD staining. Cells cult ured without GM-CSF rapidl y died. Panel C. GR-1+ enriched splenocytes cultured with GM-C SF for seven days yielded a phenotypically diverse cell population staini ng positive for CD11c and F4/80. Panel D. Cytospin preparation of enriched GR-1+ cells 10 days after cecal ligation and puncture demonstrated immature heterogeneous my eloid phenotypes with characteristic ring shaped nuclei. (Bar, 5m.) Panel E. Colony forming units of GR-1+ enriched splenocytes cultured with G-CSF, GM-CSF or erythropoietin (EPO) for ten days in soft methylcellulose. The photographs di stinguish the nature of the colonies, reflecting primarily neutrophil and monocyt e like colonies in the G-CSF and GMCSF-treated groups, respectivel y. (Bar, 15m.) Values for panels A-C represent the mean and standard error of 5-10 animals per group. p<0.01 by between CLP and sham treated animals, by Students t-test. Hatched line indicates mean percentage from healthy control animals not subjected to CLP or sham procedures. 51

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Figure 2-9. Bone marrow and lymph node GR-1+CD11b+ cells from septic and sham mice. CLP produced a rapid increase in the numbers of GR-1+CD11b+ cells in bone marrow (Panel A) and lymph nodes (Panels B and C). A sham procedure produced a more transient modest increase. Values repres ent the mean and standard error of 5-10 animals per group. p<0.01 by between CLP and sham treated animals, by Students t-test. Hatched line indicates mean per centage from healthy control animals not subjected to CLP or sham procedures. 52

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Figure 2-10. Hematoxylin and eosin stained spleen s from septic mice ten days after CLP. Panel A. 400 x magnification with perivascular region identified as an inset. Panel B. High powered view of perivascular inset showing cuffing a nd infiltration with myeloid cells showing characteristic signet ring feat ures. Note mitotically active cell (white arrow). Panel C. High powered view of s ubcapsular region also showing infiltration with myeloid cells exhibiting characteri stic signet ring features. Panel D. CD11b+ staining of spleen from septic anim al 10 days after CLP. Panel E. CD11b+ staining of spleen from sham-treated animal 10 days after surgical proce dure. In the sham animal, CD11b+ staining is distributed in the mantel region surrounding T-cell rich follicles. After 10 days of sepsis, additional CD11b+ staining appears in the perivascular and subcapsular regions. Panel F. Higher magnification staining of perivascular region showing CD11b+ staining from a 10 day septic animal. Panel G. Higher magnification showing resoluti on of subcapsular region showing CD11b+ staining from septic animal. (Bar, 100m.) 53

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Figure 2-11. Effect of ex vivo lipopolysaccharide stimulation on cytokine expression in GR-1+ splenocytes obtained from septic mice. When GR-1+ splenocytes were harvested from seven day sham-treated or septic mice, and stimulated with 10 g/ml of bacterial lipopolysaccharide, IL-1 IL-1 IL-6, IL-10, TNF RANTES, MIP-1 and KC production were significantly incr eased in all groups stimulated with lipopolysaccharide. Importantly, GR-1+ splenocytes from septic mice secreted more IL-10, TNF RANTES and MIP-1 production after lipopolysaccharide administration than GR-1+ splenocytes from sham-treated animals. Values represent the mean ( S.E.M.) of between four and six samples. p<0.05 by Students t-test. 54

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Figure 2-12. Immunoglobulin production following NP-KLH immunization in sham, septic and septic mice depleted of GR-1+ cells. Nine and ten days after induction of sepsis by CLP, mice were depleted of GR-1+ cells by the intraperitone al administration of RB68C5 anti-GR-1 antibody, as described in the Methods. Mice were then immunized with NP-KLH. Seven days later, mice were bled and serum IgM (Panel A), total IgG (Panel B), IgG1 (Panel C) and IgG2a (Panel D) responses to NP-KLH immunization were determined. Sepsis produced no difference in the IgM response while concomitantly producing an increase in the total serum IgG and IgG1 and a decrease in the serum IgG2a response consistent with a TH2 type polarization. The total IgG, IgG1, and IgG2a responses after sepsis were prevented by depletion of the GR-1+ cells. p<0.01 by ANOVA and Student-Newman Keuls multiple range test. 55

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Figure 2-13. Effect of GR -1 antibody depletion on CD11b+GR-1+ populations in the spleen and lymph nodes. Nine days after a cecal li gation and puncture, mice were treated with the intraperitoneal injection of 500 g of purified rat, antimouse GR-1 IgG, followed by a second intraperitone al injection of 250 g the next day, or equivalent quantities of an isotype control (IgG3). Twenty-four hours later, the mice were killed and GR1+CD11b+ cells in the spleen a nd lymph nodes were determined. Results were also compared to the numbers of GR-1+CD11b+ cells obtained in the same organs from a sham animal. GR-1+ produced a significant decrease in the relative and absolute numbers of GR-1+CD11b+ cells in the spleens of th e septic animals. Although depletion was not complete, the number of remaining GR-1+CD11b+ cells were only modestly increased, albeit not significantl y, from the number of cells in the sham animals. Values represent the mean of three to five samples, and differences were determined by the Students t-test (*p<0.05). 56

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Figure 2-14. Immunoglobulin production following NP-KLH immunization in sham, and septic mice. Nine and ten days after induction of sepsis by CLP, mice were depleted of CD4+ cells by the intraperitoneal admini stration of an an ti-CD4 antibody (GK1.5 hybridoma). Mice were then immunized with NP-KLH. Seven days later, mice were bled and serum IgM (Panel A) total IgG (Panel B), IgG1 (Panel C) and IgG2a (Panel D) responses to NP-KLH immunization were determined. Sepsis produced no difference in the IgM response while concom itantly producing an in crease in the total serum IgG and IgG1 and a decrease in the serum IgG2a response consistent with a TH2 type polarization. However depletion of CD4+ cells significantly attenuated the IgM responses and completely prevented the Ig G class switching in the septic animals (Panels A, B, C, and D). p<0.01 by ANOVA and Student-Newman Keuls multiple range test. 57

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Figure 2-15. Effect of GR-1+ cells from septic mice on antigen specific CD4+ T-cell proliferation and CD8+ T-cell IFN responses, Mice were treated as described in the Materials and Methods. GR-1+ cells from septic animal s markedly attenuated the IFN production (determined by ELISpot) by OT-1 splenocytes stimulated with either control peptide or OVA-derive d peptide SIINFEKL ex vivo following administration and immunization in C57BL/6 mice. In contrast, injection of GR-1+ splenocytes from ten day septic mice had only a minimal but significant suppressive effect on antigen specific and nonspecific CD4+ T-cell proliferative response in OVA antigen specific, D011.10 mice (Panel B). Values represent the mean and standard error of 5 animals per group. The experiment was repeated twice and values presented are from one of the representative experiments. p<0.05 by ANOVA and Students-Newman-Keuls multiple range test. 58

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Figure 2-16. Effects of lipopolysaccharide (L PS) and transgenic mice on expansion of the splenic GR-1+CD11b+ population ten days after CLP. (Panel A) Mice received either nothing or the intraperitoneal inject ion of 5 mg/kg BW of bacterial lipopolysaccharide, and were s acrificed at inte rvals thereafter. Lipopolysaccharide injection increased the percentage of GR-1+CD11b+ cells in the spleen within one day, and expansion of this cell population remained for about seven days. CLP was induced in C3H/HeJ (TLR4 mutant) (Panel B.), IFN / R-/(Panel C), MyD88-/(B6x129)(Panel D) and TRIF-/(Panel E) as described in the Methods. At one and seven days later, splenic GR-1+CD11B+ populations were examined in the spleens of knockout mice and their appropriate background controls. Normal expansion of the GR-1+CD11b+ splenocytes was seen in all mice at seven days with the exception of the MyD88-/mice that failed to demonstrate an increase in their GR-1+CD11b+ population. Values represent the mean and standard error of 5 animals per group. *p<0.01 versus control at the same time point, as determined by Students t-test. 59

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Figure 2-17. Requirement for MyD88-/signaling in the expansion of GR-1+CD11b+ splenocytes in response to prolonged sepsis. MyD88-/mice on a C57Bl/6 ba ckground and wildtype controls underwent a cecal ligation a nd puncture, and representative animals were sacrificed at seven and 14 days af ter induction of sepsis. The relative and absolute numbers of GR-1+CD11b+ splenocytes were determined, as previously described. As seen in the MyD88-/B6x129 animals, there was no increase in the GR-1+CD11b+ splenocyte population seven days afte r sepsis. However, at 14 days, there was an increase in the GR-1+CD11b+ population, although it was still less compared to the wild-type animals. *p<0.01 ve rsus control at the same time point, as determined by Students t-test. 60

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61 Figure 2-18. Immunoglobulin production following NP-KLH immunization in sham, and septic mice 12 weeks after cecal ligation and puncture. Mice were immunized with NPKLH with alum 12 weeks afte r cecal ligation and puncture. Seven days later, mice were bled and serum IgG2a (Panel A) responses to NP-KLH immunization were determined. Sepsis produced a decrease in the serum IgG2a response consistent with a TH2 type polarization. However, in mice that exhibited resolved abscesses the IgG2a responses were more elevated comp ared with mice with ongoing abscesses (Panel B). p<0.01 by ANOVA and Student -Newman Keuls multiple range test.

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CHAPTER 3 IL-4, IL-10 AND NITRIC OXIDE SIGNALING DO NOT CONTRIBUTE TO THE MYELOID DERIVED SUPPRESSOR CELL EXPANSIO N OR SUPPRESSOR FUNCTION DURING POLYMICROBIAL SEPSIS Specific Aim 2 The goal of Specific Aim 2 aim was to determ ine the effect of MDSC expansion on the adaptive immune system using a prolonged model of polymicrobial sepsis The suppressor cell function was evaluated by incorporating the MDSCs in vivo with T-cell recep tor specific CD8+ lymphocytes and measuring the T lymphocyte specifi c responses to antigen specific proliferative signals. Since the MDSCs must be in physical proximity with other cells to impart their immunosuppressive properties (56, 74, 75), and are known to secrete IL-4, IL-10 (65) and nitric oxide (NO) (49), we examined whether these mediators contribute directly to the MDSC suppression in sepsis by genera ting MDSCs from IL-4, IL-10 a nd iNOS (NOS2) null animals. Introduction Sepsis is the result of a severe microbial infection that leads to both an exaggerated systemic inflammatory response and immune suppr ession (4, 40, 76), as evidenced by a failure to eradicate primary infections (12), a predisposition to develop secondary nosocomial infections (12, 13), and an attenuated delayed type hypersens itivity response (11). The etiology of sepsis associated immune suppression is thought to be multifactoral, with defects in antigenpresentation (19), apoptosis of B cells, T cells, and dendritic cells (20, 77) development of an anti-inflammatory response (97), and the increased presence of regulatory cell populations (85, 98). Expansion of the myeloid derived suppressor cell (MDSC) population (91) has also been observed in the spleens and tumors of mice with tr ansplantable tumors (48, 80), and in models of chronic inflammation (50). In tumor-bearing mi ce, these cells contribute to tumor-associated 62

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antigen specific T cell dysfuncti on and tolerance (74, 80, 81, 99). We have recently described a similar heterogenous population of GR-1+CD11b+ immature myeloid cells whose numbers dramatically increase in the spleen, lymph nodes and bone marrow during polymicrobial sepsis. These MDSCs are capable of i nhibiting antigen-specific CD8+ T cell interferonproduction and antigen nonspecific CD4+ T cell proliferation during sepsis, an d the polarization of the T helper cell response from a TH1 to a TH2 profile. MDSCs are known to secrete a number of biologically active mediator s, including IL-10, TGF chemokines, oxygen free radicals, nitric oxide and peroxynitrites ( 48). However, the specific mediators responsible for either the sepsisinduced myeloid cell expansion or the associated T cell suppre ssor activity of these immature myeloid cells are yet unknown. Previously, we established that the sepsis -induced MDSC population is an immature, heterogenous group of cells capable of polarizing T and B cell re sponses during sepsis (99). Bronte and colleagues reported that MDSCs play an important role in inhibiting T cell activation during inflammatory responses (80). Suppressor ac tivity requires the MDSC s to be in immediate proximity to the effector cells (54, 87), suggesting that the MDSC suppressor activity is mediated through a paracrine or ju xtacrine mechanism. A variety of candidate molecules have been proposed, including nitric oxi de, reactive oxygen species, arginine depletion, TGF IL-4 or IL10 (49). During sepsis, increased IL-4 and IL-10 production have been associated with an antiinflammatory response and a T-helper cell TH2 polarization (4). Sin ce the MDSC population is capable of producing increased levels of IL-10, a nd to a lesser extent IL -4 during sepsis, MDSCs may utilize these cytokines to medi ate their suppressive effects. Here, we observed that an ongoing septic pro cess produces a dramatic expansion of GR1+CD11b+ cells in the spleen that is not dependent on IL-10, IL-4 or nitric oxide signaling. 63

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Although MDSCs express large quantities of IL-10, and lesser quant ities of IL-4, the suppression of antigen-specific CD8+ T-cell interferonproduction is also not dependent on MDSC expression of either mediator in vivo Results MDSCs are Capable of Immune Modulatory Cytokine Production Since myeloid derived suppressor cells obtaine d from tumor bearing hosts are known to be immune modulatory (48, 80), we sought to exam ine whether myeloid derived suppressor cells obtained from septic mice could produce immune modulatory mediators responsible for T-cell suppression and polarization during sepsis. When GR-1+ enriched splenocytes from septic mice were cultured ex vivo with bacterial lipopolysaccharide, the GR-1+ cells from septic mice produced significantly greater amounts (>5 fold) of IL-10 than similar GR-1+ cells from shamtreated animals (Figure 3-1 Panel B). They also produced increased quantities of IL-4 and MIP1 (Figure 3-1 Panel A and C). In order to confirm the ability of the spleni c myeloid cells to produ ce increased levels of immune modulatory cytokines, we used anti-IL-4 and anti-IL-10 staining antibodies and flow cytometry analysis to confirm the increased prod uction of both cytokines. Intracellular staining revealed that the total percentage and absolute numbers of splenic IL-10 or IL-4 expressing GR1+CD11b+ cells increased seven days after seps is (Figure 3-2 Pane ls A-D), although the proportion of IL-10 or IL-4 expressi ng cells remained relatively small. MDSCs Inhibit Antigen Specific CD8+ T-Cell IFNProduction Independent of Interleukin-4, Interleukin-10, and iNOS Production By determining the effect of GR-1+ cells on the CD8+ T-cell IFNresponse by splenocytes from OT-I TCR-transgenic mice (C57BL/6-Tg(TCR TCR )1100mjb) immunized with OVA-derived peptide (H-2Kb restricted aa 257, SIINFEKL), we were able to 64

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demonstrate that GR-1+ splenocytes inhibit CD8+ T-cell IFNproduction ex vivo(99) However, the specific mechanism of GR-1+ splenocyte suppression of CD8+ Tcell IFNproduction is yet unknown. In light of the myel oid derived suppressor cells ability to produce immune modulatory cytokines, we postulated that IL-4 and IL-10 may be responsible for the observed myeloid cell induced CD8+ T-cell tolerance. GR-1+ splenocytes from IL-4 and IL-10 null mice were obtained either 10 days after seps is or a sham procedure. As shown in Figure 33 Panels A-C, greater than 20 million GR-1+ cells/spleen were obtained from the IL-4 null (23 million GR-1+ cells/spleen), IL-10 null (24 million GR-1+ cells/spleen), iNOS ( NOS2 ) null ( data not shown) and wild type (22 million GR-1+ cells/spleen) mice indicating that the deficit in IL10, IL-4, or iNOS production did not hinder the expansion of the GR-1+ population. Next, the GR-1+ cells were adoptively transferred into C57B L/6 mice that had previously received CD8+ T-cells from OT-1 TCR-transgenic mice, a nd were simultaneously immunized with OVAderived specific peptide. Ten days later, th e spleens from these animals were harvested, and IFNresponses to ex vivo stimulation with OVA-de rived specific peptide were examined. IFNproduction was markedly reduced when the animals were administered GR-1+ splenocytes from septic IL-10 null, IL-4 null, iNOS null, an d wild type C57BL/6 animals, when compared to sham-treated mice (Figure 3-4 Panels A-C), indicating that the MDSC-induced CD8+ T-cell suppression is not IL-4, IL -10 or iNOS dependent. MDSCs Inhibit Antigen Specific CD8+ T-cell Cytotoxicity Although the signaling pathways imp acting the antigen specific CD8+ T cell IFN secretion reduction induced by MD SCs are unclear and do not involve IL-4, IL-10 or iNOS, the other uncertainty that exists is the direct effect of the MDSCs on T cell cytotoxic function. Since antigen specific cytokine secre tion is only an indirect method or ascertaining T cell function, we sought to find a more definitive assessment of the impact of MDSC s on the function of CD8+ T 65

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cells. We incorporated an antigen specifi c, non radioactive cytotoxicixty assay (see Materials and Methods ) to directly determine th e cytotoxic function of CD8+ T cells that have been cultured with GR-1+ cells from either sham or CLP treated mice. As Figure 3-5 demonstrates, there was an appreciable reduction in CD8+ T cell cytotoxic ability when CD8+ T cells were cultured with GR-1+ cells taken from septic animals when compared to sham controls. This in vitro cytotoxic reduction indicates that the MDSC effect on CD8+ cells may also occur in vivo and predispose the host to invasion from organisms that require CD8+ T cells to mount an immune response. Discussion Although the etiology of sepsis -induced immune dysfunction is unknown, recent interest has focused on the effects of regulatory cell popul ations responsible for the shift from a TH1 to a TH2 immune profile that results in a state of T cell anergy and immune suppression (42, 85, 98). We have previously described a substantial expansion of a GR-1+CD11b+ myeloid population with an immature phenotype capable of antigen specific CD8+ T-cell suppression and TH1 to TH2 immune polarization. However, the mechanisms driving this immature myeloid cell expansion and antigen specific T-cell suppress ion remain elusive. In the cu rrent study, we observed that an ongoing septic process produces a dramatic expansion of GR-1+CD11b+ cells in the spleen that is independent of IL-4, IL-10, and iNOS. A lthough MDSCs produce IL-10 and IL-4, their suppression of antigen-specific CD8+ T-cell IFNproduction is also not dependent on either cytokine. T cell apoptosis and loss during sepsis significantly contribute to immune dysfunction and mortality (4, 17, 88). Recently, Monneret sugg ested that T regulatory cell inhibition of T effector cells may account for the CD4+ T cell dysfunction during seps is (42), although our more recent studies, and those of Ayala, have recently questioned the role of T-regulatory cells in 66

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outcome to polymicrobial sepsis (95, 98). Other investigators (54, 74, 80, 82, 89) have demonstrated that immature myeloid cells are ab le to induce T cell tolera nce through a variety of mediators including nitric oxide, reactive oxygen species, arginine depletion, TGF IL-4 and IL10 (49). Due to the fact that sepsis induced myeloid cells are capable of producing increased levels of IL-10, and IL-4 upon ex vivo stimula tion with endotoxin (Figur e 3-1 Panels A and B), we examined whether these cytokines are re sponsible for the myeloid cell induced CD8+ T cell suppression. Despite the fact that Serafini and colleagues demonstr ated in vitro that both IL-10 and IL-4 are essential to the s uppressor function of MDSCs in can cer (49), our data suggest that neither IL-4 nor IL-10 production by MDSCs is n ecessary for the suppression of antigen specific CD8+ T-cell IFNproduction commonly exhibited by MDSCs in vivo IL-10-mediated T-cell tolerance in sepsis has been repeatedly observed and is one of the most accepted mechanisms of T cell suppression during sepsis ( 12). We can only speculate whether the increased IL-10 and IL-4 production by MDSCs may contribute to other components of the immune tolerance seen in sepsis, but we can conclude that IL-10 and IL -4 do not contribute to the suppression of CD8+ Tcell IFNproduction by sepsis derived MDSCs. An alternative explanation is that MDSC indu ced T cell tolerance is due to the increased production of oxygen free radicals and peroxynitrites. Recentl y, Nagar and colleagues found that MDSC inhibition of CD8+ T-cells results in part from the nitrosylation of the MHCII complex which inhibits the binding of the CD8+ T-cell receptor with the specific peptide MHC II complex (75). In conclusion, we observed that an ongoing se ptic process produces a dramatic expansion of GR-1+CD11b+ cells in the spleen that is likely inde pendent of G-CSF and neutrophil elastase activity, and is also independent of IL-10, a nd IL-4 signaling. Although MDSCs express large 67

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quantities of IL-10, and lesser quantities of IL-4, the suppression of antigen-specific CD8+ T-cell interferonproduction is also not depe ndent on MDSC expression of either mediator in vivo. Materials and Methods Mice All experiments were approved by the Institutional Animal Care and Use Committee at the University of Florida College of Medicine. Specific pathogen-free C57BL/6 mice, OT-1 TCR transgenic mice [C57BL/6-Tg(Tcr Tcr )1100Mjb/J], IL-4 (C57BL/ 6-Il4tm1Nnt/J) and IL-10 (B6.129P2-Il10tm1Cgn/J) null mice (on a C57BL/6 background), mice were all purchased from The Jackson Laboratory (Bar Har bor, ME). iNOS null mice (NOS2-/-) were a gift from Dr Edward Scott at the University of Florida. All mice were maintained at the University of Florida College of Medicine and were studied between 6-12 weeks of age. Cecal Ligation and Puncture For induction of polymicrobial sepsis, mice underwent sham laparotomy or cecal ligation and puncture induced by ligation of the cecum and a double enterotomy created with a 27 gauge needle. Mortality in this model was approxima tely 10-15%, and occurred predominantly in the first 3 days; thereafter, surviving mice develo ped abscesses surrounding the devitalized cecum as previously described (77, 95, 99). Flow Cytometry Spleens and bone marrow cells were analyzed by flow cytometry as previously described (95, 99). Antibodies included an ti-GR-1 (Ly6G and Ly6C (RB6-8C5)) conjugated to APC, antiCD11b (Integrin aM, chain Mac-1a chain (M1/70 )) conjugated to Pacific Blue, Fc-Block (CD16/CD32 Fc g III/II Receptor (2.4G2), and Sytox. All antibodies were purchased from BD Pharmingen. 68

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Cell Purification All magnetic bead kits were obtained from Miltenyi Biotec. Er ythrocyte-depleted splenocytes and lymphocytes were isolated usin g either anti-GR-1 (Ly6G and Ly6C (RB6-8C5)) conjugated to APC followed by anti-APC MicroBeads for GR-1+ splenocytes. Anti-CD8a (Ly2) MicroBeads were used alone for OT-1 CD8+ splenocyte isolation. Purities for either population after magnetic separation were >95% (data not shown). Ex vivo Stimulation and Cytokine Production Enriched GR-1+ cells were plated at 1 x 106 cells/well with RPMI 1640 supplemented with 10% fetal calf sera, 2 mM L-glutamine, 200 units /ml penicillin and 50 g/ml streptomycin, and stimulated with 10 g/ml of bacterial li popolysaccharide (E. coli 0111:B4). The culture supernatant was analyzed for cytokines usi ng Luminex technology using reagents obtained from Upstate Cell Signaling Solutions (Beadly te Mouse Multi-Cytokine Detection System) (Temecula, CA). Antigen Specific CD8+ T-Cell IFNProduction 3 x 106 purified T-cells from OT-1 TCR transgen ic mice were injected i.v. into naive C57BL/6 recipient mice. Two days later, mice were injected i.v. with 5 x 106 GR-1+ cells obtained ten days after sepsis or sham procedure. Mice were immunized subcutaneously with the specific peptide (100 g of OVA-derived peptide SIIN FEKL) mixed with Incomplete Freunds Adjuvant. Ten days later, lymph node and spleen cells we re isolated, and restimulated in vitro with specific (OVA-de rived peptide SIINFEKL) or c ontrol peptide (RAHYNIVTF), and analyzed via IFNELISpot. Enzyme-Linked Immunospot Assay (ELISpot) Millipore MultiScreen HA plates were coated with and blocked with phosphate-bufferedsaline with 1% bovine serum albumin prior to pl ating. Spleen and lymph node cells were plated 69

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at 2.5 x 105 cells/well with HL-1 (Cam brex) supplemented with 2 mM L-glutamine, 200 units/ml penicillin and 50 g/ml streptomycin. Cells were stimulated with eith er 10 M OVA257-264 or bovine serum albumin for 48 hours. The cells we re treated with 1 g/m l biotinylated antimouse-IFN ((XMG1.2) BD Bioscience), then a 1: 1000 dilution of stre pavidin-alkaline phosphatase conjugate and developed with 5-br omo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Pierce Fine Chemicals, Rockford IL). The spots per well were manually counted. CD8+ T Cell-Cytotoxicity A total of 3 x 106 purified T-cells from OT-1 TCR transgenic mice were injected i.v. into naive C57BL/6 mice. Two to th ree days later, these mice are injected intravenously with 5 x 106 GR-1+ cells obtained from C57Bl/6 mice obtained ten days after CLP or sham procedure. Within 1 hour after cell transfer, mice were immuni zed subcutaneously with the specific peptide (100 g of OVA-derived peptide SIINFEKL for OT-1 mice) mixed with Incomplete Freunds Adjuvant. Ten days later, OT-1 cells from ly mph nodes were isolated, washed and cultured with varying ratios of (1:1, 5:1, 10:1, and 20:1) E.G7-OVA (ATCC# CRL-2133TM) cells that constitutively express H-2 Kb restricted OVA 258-276 peptide as target cells. Different ratios of MDSCs:T cell responders (4:1, 2:1, 1:1, 1:2, 1:4, and 1:8) were added to each well. Cells were cultured for 24 hours, the supernatants then ha rvested, and the lactat e dehydrogenase (LDH) release measured using a Cyto Tox 96TM NonRadioactive Cytotoxic ity Assay (Promega) according to the manufacturers directions. Statistics Continuous variables were first tested for nor mality and equality of variances. Differences among groups in flow cytometric analyses were evaluated by analysis of variance for multiple groups and Students t-test for two groups. Significance was designated at the 95% confidence level. 70

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Figure 3-1. Effect of ex vivo lipopolysaccharide stimulation on cytokine expression in GR-1+ splenocytes obtained from septic mice. When GR-1+ splenocytes were harvested from seven day sham-treated or septic mice, and stimulated ex vivo with 10 g/ml of bacterial lipopolysaccharide, IL-1 IL-1 IL-6, IL-10, TNF, RANTES, MIP-1 KC, and MCP-1 production were significantly increased in all groups. Notably, GR1+ splenocytes from septic mice secreted mo re IL-4 (Panel A), IL-10 (Panel B), TNF, RANTES, MIP-1 (Panel C), and MCP-1 produc tion after lipopolysaccharide administration than GR-1+ splenocytes from sham-treated animals. Values represent the mean ( S.E.M.) of between four a nd seven samples. p<0.05 by Students ttest. Figure 3-2. Flow cytometry an alysis of IL-10 and IL-4 expr ession in response to ex vivo lipopolysaccharide stimulation. Total erythrocyte-depleted sp lenocytes from seven 71

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day septic and sham-treated mice were enriched for GR-1+ cells and then stimulated ex vivo with lipopolysaccharid e for 48 hours. The cells were then harvested and fixed in buffer containing 1% formaldehyde for 30 min, permeabilized by washing in flow buffer containing 0.5% saponin, and st ained with either anti-IL-10 (JES5-16E3) conjugated to FITC or anti-IL-4 (11B11) conjugated to PE. The absolute numbers of IL-10 (Panel A) and IL-4 (Panel B) expressing GR1+CD11b+ cells increased significantly in the septic mice. Values repres ent the mean ( S.E.M.) of five samples. *p<0.05 by Students t-test. 72

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Figure 3-3. GR-1+CD11b+ myeloid cell expansion is not depe ndent on the presence of IL-4 or IL-10 production. Wild type, IL-10 or IL-4 null animals underwent CLP or sham procedure and at 7 days after sepsis the sp leens harvested and analyzed for the total numbers of GR-1+ cells per spleen. Splenic expansion of GR-1+ cells in wild type C57BL/6 mice (Panel A) is similar to that of the IL-4 (Panel B) and IL-10 null mice (Panel C). Values represent the mean ( S.E.M.) of three to five samples. *p<0.05 by Students t-test. 73

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Figure 3-4. Effect of GR-1+ cells from septic mice on antigen specific CD8+ T-cell IFNresponses. Mice were treated as descri bed in the Materials and Methods. GR-1+ cells from septic wild type contro l C57BL/6 mice (Panel A), IL-10-/(Panel B), IL-4-/(Panel C), and iNOS-/(Panel D),markedly attenuated the IFNproduction (determined by ELISpot) by OT-1 splenocyt es stimulated with either control peptide or OVA-derived peptide SIINFEKL ex vivo following administration and immunization in C57BL/6 mice. Antigen specific CD8+ T-cell IFNproduction suppression is unaltered by GR-1+ cells lacking the ability to secret IL-4, IL-10, or iNOS implying that IL-4, IL-10 or iNOS production by GR-1+ splenocytes is not integral to immature myeloid cell CD8+ T-cell IFNproduction. Values represent the mean and standard error of five an imals per group. The experiments were each repeated twice and values pr esented are from one of the representative experiments. p<0.05 by ANOVA and Students-Newma n-Keuls multiple range test. 74

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75 Figure 3-5. Effect of GR-1+ cells from septic mice on antigen specific CD8+ T-cell cytotoxic function. Mice were treated as described in the Materials and Methods. GR-1+ cells from septic animals attenuated the ability of CD8+ T cells (Panel A) to execute their cytotoxic functions on OVA expressing ta rget cells (E.G7-OVA cells, ATCC# CRL2133TM)

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CHAPTER 4 MYELOID DERIVED SUPPRESSOR CELL EX PANSION IS DEPENDENT ON CXCL12 MEDIATED COMMON MYELOID PROGEN ITOR EXPANSION DURING SEPSIS Specific Aim 3 The goal in Specific Aim 3 was to confirm the signaling pathways that are required for the expansion of the MDSC populations in polymicrobial sepsis. Our preliminary data suggests that MyD88-/animals display a delay in the periphera l expansion of their MDSC populations in sepsis; however, the role that MyD88 signaling occupies in MDSC expansion is still not fully known. The following experimental approaches were devised to an swer the questions set forth in Specific Aim 3. Introduction Expansion of a myeloid derived suppresso r cell (MDSC) population (91) has been observed in the spleens and tumors of mice with tr ansplantable tumors (48, 80), and in models of chronic inflammation (50). In tumor-bearing mi ce, these cells contribute to tumor-associated antigen specific T cell dysfuncti on and tolerance (74, 80, 81, 99). We have recently described a similar heterogeneous population of GR-1+CD11b+ immature myeloid cells whose numbers dramatically increase in the spleen, lymph nodes and bone marrow during polymicrobial sepsis. These MDSCs are capable of i nhibiting antigen-specific CD8+ T cell interferonproduction and antigen nonspecific CD4+ T cell proliferation during sepsis, an d the polarization of the T helper cell response from a TH1 to a TH2 profile. However, the specific signaling mediators responsible for this sepsis-induced immature myeloid cell expansion in the sple en are yet unknown. Recent reports suggest that the expansion of immature myeloid cells in the bone marrow, spleen and lymph nodes is a highly conserved res ponse that occurs in a multitude of insults and may be dependent on Toll-like receptor (TLR) signaling (48, 100). Kinkade and colleagues observed this TLR dependent myeloid cell e xpansion first hand by demonstrating that 76

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hematopoietic stem cells (HSCs) in culture with TLR2 and TLR4 agonist (Pam3CSK4 and LPS respectively), undergo proliferation and preferen tial differentiation into myeloid lineage cells (101). We too, have observed a partial dependenc e of splenic immature myeloid cell expansion on the MyD88 pathway in vivo, although not as st unning as Kincade and associates saw with lipopolysaccharide, an exclusive TLR4 agonist. However after fu rther investig ation we found that MyD88-/animals exhibit only a delayed expansion of GR-1+CD11b+ cells in the spleen that approached wild type control leve ls with the progression of sepsis (99). This evidence leads us to believe that there must be other signals that regulate myeloid expa nsion during sepsis. In the oncology literature, multiple studies ha ve shown that cytokines and prostaglandins, including GM-CSF, G-CSF-1, IL-6, IL-10 and PG E2 may regulate the expansion of immature myeloid populations (49, 63). In states of inflam matory stress, hematopoietic stem cells (HSCs) continually circulate between the bone marrow and peripheral organs to maintain or expand lymphoid and myeloid populations.( 102) Although the exact mechan isms of stem cell egression and myeloid expansion are still unknown, Petit and colleagues demonstrated that granulocytecolony stimulating factor (G-CSF) facilitates bone marrow egression through the activity of bone marrow derived neutrophil elasta se on CXCL12 (stromal cell de rived factor-1;SDF-1). The authors further demonstrated that this egress ion can be inhibited by using specific elastase inhibitors and anti-CXCR4 neutralizing antibodies, which disrupt the CXCL12-CXCR4 signaling axis, reducing the number of progenitor cells found in the spleen (103) In addition, recent reports have shown that the C-C chemokine receptor-2 ( CCR2) is essential for monocyte recruitment from the bone marrow in times of b acterial infection; myeloid expansion in the spleen is reduced in infected mice devoi d of CCR2 (100, 104). Interestingly, CCR2-CCL2 (MCP-1) interactions were shown to facilitate the expansion and migra tion of myeloid derived 77

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suppressor cells in cancer patients and mice with growing tumors (105). However, as the MDSC population is heterogeneous, so are the signaling pathways involved in myeloid expansion. The cfms receptor (CD115, M-CSFR, CSF-1R), is a myel oid lineage cell marker that progressively increases from stem cell precursors to monocytes and macrophages (106). Cfms receptor ligation with its ligand M-CSF stimulates the diffe rentiation, proliferation and survival of HSCs as they undergo myeloid development (107). Several studies have demonstrated that administration of the c-fms receptor antibody AFS98 reduces the relative percentages and absolute numbers of macrophages and other myeloid derived cells after various injuries (108, 109). Taken together, these data provide poten tial targets for the amelioration of MDSC expansion during sepsis. In this study, we have demonstrated that th e overall sepsis induced immature myeloid cell population expansion is only modestly dependent on CSF-1 and CCR2 signaling with immature monocyte accumulation mainly dependent on these two pathways. In contrast, CXCL12 signaling during sepsis proved necessary to the complete expansion of the immature myeloid population in sepsis with CXCL12 blockade in hibiting nearly 70% of the overall myeloid expansion 7 days after the initia tion of sepsis. Further analysis of cells along the myeloid differentiation pathway demonstrated that CXCL 12 inhibition effectivel y reduced the splenic accumulation of the common myeloid progenitor ce ll population-the earliest and most immature cell in the myeloid differentiation pathway. Ther efore, we conclude th at CXCL12 signaling is imperative for the complete immature myeloid expansion cell expansion that occurs during polymicrobial sepsis. 78

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Results Hematopoietic Stem Cell Proliferation and Di fferentiation do not Dependent on MyD88 or TRIF Signaling in vivo We have previously reported that comple te splenic expansion of the MDSC population may have some dependence on MyD88 signaling wh ich resolves with sepsis progression beyond 7 days (99). Several reports also indicate that HSC proliferation and preferential differentiation into myeloid lineage cells are dependent on th e MyD88 signaling pathway in vitro (101, 110112). Since MDSCs are an immature heterogeneous contingent of cells at various stages of development in the myeloid differentiation path way, we speculated, based on the aforementioned data, that HSC proliferation and myeloid differentiation should be impaired using an in vivo system devoid of MyD88 signaling, and may account for the delay that we have observed in MDSC expansion in MyD88-/mice during sepsis. Us ing a low mortality (LD20) cecal ligation and puncture (CLP) model of polymicrobial se psis and incorporating wild type, MyD88-/and TRIF-/mice at 1 and 7 day periods, were able to determine the fluctuations that occur in the HSC, progenitor cell, and immature myeloid cell populations in the bone marrow after the induction of sepsis. In the wild type B6.129 mice, we found that sepsis produced a 2 fold increase in the relative percentages (Fi gure 4-1 Panel A) and absolute numbers ( data not shown ) of HSCs (Lineagenegc-KithighSca-1high) in the bone marrow within 24 hours after sepsis that persisted through 7 days in the CLP group compared to sham controls. Interestingly, and in contrast to Kincade and associ ates (101), we found that MyD88-/and TRIF-/animals exhibited the same increase in HSC numbers at 24 hours and 7 days post-sepsis as did sham and wild type control animals (Figure 4-1 Panels B and C). This finding suggests that MyD88 and TRIF signaling are not imperative for early or prolong ed HSC proliferation in vivo during sepsis. 79

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When HSCs proliferate they give rise to mu ltipotent progenitors (MPPs) that lose their ability to differentiate as they mature into cells in the lymphoid a nd myeloid lineages. MMPs differentiate into common lymphoid progenito rs (CLPs) in the lymphoid lineage and common myeloid progenitors (CMPs) which further mature into megakaryocyte erythrocyte progenitors (MEPs) and granulocyte macrophage progeni tors (GMPs)-all of which are bone marrow progenitors in the myeloid lineage. After finding a substantial elevation in the HSC numbers in MyD88-/mice within 24 hours after sepsis, we next sought to determine if this HSC increase actually translated into incr eased numbers of bone marrow pr ogenitor cells and whether the absence of MyD88 would hinder HSC differentia tion along the myeloid lineage in vivo. As shown in Figure 4-2 Panel A, we observed little change in the percentages of total bone marrow progenitors (Lineagenegc-Kithighsca-1neg) at 24 hours after sepsis; however, by 7 days we found a significant increase in the quantit y of progenitor cells in the se psis mice compared to sham controls. When we evaluated the bone marrow progenitor population in the MyD88 and TRIF mice after sepsis we found little change in pr ogenitor numbers at 24 hours; surprisingly, we found the same increase in progen itor cell numbers at seven days post sepsis compared to wild type controls (Figure 4-2 Panels B and C) This finding implies that although HSC differentiation in vitro may be dependent on My D88 mediated TLR signaling, that in vivo, HSC differentiation into progenitor cells can sti ll occur in the absen ce of MyD88 signaling. Although we did not observe a difference in the relative fluctuations in HSC and progenitor populations between wild type, MyD88-/and TRIF-/mice, we postulated that a MyD88 dependent differentiation between the progenitor population a nd more terminally differentiated myeloid cells may exist, a nd halt progenitor maturation accounting for the increases in progenitor ce ll observed in the MyD88-/and TRIF-/mice at 7 days post sepsis. In 80

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contrast, as Figure 4-3 Panels A, B and C suggest, the same immature myeloid cell (GR1+CD11b+) fluctuations occur in all three mouse t ypes at 24 hours and 7 days after sepsis initiation. The reduction in GR-1+CD11b+ cells at 24 hours is simila r in all the mouse types tested and represents immature granulocyte egression from the bone marrow in response to sepsis. By 7 days after CLP sepsis, there was an equivalent and signi ficant increase in the immature myeloid population across all three mouse types indicating that di fferentiation from the HSC population through the progenitor population and on to the immature myeloid population can occur in the absence of My D88 and TRIF signaling in vivo. MDSC Expansion During Sepsis Occurs Inde pendently of the CCR2 Signaling Pathway. Given our finding that immature myeloid cells can expand even in the absence of MyD88 signaling in the bone marrow and have only pa rtial dependence on MyD88 signaling to expand in the spleen, we began to inve stigate other potential mediators known to effect myeloid cells in other various models of inflammation. Previously, we reporte d a substantial increase in the GR1+CD11b+ myeloid population in the spleen and bone marrow beginning three days after sepsis (99). Phenotype analysis reve aled that over 40% of the GR-1+CD11b+ cells were also CD31+, a marker of immaturity, and could form colonies ex vivo when cultured with G-CSF or GM-CSF, but not erythropoietin. Given the increased number of immature myeloid cells in secondary lymphoid organs following sepsis, we sought to investigate the underlying mechanism(s) responsible for this myeloid cell expansion. Several reports indicate that the C-C chemokine receptor 2, (CCR2) signaling pathway is required for GR-1high monocyte egression from the bone marrow into the peripheral circulation during listeriosis.(104) We found that ex vivo stimulation (10 g/mL bacterial lipopolysaccharide) of enriched GR-1+ splenocytes obtained 7 days after CLP but not sham treatment demonstrated increased producti on of MCP-1, the ligand for CCR2 (Figure 4-4 81

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Panel A). Since the splenic myeloid population is capable of MCP-1 production, we sought to determine whether CCR2 is involved in the myeloid bone marrow egression and lymphoid expansion during polymicrobial sepsis. Seven da ys after sepsis, we examined the spleen and bone marrow myeloid cell numbers in wild type control C57BL/6 mice and in CCR2-/mice. As demonstrated in Figure 4-4 panels B and C, the GR-1+CD11b+ population in the spleen increased comparably in wild type and CCR2-/mice. Similar increases in the myeloid population were obtained in the bone marrow of wild type and CCR2-/mice (data not shown) We did not observe any significant differences in the abso lute numbers and relative percentages of GR1+CD11b+F4/80+ population or the total F4/80+ population between CCR2 null and control mice (data not shown) Furthermore, no differences were observed in the absolute numbers and relative percentages of Ly6Chigh cells between the CCR2 null and the wild type mice, suggesting that CCR2 signaling is not essential for immature myeloid cell expansion in the spleen during polymicrobial sepsis. Although the overall myeloid expansion in th e spleen of both wild type and CCR2 null mice was similar at 7 days post sepsis, there were some substantial differences in the expansion of the GR-1intermediateCD11b+ populations. These GR-1intermediate cell populations represent more immature monocytic cell populations. In Figure 4-5 panel A, th ere was a 50% reduction in the numbers of the GR-1intermediate population in the sham CCR2-/mice, as compared to the wild type sham mice. After induction of se psis, there was a 7-fold increase (Figure 4-5 panel B) in the GR-1intermediate splenocytes in the wild type group, comp ared to only a 3-fold increase in the CCR2-/mice, indicating that CCR2 may participate in the expansion of certa in subpopulations of splenic MSDCs. Based on CD11b, F4/80, and MH C II cell surface analysis, the four fold 82

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reduction consisted of mainly immature monocytes Analysis of this same subpopulation in the bone marrow revealed no significant di fferences between the two groups ( data not shown ). In order to explain the overall expansion of the myeloid populati on in the absence of CCR2, we examined whether the frequency of myeloid precursor cells in the bone marrow changes in response to sepsis. Broxmeyer and co lleagues demonstrated th at there are increased numbers of early myeloid progenitors in CCR2-/mice compared to wild type littermate controls (113). Thus, we postulated that in CCR2-/mice, the absence of MCP-1 signaling on the myeloid progenitor population may increase the numbers of bone marrow myeloid progenitors whose proliferation is dependent on the CCR2-MCP-1 axis. The relative percentages of lineage-ckit+sca-1bone marrow and splenic progenitor cells in CCR2-/and wild type litter mates were determined seven days after induction of sepsis (Figure 4-6, panels A and C). As depicted in Figure 4-6 panel B and D, there was no significant difference in the percentages of myeloid progenitor cells in the bone marrow and splee n, also suggesting that MCP-1-CCR2 does not participate in myeloid progen itor expansion during sepsis. M-CSF Receptor Signaling Modestly Inhi bits Immature Myeloid Cell Expansion Due to the fact that CCR2-MCP-1 inhibition pr ovided little impairment of the immature myeloid cell expansion that occurs during sepsis, we sought to inve stigate other signals that have been shown to govern myeloid cell development a nd expansion in other model systems. The cfms receptor and its ligand M-CSF have been shown to play a pivotal role in the differentiation and proliferation of monocytes and macrophages (107). AFS98, an anti-murine cfms antibody which inhibits M-CSFdependent growth and development by binding to the c -fms receptor, has been shown to have a profound effect on monocyte/macrophage peripher al expansion (114). Therefore, we hypothesized that cfms inhibition by AFS98 may ameliorate secondary lymphoid organ myeloid cell expansion. AFS98 was administered 12 hours prior to sepsis and once daily 83

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thereafter (see Materials and Methods ) for a total of six consecutive days after CLP treatment. We evaluated the bone marrow, lymph node, and splenic myeloid cell populations and determined the amount of myeloid cell expansion compared to isotype control treated septic mice. As shown in Figure 4-7 Panel A and B, AFS98 treatment produced only a modest reduction (25%) in the overall splenic GR-1+CD11b+ population with no differences seen in the bone marrow or lymph node GR-1+CD11b+ populations ( data not shown ) compared to CLP plus isotype control treatment. More detailed analysis reveal a more substantial reduction in the GR1intermediateCD11b+ population analogous to the reduction observed in the CCR2-/septic animals; however, again there was little absolute change in the cell numbers in the overall myeloid population. Further investigati on using CD11b, MHC II, F4/80, mark ers revealed that this GR1intermediateCD11b+ population consists almost entirely of immature monocytes and macrophages ( data not shown ). To evaluate the efficacy of the AFS98 anti-c-fms receptor inhibitor in our model of polymicrobial sepsis, we evaluated the levels of remaining c-fms receptor positive (CD115+) cells after AFS98 administration compared to isotype control treated animals. Interestingly, we found only minimal reduction in the triple positive (GR-1+CD11b+CD115+) cells as shown in Figure 4-7 Panel C. This finding leads to us believe that first, AFS98 inhibition is ineffectual in the setting of murine polymicrobial sepsis based on the plethora of CD115+ cells acquired after AFS98 treatment and secondly, based on the CCR2 and AFS98 findings, the myeloid expansion observed during sepsis is largely due to other expanding populations in the myeloid differentiation pathway a nd not monocytes and macrophages. MDSC Expansion During Sepsis Occurs Inde pendently of Neutroph il Elastase Activity. Since the expansion of myeloid cells occurs largely independent of CCR2MCP-1 and cfms-CSF-1 signaling pathways, we next tested wh ether the MDSC expansi on in the spleen may be the result of G-CSF mediated progenitor eg ression from the bone marrow to the secondary 84

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lymphoid organs. Petit and colle agues previously demonstrated that G-CSF is responsible for bone marrow stem cell mobilization through CX CL12-CXCR4 signaling, via the increased activity of neutrophil elastase (103). Neutrophi l elastase inhibition also decreased stem cell mobilization from the bone marrow. Therefor e, we hypothesized that neutrophil elastase inhibition may ameliorate the bone marrow egression of myeloid progenitor cells and inhibit the expansion of immature myeloid cells in the sp leen. Neutrophil elastase inhibitor (MeOSuc-AlaAla-Pro-Val-CMK 1.5 mg/day I.P) was administered on days 3, 4, 5 and 6 following sepsis. On day 7 during sepsis, we evaluated the bone marro w and splenic myeloid and myeloid progenitor cells. As demonstrated in Figur e 4-8 Panels A and B, there was no appreciable difference in the percentages of splenic GR-1+CD11b+ cells or splenic progenitor cells in mice treated with neutrophil elastase inhibitor compared to mice unde rgoing sepsis alone. Similarly, as shown in Figure 4-8 Panels C and D, there was also no appreciable difference between the numbers of bone marrow myeloid cells or bone marrow progenito rs between neutrophil elastase treated mice compared to mice undergoing sepsis alone. Although neutrophil elasta se activity may be necessary for stem cell migration from the bone ma rrow, our data would indicate that the same does not hold true for the egress of the bone marrow myeloid population. CXCL12 is Required for Complete MDSC Expansion during Sepsis. Since the expansion of the overall myeloid ce ll population is not en tirely dependent on TLR-MyD88, CCR2MCP-1 or cfms -MCSF-1 signaling pathways, we next investigated a pathway more central to immature cell traffickin g. Recent reports have shown that the CXCL12CXCR4 signaling axis is paramount to hematopoi etic progenitor cell bo ne marrow egression and splenic homing along with other cel l populations in the myeloid lin eage. Kelsoe and colleagues have identified CXCL12 signaling as a major de terminant of immature myeloid and lymphoid cell expansion in the spleen and bone marrow using immunization models (115, 116). Moreover 85

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immature myeloid cell expansion in the spleen may be the re sult of G-CSF mediated CXCL12 dependent progenitor egression from the bone ma rrow to the secondary lymphoid organs. Petit and colleagues previously demonstrated that G-CSF is responsible for HSC and progenitor cell mobilization from the bone marrow through CX CL12-CXCR4 signaling.(103). Considering these reports we next hypothesized that the CX CL12 signaling pathway may mediate the splenic myeloid expansion during sepsis. To test this hypothesis we obtained a preparation of antiCXCL12 antibodies (117) (see Materials and Methods ) that have been shown to block CXCL12 in murine models of pulmonary fibros is (118, 119). We administered 200 mg of antiCXCR12 i.p. daily beginning 12 hours prior to CLP sepsis through to 7 days and administered heat inactivated goat serum (200 mL ) i.p. as an isotype control. As shown in Figure 4-9 Panel A anti-CXCL12 treatment during se psis substantially reduced the relative percentage of GR1+CD11b+ splenocytes over 60% compared with isotype treatment and sepsis. Not only were the relative percentages reduced but also the total number of GR-1+CD11b+ splenocytes were reduced to almost the level of sham treatment (Figure 4-9 Pa nel B). In addition, the total numbers of splenic CD3+CD4+, CD3+CD8+, and B220+ cells were unaltered with anti-SDF-1 treatment ( data not shown ). Over the last decade, it has been demonstr ated that CXCL12 signaling is an integral component of not only HSC and progenitor mo bilization, but also for progenitor cell proliferation and survival (120122). When HSCs proliferate th ey give rise to MPPs that differentiate into CLPs in the lymphoid lineage and CMPs that further mature into MEPs and GMPs all of which are bone marrow progenitors in the myeloid lineage (123). We evaluated the bone marrow and spleen for each of the progenitor cell types in the myeloid lineage to determine if CXCL12 inhibition had an impact on immature myeloid cell precursors. At 7 days after 86

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treatment with daily anti-CXCL12 therapy in septic mice, we harvested the bone marrow and spleen and evaluated the relative percentage s and absolute number s of HSCs (LineagenegcKithighsca-1high), CMP (Lineagenegc-Kithighsca-1negCD34highFc Rlow), GMP(Lineagenegc-Kithighsca1negCD34highFc Rhigh), and MEPs (Lineagenegc-Kithighsca-1negCD34negFc Rlow)(123). The gating strategy employed consisted of first eliminating de bris and dead cells based on forward and side scatter and Sytox staining (Figure 4-10 Panel A). Next the lineageneg live non-debris cells were gated based on their c-Kit and sca-1 expression and the c-Kit+ cells were further evaluated based on their Fc R and CD34 expression. As s hown in Figure 4-10 Panels B, C and D, there was little difference in the splenic HSC percentages; how ever, there was a significant decrease in the percentage of total progenitors as well as the specific CMPs in the mice recei ving anti-CXCL12 treatment during sepsis (Figure 4-10 Panels C and D). There were no differences in the splenic MEP or GMP populations ( data not shown ). Inhibiting the CXCR4-CXCL12 signaling axis has been s hown to reduce bone marrow egression of progenitor cells in to the periphery (103). We hy pothesized that if CXCL12 was inhibiting bone marrow egression of progenitor cells that there could possibly be a buildup of progenitor cells in the bone marrow that are unnable to exit to the periphery during CXCL12 inhibition. We evaluated the bone marrow HS C, CMP, GMP and MEP populations, and found no increase in the anti-CXCL12 treated anim als HSC or CMP populations compared with control treatment and sepsis (Figure 4-11 Pane l A and B). Furthermore, there was also no difference in the bone marrow MEP or GMP populations ( data not shown ) between the control and the anti-CXCL12 treatment group. Discussion Although the etiology of sepsis -induced immune dysfunction is unknown, recent interest has focused on the effects of regulatory cell popul ations responsible for the shift from a TH1 to a 87

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TH2 immune profile that results in a state of T cell anergy and immune suppression (42, 85, 98). We have previously described a substantial expansion of a GR-1+CD11b+ myeloid population with an immature phenotype capable of antigen specific CD8+ T-cell suppression and TH1 to TH2 immune polarization. However, the mechanisms driving this immature myeloid cell expansion remain elusive. In the current study, we obser ved that an ongoing sept ic process produces a dramatic expansion of GR-1+CD11b+ cells in the spleen that is independent of CCR2, cfms receptor, neutrophil elastase e ffects, and independent of MyD 88 and TRIF signaling events in the bone marrow. However, what we did demonstr ate is that myeloid expansion during sepsis requires the presence of an intact CXCL12CXCR4 signaling axis to achieve full MDSC expansion. We have previously demonstrated that MDSC expansion in the spl een is delayed in the absence of MyD88 signaling; however, as seps is progresses MDSC expansion begins to approach wild type control levels implying th at MyD88 signaling may be involved in the early events mediating MDSC expansion yet MyD88 may not be mandatory for ultimate splenic MDSC expansion (99). Based on the work of Ki ncade and colleagues (101) demonstrating that HSC proliferation and preferential differentiati on toward the myeloid lineage is dependent on MyD88 signaling in vitro we believe that the absence of MyD88 signaling in vivo may account for the delay in MDSC splenic accumulation we obs erved during long term sepsis. Contrary to this supposition in this study, we found little evidence that HSC or progenitor cell proliferation early in sepsis requires MyD88 signaling. The absence of MyD88 signaling produced no deficiency in bone marrow HSCs, hematopoietic pr ogenitors, or immature myeloid cells (Figures 1, 2, and 3) suggesting to us that MyD88 si gnaling may have little effect on bone marrow myelopoesis in an in vivo model of polymicrobial sepsis Although these findings are in 88

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opposition to those of Kincade and associates, an y more meaningful conclusions are hard to formulate since the model systems used in the re spective studies were very different. While we used an in vivo model of polymicrobial sepsis to ev aluate the impact of MyD88 signaling on hematopoietic progenitors, Kincade et. al. employed an in vitro model where hematopoietic progenitors were placed in cult ure with various TLR agonists. Given the innate complexity of overlapping si gnaling pathways and redundant nature of the host response to injur y, it is not surprising that hematopoie tic cell expansion during sepsis is not dependent on one signaling mediator or pathway. Moreover the fact the MyD88-/mice displayed no reduction in numbers of HSCs, CMPs, and immature myeloid bone marrow cells suggests that there are indeed other signaling mediators involved in HSC proliferation. Two such mediators may be prostaglandin E2 (124) and 5-lipooxygenase (125) both of which have been shown to directly impact HSC proliferation and survival. Several reports have demonstrated that th e CCR2-MCP-1 interaction is necessary for myeloid cell recruitment during times of bacter ial infection (126, 127). Since MDSCs produced increased levels of MCP-1 (Figure 4-1 Panel A) in response to endotoxin stimulation, we postulated that the CCR2 pathway may play a ro le in their expansion during polymicrobial sepsis. However we found few differences in the relative percentages of GR-1+CD11b+ bone marrow and spleen in the CCR2 null mice compared to the wild type controls (Figures 4-1 and 3). The only appreciable difference was observed in the GR-1intermediate CD11b+ population which expanded ~60% less in the CCR2 null mice (Figure 4-2, Panels A and B). Although it was originally proposed that this GR-1intermediate sub-population may represent a more monocytic than granulocytic phenotype, when the GR-1intermediate sub-population was further analyzed, no difference in cell surface expression of F4/80 was found (data not shown) Peters and colleagues 89

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created CCR2-/chimeras and found a reduction in the lung recruitment of F4/80dim macrophages in response to M. tuberculosis infection (100). During L. monocytogenes infection, Sebrina et al demonstrated an accumulation of L6Chigh monocytes in the bone marrow that failed to egress into the circulation in CCR2 null mice (104). The differences in CCR2 dependency are likely due to the complexity of the polymicrobial sepsis model, when compared to a single microbial insult used in the other reports. Unlike a single microbial infection, polymic robial sepsis exposes the host to a multitude of intestinal pathogens and microbial products (PAMPS) that signal through multiple overlapping signaling pathways (TLR dependent and independent) that appear not to be dependent upon a functional CCR2. Realizing that myeloid expansion during se psis is CCR2 independent, we evaluated an alternative pathway that could account for the myel oid proliferation. Petit demonstrated that GCSF mediated neutrophil elas tase degradation of bone marrow CXCL12 was responsible for bone marrow progenitor egression into the blood (103). Postulating th at egression of bone marrow progenitor cells could be responsible fo r the splenic myeloid expansion, and since we have observed an increase in splenic progenito r cells in C57BL/6 mice (Figure 4-6 Panel C and D), we administered a neutrophi l elastase inhibitor in an attempt to block bone marrow progenitor egression at doses that were effective in earlier studies (103). The results of the experiment were equivocal implying that neut rophil elastase plays no significant role in polymicrobial sepsis MDSC expansion. CXCL12 has been demonstrated by Rafii et. al to play an essential role in hematopoietic progenitor cell mobilization and progenitor cell homing. More specifically, the authors demonstrate that in response to physiologic st ress, plasma CXCL12 levels rise and mediate hematopoietic cell mobilization and repopulation of peripheral organs. Others have found using 90

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models of inflammatory stress such as cardiac surgery (128), tuberculos is infection (129) and breast cancer tumor models (130 ) that CXCL12 is required for hematopoietic progenitor cell peripheral recruitment. In a m odel of malarial infection, de A ndrade demonstrated that splenic CXCL12 levels continued to increase until the in fection was under control in C57BL/6 mice and facilitated CD11c+ dendritic cell recruitment to the spleen (131). Our data support this notion of CXCL12 mediated recruitment gi ven that fact that CXCL12 i nhibition substantially reduced splenic MDSC accumulation (Figure 4-9). More importantly, anti-CXCL12 therapy produced an even greater reduction in the numbers of splenic CM Ps-the precursor that gi ves rise to the other myeloid lineage cells (Figure 4-10). This suggests, although with further investigation necessary, that MDSCs arise from CMPs that have migrated from the bone marrow to the spleen and undergo myelopoesis generating the dramatic numbers of myeloid cell observed in the spleen during long term sepsis. In conclusion, we observed that an ongoing se ptic process produces a dramatic expansion of GR-1+CD11b+ cells in the spleen that is independe nt of CCR2 and c-fms receptor signaling, bone marrow MyD88 signaling, and neutrophil elastase activity. In contrast we found that the splenic MDSC population is pa rtially dependent on CXCL12 signaling. CXCL12 depletion substantially reduced the accumu lation of the MDSC population in the spleen during sepsis. Furthermore, CXCL12 inhibition also reduced the CMP-the earliest myeloid precursor cell type suggesting that MDSC expansion de pends on CMP splenic expansion. Materials and Methods Mice All experiments were approved by the Institutional Animal Care and Use Committee at the University of Florida College of Medicine or Schering-Plough Biopha rma. Specific pathogenfree C57BL/6 mice and CCR2 null (B6.129S4-Ccr2tm 1Ifc/J) mice were purchased from The 91

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Jackson Laboratory (Bar Harbor, ME). All mice were maintained at the University of Florida College of Medicine and were studied between 6-12 weeks of age. MyD88-/mice on a B6x129(F1) background and TRIF-/mice were a kind gift of Dr. Shizuo Akira to ScheringPlough Biopharma, and were maintained at Schering-Plough Biopharma, Palo Alto, CA. Inhibitors When indicated, mice were injected with 500 L/day i.p polyclonal goat anti-CXCL12 antibodies beginning 12 hours prio r to the initiation of seps is. The anti-CXCL12 antibody preparation was a gift from Dr. Ro bert Strieter at the University of Virginia, Charlottesville, VA (117). As a control heat inactiv ated polyclonal goat serum (Sigma) was used (500 L/day i.p). AFS98, a rat monoclonal anti-murine cfms antibody (IgG2a), which inhibits M-CSFdependent growth and development by binding of M-CSF to its receptor (114) was also used when indicated. The AFS98 hybridoma cell line was a gift from Dr E. Rich ard Stanley at Albert Einstein College of Medicine, Bronx, NY. The AFS98 hybridoma was cultured and the AFS98 antibody harvested and purified by Klaus Lubbe (Bio Express, Inc. West Lebanon, NH). Two milligrams of AFS98 in 200 L of PBS were administered i.p. per mouse daily, PBS or 2 mg (200 L/i.p. daily) of an irreleva nt isotype-matched rat IgG used as described (108, 109) as a control. Mice were also treated w ith the neutrophil elastase inhibitor, MeOSuc-Ala-Ala-ProVal-CMK (1.5 mg/day) by intraper itoneal injection (Calbiochem, La Jolla, CA) on days 3, 4, 5 and 6 following the induction of sepsis (103). A ll inhibitors were injected 12 hrs before the induction of sepsis and continued daily as described for 6 consecutive days. Cecal Ligation and Puncture For induction of polymicrobial sepsis, mice underwent sham laparotomy or cecal ligation and puncture induced by ligation of the cecum and a double enterotomy created with a 27 gauge needle. Mortality in this model was approxima tely 10-15%, and occurred predominantly in the 92

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first 3 days; thereafter, surviving mice develo ped abscesses surrounding the devitalized cecum as previously described (77, 95, 99). Flow Cytometry Spleens and bone marrow cells were analyzed by flow cytometry as previously described (95, 99). Antibodies included an ti-GR-1 (Ly6G and Ly6C (RB6-8C5)) conjugated to APC, antiCD11b (Integrin aM, chain Mac-1a chain (M1/70)) conjugated to Pacifi c Blue, anti-MHC II (IA/I-E (2G9)) conjugated to FITC, anti-F4/ 80 Antigen (Pan Macrophage Marker (BM8)) conjugated to PE, Fc-Block (CD16/CD32 Fc g III/II Receptor (2.4G2), Lineage Cocktail conjugated to biotin [CD3e(145-2C11), CD 11b(M1/70), CD45R/B220(RA3-6B2), Ly6G and Ly6C(RB6-8C5), TER-119(TER-119)], Sca-1 conjugate d to either PE (D7) c-Kit conjugated to either FITC or APC (2B8). CD34 c onjugated to Alexa Fluor 647 (RAM34), Fc R conjugated to Pac Blue (CD16/32 clone 93), and Syt ox Blue. F4/80, CD11c, CD34, and Fc R specific antibodies were purchased from eBioscience a nd all other antibodies we re purchased from BD Pharmingen. Spleens, peripheral blood, and bone marrow were harvested after either CLP or Sham surgery and single cell suspensions were created by passing the cells through 70 m pore sized cell strainers (Falcon). Er ythrocytes were then lysed usi ng ammonium chloride lysis buffer and washed two times using PBS without calci um, phenol red, or magnesium. Samples were acquired and analyzed using a LSRII flow cyto meter (BD Biosciences). A minimum of 5 x 104 live non debris cells (Sytoxnegative) were collected and analyzed. Ex vivo Stimulation and Cytokine Production Enriched GR-1+ cells were plated at 1 x 106 cells/well with RPMI 1640 supplemented with 10% fetal calf sera, 2 mM L-glutamin e, 200 units/ml penicillin and 50 g/ml streptomycin, and stimulated with 10 g/ml of bacterial lipopolysaccharide ( E. coli 0111:B4) The culture supernatant was analyzed for cytokines usi ng Luminex technology using reagents obtained 93

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from Upstate Cell Signaling Solutions (Beadly te Mouse Multi-Cytokine Detection System) (Temecula, CA). Statistics Continuous variables were first tested for nor mality and equality of variances. Differences among groups in flow cytometric analyses were evaluated by analysis of variance for multiple groups and Students t-test for two groups. Significance was designated at the 95% confidence level. 94

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Figure 4-1. The absence of MyD88 and TRIF signaling pathways does not impact HSC expansion during sepsis. MyD88-/-, TRIF-/-, and B6.129 wild type mice underwent either sham or CLP induced sepsis. At da ys 1 and 7 after seps is, induction total bone marrow cells were harvested and analyzed for HSCs via flow cytometry. Panel A. The relative percentage of HSCs (Lineagenegc-kithighsca-1high) is elevated at 24 hours and remains elevated through 7 days after se psis initiation in wild type B6.129 mice. Panels B and C. MyD88-/and TRIF-/mice demonstrated the same increase in HSCs as did the wild type mice in Panel A at bot h 1 and 7 days after sepsis induction. This implies that HSC proliferation occurs independently of both MyD88 and TRIF signaling during sepsis. Values represent th e mean ( S.E.M.) of between three and five samples. p< 0.05 by Students t-test. 95

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Figure 4-2. MyD88 and TRIF signaling do not impact bone marrow progenitor expansion during sepsis. MyD88-/-, TRIF-/-, and B6.129 wild type mice unde rwent either sham or CLP induced sepsis. At days 1 and 7 after sepsis induction total bone marrow cells were harvested and analyzed for bone marrow proge nitor cells via flow cytometry. Panel A. The relative percentage of bone marrow progenitors (Lineagenegc-kithighsca-1neg) is unaltered at 24 hours, however increases 2 fold over sham levels at 7 days after sepsis initiation in wild type B6.129 mice. Panels B and C. MyD88-/and TRIF-/mice demonstrate the same unaltered levels of bone marrow progenitors as do the wild type mice at 24 hours after sepsis induction. Both the MyD88-/and TRIF-/animals exhibit 2 fold increases in bone marrow progeni tor levels 7 days after sepsis induction implying that bone marrow progenitor expansion occurs independently of both MyD88 and TRIF signaling during sepsis. Va lues represent the mean ( S.E.M.) of between three and five samples. p<0.05 by Students t-test. 96

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Figure 4-3. MyD88 and TRIF signaling do not eff ect immature myeloid cell expansion in the bone marrow during sepsis. MyD88-/-, TRIF-/-, and B6.129 wild type mice underwent either sham or CLP induced sepsis. At da ys 1 and 7 after seps is, induction total bone marrow cells were harvested and analyzed for immature myeloid cells via flow cytometry. Panel A. The relative per centage of immature myeloid cells (GR1+CD11b+) is reduced at 24 hours as a result of bone marrow degra nulation into the circulation, however increases by 30% over sham levels at 7 days after sepsis initiation in wild type B6.129 mice. Panels B and C. MyD88-/and TRIF-/mice demonstrate the same reductions in immature myeloid cells as do the wild type mice 24 hours after sepsis. Both the MyD88-/and TRIF-/animals exhibit significant increases in bone marrow immature myeloid cell levels 7 days after sepsis induction. This data implies that bone marrow i mmature myeloid cell expansion occurs independently of both MyD88 and TRIF signaling during sepsis. Values represent the mean ( S.E.M.) of between three and five samples. p< 0.05 by Students t-test. 97

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Figure 4-4. Splenic and bone marrow MDSC ex pansion is not dependent on CCR2 receptor signaling pathway. Panel A. GR-1+ splenocytes harvested seven days after CLP or sham treatment produce MCP-1 upon ex vivo stimulation with 10 g/ml of bacterial lipopolysaccharide. Panel B. Flow cy tometry dot plot of viable, GR-1+CD11b+ total splenocytes from wild type C57BL/6 or CCR2 null mice 7 days after sham or CLP treatment. Panel C and Panel D. Percentage of GR-1+CD11b+ total splenocytes and bone marrow cells recovered fromC57BL/6 wild type or CCR2 null mice at 1 and 7 days after CLP or sham treatment. The absence of a functional CCR2 receptor has no significant effect on the expansion of the GR-1+CD11b+ population of immature myeloid cells in the spleen or bone marrow. Values represent the mean ( S.E.M.) of between three and five samples. p<0.05 by Students t-test. 98

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Figure 4-5. CCR2-/mice exhibit fewer GR-1intermediate cells. Panel A. Although the same number of total events was collected in each pl ot, the CCR2 null animal s displayed fewer GR1intermediate cells at base line and at 7 days after sepsis treatment. Panel B. While there was a 7 fold increase in the GR-1intermediate population in the wild type animals there was only a 3 fold increase in the CCR2 null mice. Values represent the mean ( S.E.M.) of between 3 and five samp les. p<0.05 by Students t-test. 99

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Figure 4-6. Splenic and bone ma rrow expansion of progenitor cel ls is not dependent on CCR2 receptor signaling pathway. Total bone marro w and spleen cells were harvested at 1 and 7 days after CLP or sham treatment and the percentage of bone marrow and splenic progenitor cells were determin ed by immune phenotyping the Lineage-ckit+sca-1fraction of viable cells using flow cytometry analysis. Panel A. Flow cytometry dot plot of the c-kit+sca-1bone marrow progenitor cells (gated off of the Lineagecells) from wildtype (C57/BL6) and CCR2 null mice 7 days after CLP and sham treatment. Panel B and C. The absence of a functional CCR2 receptor (CCR2 null mice) has no effect on the percentage of bone marrow progenitors seven days after sham and CLP treatment. Panel C. Although significance between sham and CLP treated animals was achieved, ther e was no significant difference in the expansion of the splenic progenitor populati on 7 days after sepsis treatment between the CCR2 null animals and the wild type c ontrol (C57BL/6) animals. Both the CCR2 null and the C57BL/6 wild type control mice exhibited an equivalent 3 fold increase in the percentage of Lin-c-kit+Sca-1cell fraction. Values represent the mean ( S.E.M.) of between 3 and five samp les. *p<0.05 by Students t-test. 100

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Figure 4-7. Splenic MDSC expa nsion is not dependent on cfms receptor signaling. Panel A. Flow cytometry dot plot of viable, GR-1+CD11b+ total splenocytes from mice that underwent sham, CLP alone or CLP with AFS98 (ant-cfms receptor) treatment daliy for 7 days. Panel B. Graphic representati on of the modest reduction in the relative percentages of immature myeloid cells observed after CLP plus daily AFS98 treatment compared to sham and CLP alone. Panel C. Although AFS98 is an anti-cfms receptor inhibitor, AFS98 treatment only minimally reduced the percentage of cfms (CD115) positive (GR-1+CD11b+CD115+) cells in the spleen 7 days after the initiation of sepsis. Values represent the mean ( S.E.M. ) of between three and five samples. p<0.05 by Students t-test. 101

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Figure 4-8. Splenic and bone marrow expansio n of immature myeloid and bone marrow progenitor cells is not dependent on neutrophil elastase activity. Total bone marrow and spleen cells were harvested 7 days after CLP or sham treatment and the percentages of bone marrow and splenic immature myeloid cells and progenitor cells were determined by phenotyping the viable cell population using flow cytometry analysis. Bone marrow and splenic progenitors were defined as c-kit+sca-1cells gated off of the lineagebone marrow and spleen populations. Panel A and Panel B represent the immature myeloid and progeni tor populations from the spleens of mice 7 days after either CLP treatment alone or CLP treatment with neutrophil elastase inhibitor administration. Neutrophil elasta se inhibition had no significant effect on the expansion of splenic pr ogenitor or immature myeloid cell populations. Panel C and Panel D represent the immature myel oid and progenitor populations from the spleens of mice 7 days after either CLP treatment or CLP treatment with neutrophil elastase inhibitor administ ration. Neutrophil elastase inhibition had no significant effect on the expansion of splenic progen itor or immature myeloid cell populations. Panel C and Panel D represent the immature myeloid and progenitor populations from the bone marrow of mice 7 days after CL P treatment or CLP treatment with neutrophil elastase inhibitor administrati on. Neutrophil elastase inhibition had no significant effect on the expansion of bone marrow progenitor or immature myeloid cell populations. Values represen t the mean ( S.E.M.) of between 3 and 5 samples. *p<0.05 by Students t-test. 102

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Figure 4-9. Splenic expansion of myeloid derived suppressor cells is dependent on CXCL12 signaling. Total spleen cells were harvested 7 days after CLP or sham treatment and the percentages of splenic MDSCs dete rmined by phenotyping the viable cell population using flow cytometry analysis. Splenic MDSCs were defined as GR1+CD11b+ cells gated off spleen populations. Panel A and Panel B represent the immature myeloid cells from the spleens of mice 7 days after either CLP treatment alone or CLP treatment with anti-CXCL12 treatment. CXCL12 depletion reduced the splenic GR-1+CD11b+ immature myeloid cell populat ion by over 60% compared with CLP alone. Values represent the mean ( S.E.M.) of between 3 and 5 samples. *p<0.05 by Students t-test. 103

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Figure 410. CXCL12 inhibited the spleni c accumulation of common myeloid progenitors during sepsis. Total spleen cells were ha rvested at 7 days after CLP or CLP with anti-CXCL12 treatment and the perc entage of splenic HSCs (Lineagenegc-kithighsca1high), total progenitor cells (Lineagenegc-kithighsca-1neg), and CMPs (Lineagenegckithighsca-1negCD34highFc Rlow) were determined by immune phenotyping the fraction of viable cells using flow cytometry an alysis. Panel A. Flow cytometry gating strategy. The live non debris cells were gated based on forward side scatter and Sytoxneg. The Lineage negative cells were th en gated based on c-Kit and sca-1 expression and the Lineagenegc-Kithighsca-1neg cells were further analyzed based on their CD34 and Fc R expression. Panel B depicts the in crease in splenic HSCs that occurs during sepsis and is unaltered by anti-CXCL12 treatment. Panel C illustrates the overall reduction in the splenic progen itor population after 7 days of CXCL12 inhibition. Panel D shows the reduction in the specific CMP population in the spleen at 7 days after CXCL12 depletion. Valu es represent the mean ( S.E.M.) of between 3 and five samples. *p<0.05 by Students t-test. 104

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105 Figure 4-11. Bone marrow HSC and CMP expansion does not depend on CXCL12 during sepsis. Seven days after sham, CLP or CLP with anti-CXCL12 tr eatment, total bone marrow cells were harvested and analyzed via flow cytomery for the quantity of HSCs (Lineagenegc-kithighsca-1high) and CMPs (Lineagenegc-kithighsca1negCD34highFc Rlow). Panel A demonstrates th at there is no impact of the percentages of HSCs in the bone marrow w ith anti-CXCL12 treatment, however there is a significant increase in the percentage of HSCs during sepsis compared to sham treatment. Panel B. Although there was a s ubstantial increase in the percentage of CMPs after sepsis initiation, no effect on the CMP population was observed after anti-CXCL12 treatment compared to sepsis alone. Values represent the mean ( S.E.M.) of between three and five sa mples. p<0.05 by Students t-test.

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CHAPTER 5 MYELOID-DERIVED SUPPRESSOR CELLS AND THEIR CONTRIBUTION TO POSTINJURY AND SEPSIS IMMUNE SUPPRESSION Introduction Sepsis occurs when an overwhelming microbial infection leads to a systemic inflammatory response, manifesting clinically as fever, leuko cytosis, and reduced vasc ular resistance, often leading to multi-system organ failure and death. Despite progress over the past 2 decades sepsis remains the leading cause of death in the intensive care un it with over 750,000 cases and 210,000 deaths annually in the United States (1, 2) Many significant advancements in sepsis pathophysiology have occurred (3, 4), but unfortunately, this pr ogress has had only a minimal impact on the mortality rate (1, 2). A number of approaches, including anti-tumor necrosis factor(TNF) therapies, corticos teroids, antibodies against endotoxin, inhibitors of prostaglandins, bradykinins, and interleukin (IL)-1 receptor antagonist, have all failed in clinical trials (5). The only efficacious se psis-related therapies currently available are activated protein C administration (XigrisTM) (6), replacement steroids for sepsis-associated adrenal insufficiency (7), and insulin therapy for blood glucose mainte nance (8). However, as a monotherapy or in combination, these approaches still only modestly improve outcome (5). Sepsis and Immune Dysfunction Much of the early work on sepsis-induced immune dysfunction focused on inflammation and the cytokine storm that characterized the ear ly response to microbial invasion. It has also been known for several decades that both severe in jury and sepsis simultaneously produce a state of immune suppression illustrated by a loss of delayed type hypersensitivity (11), an inability to eradicate primary infections (12), a predispositio n to develop secondary nosocomial infections (12, 13), and a failure to respond to skin testing with specific antigens (11, 132). Furthermore, animal models of sepsis indica te that acquired immune dysfuncti on is an intrinsic property of 106

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sepsis, and results from defects in both the innate and acquired immune responses. Monocyte deactivation (133, 134), apoptosis induced depletion of CD4+ T cells (20) and dendritic cells (21, 135), dendritic cell exhaustion or paralysis (22), suppression of T-cell proliferative responses (24, 26, 136), and reduced inflammatory and TH1 cytokine production by monocytes and tissue macrophages (28), all contribute to immunologic compromise during sepsis, and culminate in a shift from a more proinflammatory TH1 to a more anti-inflammatory TH2 immune profile (4). Moreover, attention has focused on the TH1 to TH2 immune profile shift as an explanation for post-sepsis immune suppression (34, 137); however, the underlying mechanisms that orchestrate the shift in immune polarization during sepsis are still unknown. Myeloid Derived Suppressor Cells Play a Ro le in Injury, Sepsis and Trauma In recent years, there has been increasing interest in the role that regulatory cell populations play in the immune suppression that accompanies sepsis. There are a number of different regulatory cell populations that could potentially be important for the development of sepsis-induced immune suppression, including regulatory T cells (natural Tregs, TH3 and TR1 cells), regulatory dendritic cells (DC3 cells), and myeloid-derived s uppressor cell (MDSC) populations. Natural regulatory T cells have been recently hypothesized to contribute to the acquired immune deficits that occur in human sepsis (42). Recent studies have demonstrated that after a burn injury in rodents (43, 138), or after trauma in human subject s (139), there is an increased number of natural regulatory T cells in the draining lymph nodes and blood, respectively. Oppenheim has recently reported that depletion of regulatory T cells wi th an anti-CD25 antibody reduced mortality to polymicrobial sepsis (140 ). Those findings, however, have not been confirmed by other investigators. We and the Ayala lab have independent ly demonstrated that natural regulatory T cells are onl y transiently increased in murine polymicrobial sepsis, but both 107

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of us were unable to demons trate that this expansion of endogenous regulatory T cells contributes significantly to th e immune suppression or outcome in this model (86, 141). Due to the inability of T regulatory and re gulatory dendritic cell populations to fully explain the observed post-injur y and post sepsis immune suppre ssion, investigators have begun to search for other immunologica lly active cell types that may be responsible for mediating the observed post-injury induced immune dysfunction. One of the more recent candidate cell types focused on by investigators is a heterogeneous group of myeloid derived suppressor cells (MDSCs)(91). Currently very little is known about the origins and suppressor function of MDSCs. Much of what we know about these cel l populations comes from the cancer literature, although in the past two years, th ere has been a dramatic increase in the interest in these cell populations in surgical trauma (68), se psis (99), and burn injury (142-144). We do know that MDSCs are a heterogeneous subpopulation of immature cells of the myeloid lineage that are probably both de rived from the bone marrow and/or develop independently in secondary lymphoid organs-mainly the spleen but also the lymphnodes to a lesser degree. Hematopoiesis occurs normally in the bone marrow, but after acute inflammatory conditions, such as in burns, trauma and se psis, extramedullary hematopoiesis and more specifically, myelopoiesis, increases dramatical ly in the spleen.(45, 145, 146) In mice, for example, it is well known that burn injury induce s increased numbers of myeloid progenitors in the spleen, with maximal levels occurring after one week (143). We have also seen increased numbers of myeloid progenitors in the bone marrow of mice early after a se ptic event (99). The expansion of the MDSC population a ppears to be secondary to the normal reprioritization of the bone marrow and spleen in the injured or septic host to promote myelopoies is at the expense of both lymphopoiesis and erythropo iesis (101). When HSCs proliferate they give rise to 108

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multipotent progenitors (MPPs) that lose their ability to differentiate as they mature into cells in the lymphoid and myeloid lineages. MMPs diffe rentiate into common lymphoid progenitors (CLPs) in the lymphoid lineage and common myeloi d progenitors (CMPs) which further mature into megakaryocyte erythrocyte progenitors (M EPs) and granulocyte macrophage progenitors (GMPs)-all of which are bone marro w progenitors in the myeloid lineage (123). Under times of trauma, burns and immune stress hematopoietic progenitors from the bone marrow egress into the circulation and home to periphera l organs to initiate repair and regenerative processes (102). We see this process first hand in the spleen 7 da ys after sepsis with a substantial increase in HSCs, CMP, GMPs, MEPs, and GR-1+CD11b+immature myeloid cells illustrating the shift toward the myeloid lineage and the simultaneous contribution that both the bone marrow and spleen make to this post-injury myelopoesis. In the past, these cells in the spleen and ly mph nodes have been referred to as: natural suppressor cells (44), myeloid derived suppressor cells (45), early myeloid cells (46), and inhibitory macrophages (47). Phenotypically, these cells exhi bit a high expression of cell surface markers CD11b and GR-1. However, other ce lls of myeloid lineage can also express low levels of these receptors, such as more mature macrophages and neutrophil s. Figure 5-1 shows a cytospin preparation of GR-1+ cells obtained from the spleen of a mouse ten days post-induction of a cecal ligation and puncture. As is evid ent from a simple Wright stain, the cells are phenotypically diverse with groups of both im mature monocyte-like and neutrophil like cell forms. Other cell surface markers that these MD SCs express include ER-MP54, ER-MP58, CD115, CD31, F4/80, c-Kit, and Fc R (80, 83, 99, 116, 123, 147). Table 5-1 represents several recent attempts by various authors to define the cell surface phenotype of the MDSC population 109

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in three different injury models It is evident that the popula tion is heterogenous and immature; however, there are many similarities in the cel l surface markers between models especially the markers GR-1 and CD11b. We do know, however, that a large proportion of these cells are immature, proliferating, committed to a myeloid lineage, and responsive to growth factor stimulation. As shown in Table 5-2, when GR-1+ cells were harvested from the spleen of a sham mice and cultured with either GM-CSF, G-CSF or erythrop oietin (99), there was no eviden ce of either proliferation or the generation of myeloid col onies. Conversely, when GR-1+ cells were harvested from the spleen of a mouse 10 days after polymicrobial seps is, a large number of colonies were generated in response to culture with GM-CSF and GCSF, but not to erythropoietin (99, 144). Approximately 22% of the enriched GR-1+ placed in culture with GM-CSF became CD11c+ conventional dendritic cells, and 18% differentiated into F4/80+ macrophages. Similar results were reported by Ogle and colleagues in which splenocytes from eight day burned mice (but not sham controls) were incubated with either M-CSF (CSF-1), GM-CSF or G-CSF, and dramatic increases in the numbers of col onies were observed (142). Under in vivo conditions, we have also observed that treatment of septic mice with the nucleosid e analogue, gemcitibine (Figure 52), completely prevented the expansion of this CD11b+GR-1+ population in the spleen and lymph nodes, confirming that under in vivo conditi ons, these cells are rapidly dividing. In addition, we are beginning to resolve the signaling pathways which drive the expansion of MDSCs in sepsis. In one of the earliest studies looking at these my eloid derived suppressor cells, Holda demonstrated that very modest inj ections of lipopolysaccharide markedly enhanced what he termed natural suppressor cell activity ( 66). We also showed that administration of lipopolysaccharide and the TLR5 agonist, flagellin, significan tly increased the number of 110

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CD11b+GR-1+ cells in the spleen and lymph nodes ((99) and unpublished findings ). Almost four years ago, Murphey, Sherwood and colleagues repor ted that five days following polymicrobial sepsis, there was a near two-fold increase in the number of what they te rmed natural suppressor cells in the spleens of mice (6 7), although they did not look at suppressor activity directly. Around the same time Cora Ogles group in Cinci nnati reported that macrophage progenitors increased in the spleen 10-20 fold eight days after a scald burn (143). In 2006, Dr. Juan Ochoa and colleagues at the University of Pittsburgh reported a seven fold increase in the CD11b+GR1+cell population in the spleens of mice 12 hours following an experimental laparotomy (68). All of these studies sugg est that expansion of an immature myeloid suppressor cell population is a common finding in a number of acute inflammato ry states, including su rgical injury, trauma, burns and sepsis. Over the past couple of years, we too have been exploring under what inflammatory conditions does this expansion of this immatu re myeloid cell population occurs, and whether the expansion of this cell population is an integral component of the injury response. One of the most striking findings is that rather modest noninfectious infl ammatory states produce rather immediate and significant increases in the numbers of these cell populations. As previously noted, Ochoa and colleagues showed that a surgical injury dramatically increased the numbers of these cell populations in the spleen within 12 hours (68). In our own studies where we performed a sham surgical procedure to mobili ze the bowel, and then cl osed the peritoneal cavity, we observed a 50% increase in the CD11b+GR-1+ population after 3 days. (Table 5-3) We also saw a similar expansion of the populatio n in mice that underwent a surgical procedure and 45 minutes of warm ischemia to the liver ( Unpublished observations of OMalley and Moldawer) Similar to the findings of Ogle and colle agues, a scald burn in the mouse produced a 111

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significant expansion of the CD11b+GR-1+ cells in both the spleen and the draining lymph nodes that increased with th e size of the burns ( Unpublished data of Moreno and Moldawer) Such data suggest that surgical inflammation and noni nfectious stimuli can produce expansion of these immature cell populations. With that said, however, the most dramatic changes in the expansion of these cell populations occur in sepsis or in microbial infections. Using e ither a cecal liga tion and puncture or a fecal peritonitis model of polymicrobial seps is (148), the increases in the expansion of these cell populations were dramatic and often resulted in over 40% of the sp leen cellularity being MDSCs. In addition, when Pseudomonas was injected subcutaneous ly in healthy and scald burned mice, increased numbers of CD11b+GR-1+ cells were observed in both spleen and lymph nodes, and when Pseudomonas was superimposed on the burn injury, the numbers of CD11b+ GR-1+ cells increased dramatically ( Unpublished data of Moreno and Moldawer) According to the data presented in Table 3 from injury models in our own laboratory and the laboratories of other investigators, it is apparent that although non-infectious insu lts such as organ ischemia or acute trauma produce significant increases in the MDSC population, the most dramatic expansion in the MDSC population is produced from infectious stimuli. Furthermore, the longer the infection continues, the more substantial the myeloid cell accumulation in the primary and secondary lymphoid organs. These findings suggest that although both noni nfectious and infectious challenges can increase the expansion of thes e cell populations in the spleen and lymph node, infectious challenges, and those that ar e ongoing and sustained, generally produce the greatest and most profound expansion. Along the same lines, polymicr obial sepsis appears to produce a delayed expansion of these cell populations, as after surgical injury, these CD11b+GR-1+ cells peaked in 112

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the spleen 12-48 hours after the insult, whereas in the cecal ligation and puncture models of sepsis, total CD11b+GR-1+ cells decreased in the first 24 hours, and peaked at 7-10 days. There is, however, an important caveat, and that is that we are dealing with heterogenous cell populations that may not be identical with the di fferent injury models. For example, we have reported that the CD11b+GR-1+ cells recovered from the spleens of septic mice express extremely low levels of MHC class II (<5%), and are localized in periarterial and subcapsular regions of the spleen (99), whereas the CD11b+GR-1+ cells identified by Ochoa and colleagues have a much higher proportion of MHC class II expression and are located in the mantle surrounding the lymphoid follicles (68). Such findings suggest that the heterogenous populations may be similar, but still phe notypically and functionally distinct. Since the expansion of the MDSC popul ation appears to be a highly conserved phenomenon that occurs in response to both infe ctious and non-infecti ous stimuli, we also examined the signaling pathways responsible for their invocation. Because both bacterial lipopolysaccharide and flagellin c ould increase the expansion of this cell populat ion, a natural first focus was on signaling pathways derived from the family of TLR receptors and mediated through MyD88 and TRIF signaling pathways. There is growing appreciation that the ligands for TLR signaling involve not onl y microbial products, but also endogenous products, such as heat-shock proteins, mammalian nucleic acids and HMGB1, that can serve as endogenous danger signals (149). Simultaneously, since the pr oduction of type I interferon is a consequence of TLR activation, we examined whether thes e signaling pathways were required for the expansion of this cell population. Surprisingly, we found that mice defective in either TLR4 signaling, TRIF or type I interferon had no attenuation in their ability to increase the numbers of MDSCs in the bone 113

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marrow, or in secondary lymphoid organs, in response to a cecal ligation and puncture (99). Similarly, mice deficient in IL-10, IL-4, and IL-6 (data not shown) also have a normal expansion of their MDSC population. Despite the fact that a TLR4 agonist could induce the expansion of this cell population, the absence of TLR4 did not prevent any increase in response to sepsis, suggesting that TLR4 signaling could induce expansi on of this population, but wasnt required in polymicrobial sepsis. In contrast, the absence of MyD88 signaling appeared to delay the expansion of these cells over the first seven days of a cecal ligation and puncture, which began to approach wild type levels at 14 days. Since My D88 signaling is linked to both the recognition of microbial products and endogenous danger signals, the requirement for MyD88 would suggest that the expansion of the MDSC population is a fundamental compone nt of the host response. So the question remains: do MDSCs themselv es require MyD88 signaling necessary to either emigrate from the bone marrow to the secondary lymph organs, or to promote extramedulllary hematopoiesis or myelopoesis, or is MyD88 signaling required to facilitate the mediators that do control myel opoesis? Bronte administered GM-CSF to healthy mice and showed a modest expansion of the MDSC popu lation, suggesting that GM-CSF may contribute, but cannot recapitulate in its entirety, the expa nsion seen in inflammation (150). Ramphal and associates demonstrated that MyD88 null mi ce lack the ability to produce G-CSF after Pseudomonas infection which directly reduced neutr ophil recruitment to the lung and increased mortality(151). The expansion of these MDSCs ma y also simply be a component of the larger extramedullary hematopoietic response that is c onserved in many animal models of acute injury. We definitely witness evidence of extramedullary hematopoiesis be ginning at three days after the initiation of sepsis, and as seen in Figure 5-3; by 10 days there is a fourfold increase in the number of megakaryocytes in the spleens of se ptic mice. Whether the myeloid expansion is a 114

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required component of extramedullary hematopoies is or occurs independently remains to be seen. We have previously reported that complete splenic expansion of the MDSC population has some dependence on MyD88 signaling which diminishes with sepsis progression beyond 7 days (99). Several reports also indicate that HSC pr oliferation and preferenti al differentiation into myeloid lineage cells is dependent on the My D88 signaling pathway in vitro (101, 110-112). Since MDSCs are an immature heterogeneous co ntingent of cells at various stages of development in the myeloid differentiation path way we speculated, that HSC proliferation and myeloid differentiation should be impaired using an in vivo system devoid of MyD88 signaling, and may account for the modest delay that we have observed in MDSC expansion in MyD88-/mice during sepsis. Much to our surprise, we discovered that there was no MyD88 dependency on HSCs (Lineagenegc-KithighSca-1high) proliferation in the bone marrow after sepsis. More importantly and in contrast to Kincade and associates (101), we found that MyD88-/and TRIF-/animals exhibited the same increase in HSC numb ers at 24 hours and 7 days post-sepsis as did sham and wild type control animals. This finding suggests that MyD88 and TRIF signaling are not essential for early or prolonged HS C proliferation in vivo during sepsis. When HSCs proliferate they give rise to multipotent progenitors (MPPs) that lose their ability to differentiate as they mature into cells in the lymphoid a nd myeloid lineages. MMPs differentiate into common lymphoid progenito rs (CLPs) in the lymphoid lineage and common myeloid progenitors (CMPs) which further mature into megakaryocyte erythrocyte progenitors (MEPs) and granulocyte macrophage progeni tors (GMPs)-all of which are bone marrow progenitors in the myeloid lineage. After finding a substantial elevation in the HSC numbers in MyD88-/mice within 24 hours after sepsis, we next sought to determine if this HSC increase 115

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actually translated into incr eased numbers of bone marrow pr ogenitor cells and whether the absence of MyD88 would hinder HSC differentiation along the myeloid lineage in vivo. When we evaluated the bone marrow progenitor population (Lineagenegc-Kithighsca-1neg) in the MyD88 and TRIF mice after sepsis, we f ound little change in progenitor num bers at 7 days post sepsis compared to wild type controls. This finding implies that although HSC differentiation in vitro may be dependent on MyD88 mediated TLR signaling, that in vivo HSC differentiation into progenitor cells can occur in the absence of MyD88 signaling. Although we did not observe a difference in the relative fluctuations in HSC and progenitor populations between wild type, MyD88-/and TRIF-/mice, we postulated that a MyD88 dependent differentiation between the progenitor population a nd more terminally differentiated myeloid cells may exits and halt progenitor maturation accounting for the increases in progenitor cell obs erved in the MyD88-/and TRIF-/mice at 7 days post sepsis. In contrast, the same immature myeloid cell (GR-1+CD11b+) fluctuations occur in all three mouse types at 24 hours and 7 days after sepsis initiation. By 7 days after CLP sepsis, there was an equivalent and significant increase in the immature myeloi d population in wild type, MyD88 and TRIF null mice indicating that differentiation from the HSC population through th e progenitor population and on to the immature myeloid population can occur in the absen ce of MyD88 and TRIF signaling in vivo. We have demonstrated that myeloid e xpansion during sepsis is modestly CCR2 independent, and evaluated some alternative path ways that could account for the splenic myeloid expansion. Petit demonstrated that G-CSF medi ated neutrophil elastase degradation of bone marrow CXCL12 was responsible for bone marrow pr ogenitor egression into the blood (103). Postulating that egression of bone marrow progen itor cells could be resp onsible for the splenic 116

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myeloid expansion, and since we have observed an increase in splenic progenitor cells in C57BL/6 mice, we administered a neutrophil elas tase inhibitor in an attempt to block bone marrow progenitor egression at doses that were effective in earlier studies (103). However, the results of the experiment were equivocal implyi ng that neutrophil elastase plays no role in polymicrobial sepsis MDSC expansion. Still other signals that have been shown to govern myeloid cell development and expansion in other model systems. The cfms receptor and its lig and M-CSF (CSF-1) have been shown to play a pivotal role in the di fferentiation and proliferation of monocytes and macrophages (107). AFS98, an anti-murine cfms antibody which inhibits MCSFdependent growth and development by binding to the c -fms receptor, has been shown to have a profound effect on monocyte/macrophage peripheral expansi on (114). We hypothesized that cfms inhibition by AFS98 may ameliorate secondary lymphoid organ myeloid cell expansion. AFS98 treatment produced only a modest reduction (2 5%) in the overall splenic GR-1+CD11b+ population with no differences seen in the bone marrow of lymph nodes GR-1+CD11b+ populations ( data not shown) compared to CLP plus isotype control treatme nt. More detailed analysis reveals a more substantial reduction in the GR-1intermediateCD11b+ population analogous to the reduction observed in the CCR2-/septic animals; however, again ther e was little absolute change in the cell numbers in the overall myeloid population. Further investigati on using CD11b, MHC II, F4/80, markers revealed that this GR-1intermediateCD11b+ population consists almost entirely of immature monocytes and macrophages (data not shown ). To evaluate the efficacy of the AFS98 anti-c-fms receptor inhibitor in our model of polymicrobial sepsis we evaluated the levels of remaining c-fms receptor positive (CD115+) cells after AFS98 administration compared to isotype control treated animals. Interestingl y, we found only minimal reduction in the triple 117

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positive (GR-1+CD11b+CD115+) cells. This finding leads to us believe that first, AFS98 inhibition is ineffectual in the setting of murine polymicrobial sepsis based on the plethora of CD115+ cells acquired after AFS98 treatment, and secondly, based on the CCR2 and AFS98 findings, the myeloid expansion observed during sepsis is larg ely due to other expanding populations in the myeloid differe ntiation pathway and not monocyt es and macrophages. In two other independent studies Jose (108) and Murayama (109) found th at AFS98 inhibition of c-fms signaling inhibited mainly monocytes and macr ophages using chronic models of inflammation and not acute infection. This finding is not entirely surprising gi ven the fact that cfms expression progressively increa ses from stem cell precursors to monocytes and macrophages (106). Since our GR-1+CD11b+ cells are more immature than most monocytes and macrophages they probably have a lower cell surface density of the cfms receptor, making cfms inhibition a minimal factor in immature myeloid cell survival. The chemokine CXCL12 (stromal cell derived factor-1) has been demonstrated by Rafii et. al to play an essential role in hematopoietic progenitor cell mobiliza tion and progenitor cell homing to peripheral organs during times of st ress. Others have found using models of inflammatory stress such as cardiac surgery (128), tuberculosis infection (129) and breast cancer tumor models (130) that CXCL12 is imperative for hematopoietic progenitor cell peripheral recruitment. In a model of ma larial infection, de Andrade de monstrated that splenic CXCL12 levels continued to increase unt il the infection was unde r control in C57BL/6 mice and facilitated CD11c+ dendritic cell recruitment to the spleen (131). Applyi ng the knowledge of the MDSCs that we have available, namely that they are immature, rapidly prolifer ating, precursors to more mature phenotypes in the myeloid differentiation pathway and are ul timately derived from common myeloid progenitors, it is no t hard for us to see the inex tricable link between peripheral 118

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expansion of these cells with CXCL12 and he matopoietic progenitor cell homing. Our data support this notion of CXCL12 me diated recruitment given that fact that CXCL12 inhibition substantially reduced splenic MDSC accumula tion. More importantly, anti-CXCL12 therapy produced an even greater reduction in the numbers of splenic CMPs-the precursor that gives rise to the other myeloid lineage cells. This sugge st, although with further investigation necessary, that MDSCs arise from CMPs that have migr ated from the bone marrow to the spleen and undergo myelopoesis generating the dramatic numbers of myeloid cell observed in the spleen during long term sepsis. The is data suggest that an ongoing septic pr ocess produces a dramatic expansion of GR1+CD11b+ cells in the spleen that is independent of CCR2 and cfms receptor signaling, bone marrow MyD88 and TRIF signaling, and neutrophil elastase activity. In contrast, we have demonstrated that the splenic MDSC populati on is dependent on CXCL 12 signaling. CXCL12 depletion substantially reduced the accumulation of the MDSC population in the spleen during sepsis. Furthermore, CXCL12 inhibition also re duced the CMP-the earliest myeloid precursor cell type suggesting that MDSC expans ion depends on CMP splenic expansion. MDSCs and Sepsis-induced Immune Suppression The inevitable question is: what are the immunological consequences of this cell population in terms of the host response to severe in jury or sepsis? Do these cells contribute to the immune suppression and TH2 polarization that are seen in seps is? The rather late appearance (days 3-8) of these cells in burns and sepsis ( 99, 142, 143) suggest that they probably play little role in the early inflammatory response, and more likely contribute to the later responses seen in animals that survive the early cytokine storm. It is easy to speculate that any cell population which contributes as much as 40% of the cells in the spleen, 10% in the lymph nodes and 90% in the bone marrow is very likely to have some impact on the immunological response, although 119

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this has not been definitively shown. Studies from our own laboratory and those of Cora Ogle reveal that these cells obtained from septic and burned mice, respectively, produce a large number of inflammatory mediators, including TNF, MCP1, and MIP1 (99, 142). These cells also produce a large number of mediators and cy tokines that are immune suppressive, including reactive oxygen speci es, nitric oxide, peroxynitrites, TGF IL-4 and IL-10. There is also a significant amount of indirect, a ssociative data sugge sting that the expansion of these cell populations is temporally related to the deve lopment of immune suppression and increased susceptibility to secondary infect ions. Murphey and colleagues, fo r example, showed in septic mice that the increased number of what they termed suppressive macrophages occurred simultaneously with the redu ced bacterial clearance to a Pseudomonas infection (67). Similarly, the increased appearance of these CD11b+GR-1+ cells after a burn injury coincides with increased lethality to a secondary Pseudomonas pneumonia. Of course, association is not cause and effect, and studies to date have not convincingly demonstrated that these defects in acquired imm unity are due to the pres ence of these suppressor cells in sepsis. Much of the difficulty has to do with the heterogeneity of the cell population and the lack of experimental approaches to selectively deplete the host of these CD11b+ GR-1+ cells. For example, we have used a GR-1+ depleting antibody to preven t the expansion of these immature cell populations, and can successfully prev ent their increase in sepsis (99). However, mature neutrophils also express GR-1+, and a depleting antibody i ndiscriminately removes both immature MDSCs and terminally differentiated ne utrophils. Thus, the effects on survival to a bacterial infection in the presence of GR-1+ depletion cannot easily di stinguish between MDSCs and neutrophils. Similar concerns exist with gemcitibine, a nucleoside analog that inhibits rapidly dividing cells, which not only kills replic ating MDSCs, but also other proliferating cells 120

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required for a successful response to microbial infection (Figure 5-2) There are, however, other approaches under consideration. Gabrilovich and colleagues ha ve used the subcutaneous implantation of pellets containing all trans retinoic acid (ATRA) to force the differentiation of MDSCs in tumor-bearing mice (59). Recent studie s suggest, however, that this approach may also lack some specificity, since ATRA also will stimulate the expansion of some regulatory DC populations (152). Other approa ches under consideration involve the use of specific tyrosine kinase inhibitors involved in gr owth factor signaling (153). These prevent the expansion of myeloid derived suppressor cells by blocking growth factor signaling, and can be used short term to block the expansion of these cell populations. With this said, however, there is actually considerable information known about the immune suppressive phenotype of MD SCs. However, very little of that information comes from the sepsis and injury field, while most comes from the oncology literatur e. Because of the heterogeneity of the myeloid populations and th e probability that chronic tumor growth may stimulate the expansion of a similar but distin ct MDSC population as s een in sepsis, caution should be employed when directly comparing the populations. Wh at we do know from both sets of literature, however, is that these cells are qui te immunologically active. Because of their relative undifferentiated phenotype, there is consider able functional plasticity of these cells with a suppressive phenotype elicited upon continued exposure to TH2 cytokines (IL-4, IL-10 and TGF ) often increased in sepsis (47, 53). On the other hand expos ure of MDSCs to TH1 cytokines (TNF ) stimulates differentiation along macr ophage pathways, and enhances T cell cytotoxic responses. For example, when MD SCs obtained from tumor bearing mice were administered to healthy nave animals, they differentiated into normal dendritic cells and 121

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macrophages, but when administered into other tumor bearing animals, they maintained their suppressor cell phenotype (54). In mouse tumor models, the immune suppression to growing tumors could be ameliorated by CD11b+GR-1+ cell depletion with all trans retinoic acid, thereby restoring T-cell responses (58, 59). MDSCs can inhibit T cell activa tion through cell-cell co ntact or immediate juxtaposition. The mechanisms of MDSC T-lymphocyte inhibiti on are not yet fully understood; however, they appear to depend in part on Larginine metabolism to decrease T lymphocyte responsiveness to subsequent an tigen stimulation (48, 49). Furt hermore induction of iNOS with NO release and peroxynitrite formation can account for some of the T cell unresponsiveness in tumor models (60-62). Over production of argi nase I by MDSCs can result in local arginine starvation that can inhibit T-lymphocyte proliferation (63, 64). In addition, GR-1+ cells also secrete IL-4 and IL-10 (65), reactive oxygen species, and TGF all of which can have immunosuppressive properties. Recent studies by Gabrilo vich have proposed a novel mechanism by which MDSCs can suppress CD8+ T-cell function. They had previously shown that much of the tumor evasion of host imm unity was mediated by MDSC abrogation of CD8+ T cell function, including cytolytic killi ng of tumor cells, proliferation and IFN production (55). They reported that increased peroxynitrite produ ction by MDSCs could dire ctly nitrosylate the TCR complex and prevent MHC complex dimers-T cell receptor interactions (154). In contrast, little is known about the poten tial mechanisms by which MDSCs can produce immune suppression and TH2 polarization in severe injury and sepsis. Ochoa examined the functionality of these CD11b+GR-1+ cells recovered from the spleen of mice after traumatic injury, and showed that these cells express large quantities of arginase (68). These CD11b+GR1+ cells obtained from the spleen of trau matized mice significantly inhibited CD3/CD28122

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mediated T cell proliferation, TCR zeta-chain e xpression, and IL-2 prod uction. The suppressive effects could be overcome by bloc king arginase activity or by s upplementation of medium with L-arginine. To examine whether these cells can affect adaptive immune responses in sepsis, shamtreated and septic mice were immunized with NP-K LH using alum as an adjuvant and the serum immunoglobulin response determined ten days la ter (99). The serum immunoglobulin response to NP-KLH with alum can be used to determine the predilection of the CD4+ T-cell response (92). Polymicrobial sepsis was not associated with any disturbances in the total serum IgG and IgM responses in the mice immunized with NP-KLH with alum, but the serum IgG2a response was significantly decreased while the IgG1 response increased in the septic mice, consistent with a shift from a TH1 to a TH2 T-cell response. When the septic mice were depleted of their total GR-1+ cells by antibody treatment, the sepsis-induced increase in IgG1 and the decrease in IgG2a response were abolished, suggesting that GR-1+ cells were contributing to this TH2polarizing response (99). To further confirm the in vivo role that thes e cells play in suppressing an antigenic T-cell response, the effect of GR-1+ cells on the cytotoxic T-cell IFN response by splenocytes from OT-I TCR-transgenic mice immunized with OVA-derived peptide was examined. GR-1+ cells were obtained from either ten day septic or sh am-treated mice, and were infused into C57BL/6 mice that had previously received CD8+ T-cells from OT-1 TCR-transgenic mice, and simultaneously immunized with OVA-derived specific peptide. Ten days later, the spleens from these animals were removed, and IFNresponses to ex vivo stimulation with OVA-derived specific peptide were examined. The IFN(99) production and cytotoxicity were markedly reduced when the animals were administered GR-1+ splenocytes from septic animals when 123

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compared to GR-1+ splenocytes from sham-treated mice, confirming that GR-1+ splenocytes from these septic mice could suppr ess antigen specific T-cell IFN and cell cytotoxicity responses. To determine whether GR-1+ cells could directly suppre ss an antigen specific or nonspecific CD4+ T-cell proliferative response, D011.10 OVA-TCR transgenic mice were made septic and at 10 days, CD4+ splenocytes from septic mice were cultured with irradiated GR-1+ containing antigen presenting cells from the spleen s of 10 day septic or sham-treated mice, and with either OVA peptide, bovine serum albumin or on CD3/CD28 coated plates. Culturing CD4+ cells with irradiated GR-1+ cells from septic mice only modestly, but still significantly, reduced both the antigen-specific (OVA) and n on-specific (CD3/CD28) proliferative responses (99). Since these MDSCs secrete increased quanti ties of IL-10 and IL-4, we asked whether MDSCs obtained from IL-10 and IL-4 null mi ce had the same phenotype as MDSCs obtained from wild-type mice. Sepsis was induced in C57BL/6 IL-10 and IL-4 null mice, and these animals showed a similar expansion of their MD SC population in the spl eens and lymph nodes. GR-1+ splenocytes from 10 day septic IL-10 or IL-4 null mice were injected into nave B6 mice along with OT-1 OVA-specific CD4+ T cells and then immunized with OVA peptide, their ability to suppress a CD8+ T cell IFNresponse was comparable to that seen from GR-1+ cells obtained from a septic, wild-type mouse. Such findings suggest that the suppressive phenotype is seen in MDSCs from these null animals and that their suppressive effects maybe mediated through other signaling pathways. As summarized in Figure 5-4, there are a number of mechanisms by which these MDSCs could directly impact both innate and acquired immune responses. The increased production of a 124

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number of inflammatory mediators, such as TNF suggests that in response to a secondary infectious challenge and micr obial products, the increased nu mbers of these MDSCs could contribute to a secondary cytoki ne storm. Simultaneously, incr eased production of IL-4, IL-10 and TGF by MDSCs could promote the further devel opment of other regulat ory/inhibitory cell populations, such as TH3, TR1 and regulatory dendritic cells as well as directly suppress TH1 effector cell populations. Interestingly, the increased production of reactive oxygen species, peroxynitrites and the depletion of arginine in the T cell microenvironment could contribute to both suppressed CD4+ and CD8+ proliferative and effector responses by multiple mechanisms, including inactivation of the T cell receptor comp lex by nitrosylation and amino acid starvation during proliferation. All in all, these metabolically active cells are well positioned in bone marrow, spleen and secondary lymph nodes to wreak havoc on normal acquired and innate immune responses. The sepsis state promot es the continued expansion and suppressive phenotype of these cells. Conclusions Studies to date have focused on a novel MDSC population in bone marrow, spleen and secondary lymph nodes that has previously attracted only a limited amount of research attention by the shock, trauma and sepsis communities. Although there is now convincing evidence to suggest that this cellular populat ion is dramatically increased in mice after a number of acute inflammatory challenges, including surgical trauma, burns, ischemia/reperfusion injury and polymicrobial sepsis, little is know n about either the mediators that drive this expansion, or their functional consequences. Furthermore, there is at present no informa tion whether these MDSC populations increase in septic pati ents or individuals after severe trauma or burn injury. Data from other literature suggests, however, that in creases in the MDSC popula tion are likely to have 125

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dramatic consequences on acquired immunity, simila r or greater than the effects attributed to regulatory T cell and dendritic ce lls populations. Thus, future studies are required to further resolve the role that this nove l suppressor cell population play s as a potential mechanism for immune suppression and a target for th erapeutic interven tion during sepsis. Myeloid Derived Suppresor Ce lls and Future Directions Although we have described an immature he terogenous myeloid cell population that is capable of dramatic expansion after a variety of inflammatory insults ranging from organ ischemia to bacterial infections, we still know ve ry little regarding the true phenotypes of these cells and what purpose they serv e in hematopoiesis. Kelsoe a nd colleagues have demonstrated that in the bone marrow the GR-1+CD11b+ population can actually be divided into 3 different subpopulations based on their cell surface expression of c-Kit and Fc R (115). One avenue that would benefit our understanding is to phenotype the MDSC stage of development in the myeloid differentiation pathway. In addition to cell su rface expression of GR -1, CD11b, and F4/80, we would also incorporate c-Kit and Fc R to further compare the cells we observe in the spleen and bone marrow after sepsis to the published report s. By separating out the subpopulations that comprise the GR-1+CD11b+ cohort of cells we will then be able to investigate the subpopulations myeloid transcription factors (155) and in conjuncti on with the immune phenotyping, determine exactly what stage of myeloid development these cel ls persist. Knowing this information would provide us the necessa ry understanding to better tailor therapeutic interventions to arrest MDSC e xpansion without negatively affec ting the whole myeloid lineage. Another avenue for consideration is the mechan ism of expansion of the cells during sepsis. Our CXCL12 data shows that MDSC expansio n is dependent on an uninterrupted CXCL12 signaling cascade; however, the events surround ing the CXCL12 signaling remain a mystery. Heissig demonstrated that after immune stre ss with 5-FU, bone marrow egression and splenic 126

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homing of hematopoietic progenitors is depende nt on MMP-9 mediated SDF-1 induced release of the Kit ligand (122). If we consider seps is an immune stress a nd consider the loss of lymphocytes that Hotchkiss (88) and others have shown as providing a spl een niche, then it is not so large of a stretch to imagine that all of these events happen in concert to allow the expansion of the myeloid lineage and extramedu llary hematopoiesis in the spleen. Although there are some assumptions being made on our part, based on data in other model systems, the events that are happening in sepsis are the same namely myeloid expansi on after immune stress. Consider that 90% of the bone marrow consists of immature myeloid cells by five days after sepsis and then that level begins to recede back to baseline at seven days. In the meantime, the splenic levels of immature myeloid cells are just beginning to rise and by ten days are completely expanded. The question is: what governs this initial bone ma rrow myeloid expansion and once the bone marrow levels return towards baseline what is mediating the continued splenic expansion of the myeloid lineage? The involve ment of CXCL12 only explains the bone marrow egression and possibly the spleni c homing; however, it does not e xplain the initial events that signal stem cells and bone marrow progenitors to proliferate and differentiate. These are the questions that need to be reso lved in order to truly understand the myeloid cell expansion during sepsis. Lastly, and probably most important is the establishment of a link between what we understand to be true in our mous e model of polymicrobial sepsis and what actually happens in humans during sepsis. Are we beneficially im pacting sepsis understa nding and human myeloid biology or are we just studying various differences in immune system function between various animals? Currently, aside from a few scant publi cations in the oncology literature pertaining to proposed phenotypes of MDSCs in humans (156) there have been no definitive reports. 127

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Probably the most important unders tanding that we could have w ould be whether this myeloid expansion occurs in human sepsis, and to dete rmine the cell phenotype involved. Currently we have obtained several histological sections of human spleen tissu e from septic patients. H&E staining revealed that there is indeed some in itial promise to demonstrate myeloid expansion in the periarterioler areas in the spleen. Howeve r to say these findings are analogous to what we have demonstrated in mouse sepsis are very prem ature. First off, there are no human correlates of the GR-1 marker that we use to define the population in mice. Secondly, our mouse model is a model of long term sepsis unabated by the administration of any medical care such as antibiotics or nutrition support. In human sepsis rarely is there a patient that has a chronic inflammatory process that progresses without medical attention, antibiotic treatment and nutritional support. The caveat is that the myeloid expansion we s ee in mouse sepsis could just be the product of uncontrolled inf ection that does not occur in huma n sepsis. It is this very question that needs to be investigated and answered. 128

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Figure 5-1. Panels A and B represen t a cytospin preparation of GR-1+ cells isolated from the spleen of a 10 day septic mice. Mice underwent a cecal ligation and puncture. After ten days, the mouse was euthanized and the GR-1+ splenocytes were enriched using Miltenyi columns (33). The GR-1+ cells were stained with a Wright stain and visualized at low and high magnifications. Most evident is the heterogeneity of the cell population (Panels A), with cells phenotypi cally varying from mature neutrophils and monocytes to immature ring-shaped cells that appear to be neutrophil and monocyte precursors (Panel B). 129

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Table 5-1. MDSC Phenotypes in Various Inflammation Models. GR -1 CD11b MHC I MHC II CD80 CD86 CD40 CD11c CD34 F4/80 CD16/32 CD31 Ter 119 c-kit CD115 Operative Trauma H H H L L L L L L L L L Makarenko va et al. (32) Polymicrobial Sepsis H H L L L I L L L L I Delano et al. (33) Burn Sepsis H H L Noel et al. (34) Cell surface expression of various receptors a nd markers on the MDSCs as reported from our laboratory and from other investig ators using sepsis and injury m odels. It is obvious that the GR-1 and CD11b markers broadly define the populat ion however the heterogeneity of the cells is reflected in the various expression of a wide arra y of cell surface markers. Relative cell surface expression of each marker was interpreted from the authors publishes work and classified as H for High, I for Intermediate, and L for Low surface le vels of the respective markers. Empty cells mean that the authors presented no data on the marker. 130

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Table 5-2. MDSCs Proliferate When Cultured With Growth Factors. Sham CLP Reference (cfu) (cfu) Erythropoietin 1 4 (33) GM-CSF 2 38 (33) G-CSF 2 26 (33) CSF-1/M-CSF 5 30 (34) Effect of growth factors on th e proliferation and differentiati on of immature MDSCs obtained from burned and septic mice. *Data was adapted from Neol et al.(142) based on the number of cells in their initial cultures. 131

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Table 5-3. MDSC Fold Change Increases in Various Models of Inflammation. Time (days) Fold Change: Increase Reference Burn 8 4 Noel et al. (34) Liver Ischemia 7 1.5 O'Malley, K. (unpublished data) Kidney Ischemia 7 2 Delano, M.J. (unpublished data) Acute Trauma 3 6 Makarenkova et al. (32) Polymicrobial Sepsis 10 12 Delano et al. (33) Pseudomonas Infection 10 9 Moreno, C. (unpublished data) Cecal Slurry Peritonitis 2 4 Wynn, J.L. (unpublished data) Relative fold change increases between injured and control animals observed in various injury models. The infectious injuries such as polymicrobial sepsis or Psuedomonas infection seem to more dramatically induce MDSC population expans ion compared to the non-infectious models such as organ ischemia. 132

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Figure 5-2. Gemcitibine, a nucleoside analog that inhibits rapidly proliferating cells, was administered i.p. at 120 mg/kg/mouse and wa s able to inhibit the expansion of the myeloid population after 10 days in the spl eens of septic mice (n=5) as compared to septic mice without gemcitibine treatment (n=5). P<0.05 (Students t test) between mice that underwent CLP alone and mice that underwent CLP and gemcitibine treatment (CLP + Gem). CLP, Cecal lig ation and puncture, Gem, Gemcitibine. 133

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Figure 5-3. H&E preparations of mouse spleens 10 days after either sham or sepsis treatment. Panels A and B are low power views of sham and sepsis treated mouse spleens. The arrows indicate the presence of megakaryoc ytes. There is a 4 fold increase per low power field in the number of megakaryocytes in the septic spleen compared to the sham spleen. Panel C is a high power view of the megakaryocytes in the septic mouse spleens. 134

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135 Figure 5-4. Proposed model by which MDSCs can impact both components of acquired and innate immunity. Expansion of the MDSC population leads to in creased numbers of myeloid derived suppressor cells in direct proximity to both CD4+ and CD8+ T cells with the ability to secrete large numbers of immunoregulatory peptides. It is presumed that the immunosuppressive phe notype is achieved through both direct contact and immediate proximity with CD4/CD 8 T cells as well as the release of both paracrine and endocrine-like mediators of inflammation. cDC, conventional dendritic cell.

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BIOGRAPHICAL SKETCH Matthew J. Delano was born in Butler, PA in 1974. He attended East Brady High School in East Brady, PA and then graduated from Ar mstrong Central High School in Kittanning, PA in 1992. He then enrolled in Saint Vincent Colle ge where he graduated with Honors with a Bachelor of Science degree in Chemistry. Matt then worked for Costello Pharmaceuticals for two years before entering Temple Medical School in Philadelphia, PA in the fall of 1998. From 2001-2002 Matt was awarded a stipend to study panc reatic cancer under th e direction of Dr. Howard Reber at the David Geffen School of Medi cine at the University of California at Los Angeles. In 2002 Matt resumed his medical sc hool curricula and graduated from Temple Medical School with a Medical Doctor degree in the spring of 2003 with a categorical residency position in general surgery in the University of Florida, College of Medicine, Department of Surgery, beginning in July of 2003. After completi ng two years of a 5 year residency in general surgery, Matt entered the laboratory of Dr. Ly le Moldawer and gained acceptance to the Immunology Microbiology Advanced Concentration in the IDP program at the University of Florida. Matts plans are to fini sh his general surgery residency a nd to pursue a career structured around inflammation biology and its impact on post surgical immune suppression. 151