Fibrinogen Like Protein 2 (FGL2): a Novel Regulator of Macrophage M1 Polarization

Fibrinogen Like Protein 2 (FGL2): A Novel Regulator of Macrophage M1 Polarization

Kaveh Farrokhi

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Immunology University of Toronto

Fibrinogen Like Protein 2 (FGL2): A Novel Regulator of Macrophage M1 Polarization

Kaveh Farrokhi

Master of Science

Department of Immunology University of Toronto

2018 Abstract

Fibrinogen-like protein 2 (FGL2) is a potent immunosuppressive molecule. The effects of FGL2 on macrophages, however, has not been studied. Peritoneal cell accumulation in fgl2-/-, fgl2+/+ and fgl2Tg mice in response to thioglycollate was measured. Significantly fewer cells were recovered from the peritoneal cavity of fgl2Tg mice compared to fgl2+/+ or fgl2-/- mice after thioglycollate injection. The difference in cell numbers was found to be due to decreased eosinophil accumulation in fgl2Tg mice. The effect of FGL2 on macrophage M1 and M2 polarization was next examined. IL-12 was significantly reduced in macrophages isolated from fgl2Tg mice. Addition of recombinant FGL2 protein to fgl2+/+ macrophages led to suppression of IL-12 production in a dose-dependent manner. Finally, Flow cytometric analysis of fgl2Tg macrophages revealed reduced expression of TLR4 as well as decreased phagocytosis. These data demonstrate that FGL2 inhibits the proinflammatory activity of macrophages and provides a rationale for developing anti-FGL2 therapies.

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Acknowledgments

I would like to sincerely thank my supervisors, Dr. Gary Levy and Dr. Nazia Selzner for their support and guidance throughout this degree. They have served as excellent mentors and always gave me the support I needed. Their guidance has been greatly appreciated and I take the lessons they have taught with me throughout my life.

I am also very appreciative and would also like to sincerely thank Dr. Andrzej Chruscinski for his guidance and help in interpreting experimental results, planning experiments and editing this thesis. This thesis would not have been possible without Dr. Chruscinski’s help and support.

I would also like to thank my committee members Dr. Reginald Gorczynski and Dr. Tania Watts. for their constant support and help throughout this degree. I have learned a lot from these two great scientists and am very thankful to have them on my committee.

Thank you, Mom, Dad and Kathy, for helping me through this degree and taking all those trips to come visit me, I couldn’t have done it without you.

I would like to sincerely thank Angela Li, for the late nights, the cell counting, teaching me flow, putting up with my grumpy nature and the memories. This work could not be possible without you. You made the years fly by.

Thank you to my peers, Hassan and Vanessa for being such great lab mates, friends, colleagues and pillars of support.

Thank you to William and Jianhua for helping me with anything I needed to complete this project. You two are among the hardest working and kindest people I know.

Thank you to Natalie, Lianne, Tharssun, Sarah and Arian from the flow facility for the help and answering my flow-based questions

Thank you to (in no particular order): Justin Manuel, Dario Ferri, Conan Chua, Dr. Jongstra- Bilen, Andre Seigel. Dr. Shannon Dunn, Jennifer Ahn, Dr. Micheal Julius, Dr. Jennifer Gommerman the Canadian Liver Foundation and the Department of Immunology. iv

Table of Contents

Acknowledgments...... iii

Table of Contents ...... iv

List of Abbreviations ...... vii

List of Figures ...... x

Chapter 1 Introduction ...... 1

Introduction ...... 1

1.1 Macrophages ...... 1

1.1.1 Macrophage origins and maintenance in tissue ...... 1

1.1.2 Macrophages during embryonic development ...... 2

1.1.3 The Role of the Macrophage in homeostasis ...... 3

1.1.4 Macrophages and the immune response ...... 4

1.1.5 Macrophage polarization and the M1/M2 paradigm ...... 6

1.1.6 Macrophage phenotypic complexity and re-evaluation of the M1/M2 paradigm ...... 7

1.1.7 Macrophage polarization in disease ...... 8

1.2 Fibrinogen-like protein 2 ...... 9

1.2.1 The coagulation cascade ...... 9

1.2.2 Genomic localization and protein structure of FGL2 ...... 10

1.2.3 Membrane-bound FGL2 ...... 11

1.2.4 Soluble FGL2 ...... 12

1.2.5 The role of FGL2 in disease...... 13

1.3 FGL2 and macrophage M1/M2 polarization ...... 16

1.4 Hypothesis and Objectives ...... 17

Chapter 2 Materials and Methods ...... 18

Materials and Methods ...... 18 v

2.1 Animal Care ...... 18

2.2 Generation of fgl2-/- and fgl2Tg Mice ...... 18

2.3 Peritoneal Exudate Cells ...... 18

2.4 Production and Purification of Recombinant FGL2 Protein ...... 19

2.5 Stimulation of Peritoneal Macrophages ...... 19

2.6 Griesse and Arginase Assay ...... 19

2.7 ELISA ...... 20

2.8 Flow Cytometry ...... 20

2.9 Phagocytosis Assay ...... 20

2.10 Statistics ...... 21

Chapter 3 Results ...... 22

Results ...... 22

3.1 Fewer cells are collected from the peritoneal cavity of fgl2Tg mice after injection of thioglycollate...... 22

3.2 Macrophages from fgl2-/- and fgl2Tg mice express similar levels of iNOS and arginase to macrophages from fgl2+/+ mice...... 26

3.3 Fgl2Tg macrophages produce significantly less IL-12 in response to stimulation ...... 28

3.4 Fgl2Tg macrophages express significantly lower TLR4 than fgl2+/+ or fgl2-/- macrophages ...... 30

3.5 FGL2 protein suppresses IL-12 by macrophages in a dose-dependent manner...... 32

3.6 FGL2 protein suppresses IL-12 by macrophages to a variety of stimuli ...... 34

3.7 FGL2 inhibits IL-12 at early and late timepoints after stimulation and with low and high doses of LPS...... 36

3.8 FGL2 inhibits IL-6 but not TNFα in response to LPS simulation ...... 38

3.9 Fgl2+/+, fgl2-/- and fgl2Tg macrophages express similar levels of MHCII and CD86 in response to LPS or IFNγ ...... 40

3.10 Phagocytosis of Bacteria is impaired in fgl2Tg macrophages ...... 42

Chapter 4 Discussion ...... 44 vi

Discussion ...... 44

4.1 FGL has suppressive roles in eosinophil accumulation in the peritoneal cavity in response to thioglycollate...... 44

4.2 FGL2 is a novel regulator of IL-12 expression by macrophages...... 47

4.3 FGL2 suppresses the anti-bacterial response in macrophages ...... 52

4.4 Future Directions ...... 54

4.5 Conclusions ...... 55

References or Bibliography ...... 57

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List of Abbreviations

• A700 Alexa Fluor 700, • AKT Protein Kinase B • AP-1 Activator Protein-1 • APC Allophycocyanin • BFGF Basic Fibroblast Growth Factor • BMDC Bone Marrow-Derived Dendritic cell • BMDM Bone Marrow-Derived Macrophage • BV650 Brilliant Violet 650 • CCL C-C Motif Chemokine Ligand • CD Cluster of Differentiation • CEBPα CCAAT/Enhancer Binding Protein α • CEBPβ CCAAT/Enhancer Binding Protein β • CMV Cytomegalovirus • CXCL C-X-C Motif Chemokine • DMEM Dulbecco’s Modified Eagle Media • E. coli Escherichia coli • EAE Experimental Autoimmune Encephalomyelitis • EGF Endothelial Growth Factor • eGFP Enhanced Green Fluorescent Protein • ELISA Enzyme-Linked Immunosorbent Assay • ES cell Embryonic Stem Cell • Ets Erythroblast Transformation-Specific • FBS Fetal Bovine Serum • Fc Fragment crystallizable • FGL2 Fibrinogen-Like Protein 2 • FIBCD-1 Fibrinogen C Domain Containing 1 • FITC Fluorescein Isothiocyanate • FRED Fibrinogen-related Domain viii

• G-CSF Granulocyte-Colony Stimulating Factor • HIF1a Hypoxia Inducible Factor 1 Alpha • HRP Horseradish Peroxidase • IFN-γ Interferon Gamma • IFNγR Interferon Gamma Receptor • IL Interleukin • iNOS Inducible Nitric Oxide Synthase • IRF Interferon Regulatory Factor • LacZ Lactose Operon Z • LCMV Lymphocytic Choriomeningitis • LPS Lipopolysaccharide • Ly6C Lymphocyte Antigen 6 Complex, Locus C • Ly6G Lymphocyte Antigen 6 Complex, Locus G • MARCO Macrophage Receptor with Collagenous Structure • M-CSF Macrophage-Colony Stimulating Factor • MerTK MER Proto-Oncogene, Tyrosine Kinase • MHCII Major Histocompatibility Complex Class II • MMP9 Matrix Metallopeptidase 9 • Myb Myeloblastosis Oncogene • NFAT Nuclear Factor of Activated T cells • NFκB Nuclear Factor kappa-light-chain-enhancer of Activated B cells • NO Nitric Oxide • OCT Octamer Binding Protein • PBS Phosphate Buffered Saline • PCR Polymerase Chain Reaction • PDL-1 Programmed Death-Ligand 1 • PE Phycoerythrin • PerCP Peridinin-Chlorophyll Proteins • PKG-neo Phosphoglycerate Kinase I Promoter-Neomycin Resistance • PLC-γ Phosphoinositide Phospholipase C gamma • PU.1 Spi-1 Proto-Oncogene ix

• SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis • Siglec-F Sialic Acid-binding Immunoglobulin-like Lectin F • SP1 Specificity Protein 1 • Src Proto-Oncogene Tyrosine-Protein Kinase • STAT1 Signal Transducer and Activator of Transcription 1 • TGFβ Transforming Growth Factor β • TIGIT T cell Immunoreceptor with Ig and ITIM Domains • TIM4 T-Cell Immunoglobulin and Mucin Domain Containing 4 • TLR Toll-like Receptor • TNFα Tumor Necrosis Factor α • VEGF Vascular Endothelial Growth Factor • α-ISPP α-isonitrosopropiophenone x

List of Figures

Figure 1. Total cell numbers recovered from the peritoneal cavity in response to thioglycollate in fgl2+/+, fgl2-/- and fgl2Tg mice……………………………………………………………………………………………………...Page 24.

Figure 2. Flow cytometric analysis and total numbers of cell subsets………………………………………....Page 25.

Figure 3 Nitric Oxide and Arginase expression by cultured macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice…………………………………………………………………………………………………………….Page 27.

Figure 4. IL-12 and IL-10 production by macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice…………...... Page 29.

Figure 5. Expression of IFNγR and TLR4 on macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice……...... Page 31.

Figure 6. IL-12 production is suppressed by rFGL2 in fgl2+/+ macrophages…………………………...... Page 33.

Figure 7. Examining IL-12 expression in fgl2+/+ macrophages in response to stimulation with CD40 agonist, LPS and rFGL2 protein…………………………………………………………………………………………...…Page 35.

Figure 8. IL-12 production by fgl2+/+ macrophages at early and late timepoints in response to FGL2 and lower and higher doses of LPS…………………………………………………………………………………………….Page 37.

Figure 9. TNFα and IL-6 production by fgl2+/+ macrophages at early and late timepoints in response to FGL2 and LPS……………………………………………………………………………………………………………..Page 39.

Figure 10. Expression of co-stimulatory molecules on macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice…….Page 41.

Figure 11. Phagocytic activity of macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice…………………………...Page 43.

Figure 12. Model of the effect of FGL2 on macrophage phenotype………………………………………...…Page 56. 1

Chapter 1 Introduction Introduction 1.1 Macrophages

Elia Metchnikoff first observed a cell type that was characterized by phagocytic ability in 18921. He termed these newly identified cells macrophages from the Greek “big eater”. With this discovery, Metchnikoff laid the foundations for the field of innate immunology. As our understanding of innate immunity grows, we have come to appreciate that macrophages play critical roles in health and disease. Macrophages can adopt pro-inflammatory phenotypes which help to initiate immune responses and control pathogens; they can also adopt anti-inflammatory or regulatory phenotypes to resolve inflammation and initiate repair after infection. This plasticity, which is a hallmark of macrophage function, has important outcomes in diseases such as cancer or chronic viral infections. It is thus important to understand the factors that influence macrophage phenotypes, as this may lead to the development of new therapeutics.

1.1.1 Macrophage origins and maintenance in tissue

In a seminal paper from 1968, Ralph van Furth and Zanvil Cohn showed that blood monocytes had the ability to enter tissues and differentiate into tissue macrophages2. Subsequent work supported this conclusion by demonstrating the differentiation of bone marrow precursors and monocytes into macrophages in vitro. These findings informed the paradigm that populations of macrophages in tissues were replenished by infiltration of monocytes derived from bone-marrow precursors leading to the creation of the model known as the mononuclear phagocyte system (MPS). The MPS model asserts that bone-marrow progenitors give rise to adult macrophage populations through the replenishment of tissue macrophages by bone marrow derived monocytes. Included in the MPS are monocytes, which are divided into short lived Ly6Chi CCr2hi classical monocytes (CD14+ CD16- in humans) and longer lived Ly6Clo CX3CR1hi “patrolling” monocytes (CD14- CD16+ in humans)3. Also included in the MPS are committed bone marrow progenitors termed monocyte dendritic cell precursor (MDP), which give rise to monocytes as well as dendritic cells4.

Evidence has emerged, however, which has challenged the MPS model. Several studies have demonstrated that monocytic replenishment may not be the only method by which tissue macrophage populations are maintained. For example, Langerhans cells, which are the resident macrophage population of the epidermis, have been shown to be of host and not donor origin following congenic stem cell transplantation5. Microglia, the resident macrophages of the brain, were also found to be of host origin after parabiosis and congenic bone marrow transplantation6,7. These findings were the first to indicate that macrophage populations could maintain themselves independently from circulating monocyte populations. One notable exception to this was found in the gut where a large resident macrophage population was found to be entirely dependent on monocyte replenishment8.

1.1.2 Macrophages during embryonic development

With the development of sophisticated cell lineage tracing tools, tissue macrophage populations have been identified that are of yolk-sac origin. These macrophage populations persist into adulthood and are independent of hematopoietic stem cells. RUNX1 is a transcription factor which is critical for hematopoiesis and RUNX1+ cells are expressed in the yolk sac starting at embryonic day 7 (E7.0) to E8.0 and in the precursors to definitive hematopoiesis at E8.5. The Runx1-Mer-cre-Mer model allows for the selective labeling of RUNX1 expressing cells. Cells actively expressing RUNX1 upregulate the Cre-recombinase fused to 2 modified estrogen receptors, and after the administration of Tamoxifen, the Cre-recombinase is able to induce the expression of eYFP and label RUNX1+ cells. Injection of Tamoxifen at E7.0 results in eYFP-labeling of yolk sac cells, whereas injection with tamoxifen at E8.5 results in labeling of cells arising through definitive hematopoiesis. When Tamoxifen was injected at E7.0, yolk sac- derived eYFP+ macrophages subsequently colonized all embryonic tissues9. However, the only labeled tissue macrophage population to persist into adulthood was microglia. Injection at time points later than E8.0 resulted in a dramatic decrease in labeled microglia, indicating that microglia primarily arose from yolk sac-derived precursors. Furthermore, mice deficient in c- Myb, which is essential for the regulation of hematopoietic stem cells and hematopoiesis, were examined during embryonic development10. Embryonic tissues of these mice during development showed deficiencies in CD11b+ myeloid cells; however, F4/80+ macrophages were still found to be colonize the tissue, indicating that these macrophages arise independently from 3

Myb+ hematopoietic stem cells in the yolk sac. These findings changed the understanding of macrophage ontogeny and the field of and embryonic development.

Besides the yolk sac, the fetal liver has been identified as an important source of macrophages during embryonic development and these macrophages were found to persist into adulthood and comprise the majority of tissue resident macrophage populations. By using conditional and constitutive reporter models for the chemokine receptor CX3CR1, tissue resident macrophage populations in the spleen, lung, peritoneal cavity and liver were found to be established prenatally and maintained into adulthood independently of replenishment by monocytes in the steady state11. In some organs, colonization by macrophages has been shown to occur in waves. Using a FLT3 reporter system in which cells that had passed through a FLT3+ progenitor during definitive hematopoiesis were labeled, it was found that at E14.5 there were distinct FLT3- yolk sac-derived and FLT3+ fetal liver-derived macrophages in the heart12. Similarly, macrophage colonization of the aorta was found to occur in 2 waves: the first being a CX3CR1+ precursor prenatally, and the second immediately after birth by bone marrow-derived monocytes13. In the steady state, there exist subpopulations of adult tissue macrophages, which are thought to be monocyte-derived. In the peritoneal cavity, for example, there exist at least 2 populations of resident macrophages: the large peritoneal macrophage (LPM) and the small peritoneal macrophage (SPM). LPM are characterized by high expression of F4/80 and CD11b, while SPM express lower levels of F4/80 and CD11b, but high levels of MHCII14. During steady state conditions, LPM are the majority of total macrophage population in the peritoneal cavity; however, after injection of lipopolysaccharide (LPS) or thioglycollate, there is a rapid influx of monocytes which differentiate into SPM, demonstrating the duel origins of these macrophage subsets. Similarly, the heart contains different resident macrophage subsets: some are embryonically-derived and others have a monocytic origin. At present, it is unclear if tissue macrophages derived from different origins have distinct functions and how the dynamics of these populations change after inflammation, potentially leading to different roles in disease.

1.1.3 The Role of the Macrophage in homeostasis

Macrophages have specialized functions based on the tissue compartments in which they are located and are essential in maintaining tissue homeostasis. Splenic and hepatic macrophages have important roles in the clearance of senescent erythrocytes and the recycling of iron15,16. 4

Osteoclasts and osteoblasts have specialized roles in secretion and resorption of bone, failure of which may lead to osteoporosis17. Macrophages also have important roles in wound healing and tissue remodeling. Depletion of macrophages results in delayed wound closure and delayed angiogenesis18. Depletion of macrophages also results in lower levels of growth factors including transforming growth factor beta (TGFβ) and vascular endothelial growth factor (VEGF), demonstrating the important role of macrophages in the resolution of injury18.

Macrophage survival and proliferation in tissue is dependent on macrophage colony stimulating factor (MCSF) also known as CSF-1, which signals through CFS-1R (also known as CD115). Binding of CSF-1 to CFS-1R results in phosphorylation and activation of multiple kinases including PLC-γ, Src, AKT, ERK and PI3-kinase19. The importance of the MCSF/CSF- 1R axis is demonstrated in mice which have a homozygous mutation in the Csf1 gene, rendering it inactive (Csf1op/op ). These mice have severely reduced numbers of osteoblasts and osteoclasts leading to osteoporosis, as well as reduced numbers of monocytes and other macrophage populations17. Interestingly, Csf1r-/- mice were found to have a more severe phenotype, with fewer tissue macrophages than Csf1op/op mice. This was explained by the discovery that IL-34 is also a ligand of CSF1-R and shares some redundant functions with MCSF20.

In each tissue, macrophages clear apoptotic cells and debris. Apoptotic cells release factors such as sphingosine-1-phosphate, which is detected by macrophages and activates erythropoietin (EPO) receptor signaling pathways leading to their clearence21. Macrophages also recognize apoptotic cells through several surface receptors including MerTK and TIM-4, which recognizes phosphatidylserine on the surface of apoptotic cells22. Importantly, defects in the phagocytosis of apoptotic cells is associated with the breaking of self-tolerance and autoimmunity. For example, TIM-4 knockout mice exhibit hyperactive T and B cells23, and Epor -/- mice developing lupus-like symptoms21. Macrophages have been shown to promote self- tolerance by releasing the immunoregulatory cytokines IL-10 and TGFβ during the clearance of apoptotic cells24,25.

1.1.4 Macrophages and the immune response

Macrophages have been referred to as immune “sentinels”, in that they patrol tissues and detect pathogens through several genomically encoded pattern recognition receptors (PRRs). These include cell surface receptors, such as Toll-like receptors (TLRs) or C-type lectin 5 receptors, as well as cytosolic receptors such as the NOD-like receptors26. PRRs detect highly conserved pathogen motifs called pathogen-associated molecular patterns (PAMPS) which include bacterial structural components such as LPS or flagellin, as well as molecules such as double stranded RNA, which make up viral genomes27. PRRs can also detect host-derived ligands that are associated with damage, termed damage-associated molecular patterns (DAMPs) including heat shock proteins and extracellular ATP28. Once a PAMP has been detected, a signaling cascade is initiated based on the type of PAMP. For example, TLR5 which detects bacterial flagellin, uses MYD88 as an adapter, leading to NFκB activation and the initiation of an anti-bacterial immune response. TLR3, which detects dsRNA commonly associated with viruses, signals through TRIF. This signaling activates transcription factors such as IRF3, IRF5 and IRF7, leading to the production of type-I interferon29,30. Activation of macrophages leads to the production of cytokines (e.g., IL-1, IL-6 and TNFα), which mediate inflammation, and the production of chemokines (e.g., CCL2, CCL5 and CXCL16), which recruit other inflammatory cells to the site of inflammation. Based on the tissue in which they reside, macrophages may respond differently as they encounter PAMPS. Gut-resident macrophages are continuously exposed to PAMPS from the microbiota. However, these macrophages are specialized in that they do not produce inflammatory cytokines in response to these bacterial PAMPS in the steady state31.

Macrophages, along with dendritic cells and B cells, are referred to as “professional” antigen presenting cells (APC) due to their expression of MHCII. Thus, macrophages have the ability to present antigen to T cells and initiate immune responses. Macrophages are also able to express high levels of the co-stimulatory molecules CD80/86 and CD40, which are required for the initiation of primary immune responses. The prevailing thought has been that dendritic cells are the cell type predominantly responsible for presenting antigen to naïve T cells and the initiation of the immune response; however, this is controversial. Bone-marrow-derived CD11c negative macrophages that had been pulsed with antigen are able to migrate to lymph nodes and induce CD8+ T cell proliferation in-vivo32. Likewise, after stimulation with IFNγ, antigen loaded immortalized macrophages are able to induce a primary Th1-polarized response in mice33. Another study showed that antigen-pulsed macrophages are able to prime T cells; however, as opposed to dendritic cells, these macrophages initiated Th2 responses in-vivo34. Macrophages are also involved in presenting captured antigen to B cells. Using inactivated VSV particles, which 6 initiate T-independent antibody responses, CD169+ lymph node macrophages were observed capturing viral particles and presenting them to follicular B cells35. This served to prevent systemic dissemination of the antigen while also ensuring antigen was efficiently presented.

1.1.5 Macrophage polarization and the M1/M2 paradigm

Macrophages are known to have plasticity in their phenotype based on the environmental signals they detect. In 1960s while studying Listeria monocytogenes, Mackaness described a form of resistance to listeria that was dependent on changes to the anti-microbial activity of macrophages36. They also noted that this change in anti-microbial activity was not exclusively directed to listeria as the change in activity applied to other pathogens. These findings were among the first to demonstrate the concept of macrophage activation. Later work would identify IFNγ as the key lymphocyte-derived molecule that was responsible for the observed changes to macrophage phenotype37. The effects of IFNγ on macrophages included increased antigen presentation, increased phagocytosis, production of inflammatory cytokines and the production of antimicrobial molecules such as reactive oxygen species (ROS) and nitric oxide (NO)38. Macrophage activation by IFNγ produced by T cells and NK cells became known as classical activation.

When IL-4 was discovered to be the counterpart of IFNγ, a paradigm was created which separated activated CD4+ T cells into Th1 or Th2 polarizations: Th1 associated with the control of intracellular pathogens, and Th2 associated with the control of helminths, fungi and protozoa. When examining the effects of IL-4 on macrophage function, it was observed that IL-4 could suppress the production of inflammatory cytokines and ROS, but would upregulate the expression of MHCII on the macrophage39,40. IL-4 was also found to upregulate receptors such as the mannose receptor (MRC1). With these findings, the concept of “alternative” macrophage activation began to emerge41. This was further exemplified by examining macrophages from the Th1 dominant C57Bl/6 and the Th2 dominant BALB/c strains of mice. Macrophages from C57Bl/6 mice activated by LPS or IFNγ upregulated inducible nitric oxide synthase (iNOS) and produced nitric oxide, whereas macrophages from BALB/c mice upregulated arginase, leading to ornathine synthesis42. Interestingly, C57Bl/6 macrophages caused T cells to produce IFNγ, whereas BALB/c macrophages induced TGFβ, indicating these opposing macrophage phenotypes also had the ability to modulate adaptive immune response42. From these findings, 7 the paradigm of M1/M2 was proposed, broadly dividing macrophages based on arginine metabolism. Furthermore, M1 macrophages associated with Th1 T cell responses and M2 where associated with Th2 T cell responses. M2 macrophages have also been associated with tissue repair as ornithine can promote cell proliferation. As understanding of M1/M2 polarization grew, other markers of macrophage polarization were described beyond the original iNOS and arginase. Human macrophages do not express iNOS or arginase after stimulation, which made identifying M1/M2 polarization difficult in human macrophages; however, M1 macrophages were found to express IL-12 and proinflammatory cytokines such IL-6 and TNFα, whereas M2 macrophages expressed IL-10, TGFβ as well as surface receptors such as CD206 in both mice and humans43,44.

1.1.6 Macrophage phenotypic complexity and re-evaluation of the M1/M2 paradigm

Although the M1/M2 paradigm was originally thought to explain macrophage polarization, it became clear that macrophage phenotypes are more complex. Stimuli other than IFNγ or IL-4 were found to activate macrophages and induce unique phenotypes that did not easily fit into the original M1/M2 categorization and new subdivisions had to be proposed45,46. Immune complexes binding to FcγR in combination with TLR agonists were found to activate macrophages and induce the expression of the pro-inflammatory cytokines IL-6 and TNFα as well as high levels of the immunoregulatory cytokine IL-1046,47. By cytokine expression alone, it would be difficult to classify this macrophage phenotype as either M1 or M2. These macrophages were found to be functionally immunosuppressive and adoptive transfer of macrophages activated in this fashion were able to protect mice from lethal endotoxin shock48. These macrophages were eventually classified as M2b macrophages with the traditional IL-4- derived macrophages classified as M2a. IL-10 itself can also induce a unique M2 macrophages phenotype (M2c), which is characterized by high expression of IL-10, CXCL13, CXCL4 and MARCO49. Recently there have been many new stimuli discovered that activate and polarize macrophages. GMCSF and MCSF were found to be able to polarize macrophages to proinflammatory and anti-inflammatory phenotypes, respectively50. Likewise, glucocorticoids, TGFβ, IL-25 and prostaglandin have also been implicated in macrophage polarization51-54. With an ever-expanding list of polarizing molecules, it has become evident that macrophage activation is a complex process, which is not sufficiently explained by the M1/M2 paradigm. 8

Another consideration with the M1/M2 model is that all of the described phenotypes have been identified in vitro. It is significantly more difficult to identify macrophage polarization in- vivo due to the number of stimuli that can be acting on macrophages at any given time. Macrophages may be polarized with pro- and anti-inflammatory stimuli simultaneously, resulting in phenotypes that are somewhere in-between M1 or M2. It is therefore more appropriate to think of macrophage polarization as a spectrum with M1 and M2 as extremes on the spectrum. It is important to note that unlike Th1 or Th2 cells, which once polarized do not change their polarization, macrophages have the ability to change their phenotypes through the course of an immune response55. For example, during traumatic brain injury, M1-like macrophage polarization peak at 2 hours post-injury and began to decline by 24 hours post- injury, while M2-like macrophage polarization peaked by 24 hours, demonstrating a shift from M1 to M2 polarization over time56.

1.1.7 Macrophage polarization in disease

Macrophage polarization is now thought to play an important role in disease pathogenesis and in some cases, results in ineffective immune clearance. In the LCMV Cl 13 model of chronic viral infection, IL-10 was found to promote the persistence of virus by promoting T cell exhaustion and T cell dysfunction. When IL-10 was targeted, viral clearance was enhanced and T cell function was restored57. Using Vert-X IL-10 reporter mice, the main cell types responsible for production of IL-10 were found to be dendritic cells and macrophages, which indicate that macrophages during LCMV Cl 13 infection are polarized to an M2-like phenotype58. These findings show that macrophage polarization can modulate adaptive immune responses, and that dysfunctional macrophage polarization can have negative outcomes in disease. Likewise, M2- like macrophages termed tumor-associated macrophages (TAMs) have been implicated in the persistence and progression of cancer. M2-polarizing stimuli are highly expressed in the tumor microenvironment including IL-10, TGFβ, glucocorticoids and immune complexes59. Tumor cells themselves also produce factors that influence macrophage polarization. Lactic acid produced by lung carcinoma and melanoma cells was found to polarize macrophages to an M2- like phenotype through HIF1a, which lead to the transcription of VEGF and arginase60. The hypoxic environment of tumors can induce TAMs to promote tumor progression by stimulating angiogenesis through production of MMP9, VEGF and BFGF61. TAMs can also support tumor growth by the production of ornithine through arginase which can lead to polyamines and cell 9 proliferation. Blocking arginase expression by macrophages was found to significantly reduce tumor size60. TAMs have been found to also promote metastasis through mechanisms including production of the chemokine CCL3 and EGF, while also expressing IL-10 and PDL-1 which inhibit robust anti-tumor adaptive responses62-65. Due to their important roles in cancer, TAMs have become attractive therapeutic targets. One study found the scavenger receptor MARCO to be highly expressed on suppressive TAMs. Targeting MARCO with a monoclonal antibody led to the reprogramming of TAMS to a proinflammatory phenotype and enhanced anti-tumor immune responses66.

M1-like polarization in macrophages has also been shown to contribute negatively to disease outcomes by promoting excessive inflammation. In the experimental autoimmune encephalomyelitis (EAE) mouse model, a model of multiple sclerosis, macrophages were found to be activated and producing NO67. Depletion of macrophages with clodronate liposomes has been shown to result in improvement of EAE68. Similarly, in rheumatoid arthritis, synovial fluid macrophages were shown to have M1 profiles due to TNFα-induced IFN-dependent STAT1 signaling69. Obesity and type 2 diabetes are becoming understood as chronic inflammatory diseases. In obese humans and mice, macrophages were shown to heavily accumulate in adipose tissue. These macrophages were found to express the M1 markers iNOS and TNFα, while in lean adipose tissue, macrophages have an M2-like polarization70. Inflammatory cytokines have been shown to contribute to insulin resistance, indicating that macrophage polarization may have an impact on metabolic diseases such as type-2 diabetes. These findings demonstrate that macrophages are a critical cell type in determining the course of many diseases and highlight the possibility of targeting macrophage polarization for future therapeutics.

1.2 Fibrinogen-like protein 2 1.2.1 The coagulation cascade

Coagulation factors are a series of zymogens that undergo proteolytic cleavage in response to blood vessel damage, resulting the in formation of a clot. In addition, pathogen invasion may result in the activation of the coagulation cascade, as seen in bacterial infection and sepsis71. The external arm of the coagulation cascade is activated by the transmembrane serine protease tissue factor (CD142), which is expressed on damaged endothelial cells. Activation of tissue factor results in a series of proteolytic cleavages, which eventually lead the generation of 10 thrombin. Thrombin cleaves fibrinogen to the activated form fibrin resulting in deposition of fibrin and the formation of a clot71.

It is becoming increasingly apparent that coagulation and the immune system share a common, early evolutionary origin in eukaryotes and continue to function together to maintain homeostasis. During inflammation, TNFα and IL-1 have been shown to induce the expression of tissue factor on monocytes and endothelial cells72. T cells are also thought to regulate the expression of tissue factor with Th1 cytokines upregulating and Th2 cytokines downregulating tissue factor73. Likewise, tissue factor also modulates the immune response by promoting transendothelial migration and leukocyte adhesion74. Tissue factor was also found to be proinflammatory, and caused arthritis like symptoms when injected into healthy mice75. Thrombin was also found to be pro-inflammatory and was found to upregulate expression of IL-6, IL-1, CCL2 and TNFα in monocytes and has been implicated in contributing to the pathogenesis of inflammatory diseases such as coronary thrombosis, atherosclerosis, and cancer76. Fibrinogen has been shown to activate NFκB in neutrophils and promote survival77. These examples serve to highlight the extensive cross-regulation between the coagulation and the immune systems and how they act in unison to promote the clearance of pathogens and the return to homeostasis.

Fibrinogen belongs to a family of proteins characterized by a highly-conserved Fibrinogen-related domain (FRED) on the β and γ chains. Members of this family include: tenascins, ficolins, angiopoietin, angiopoietin-related proteins, Fibrinogen-like protein 2 (FGL2) and FIBCD-1. Members of this family have diverse functions, including tissue growth and repair as well as structural functions. Members of this family have also been shown to have immunoregulatory functions. Angiopoietin was found to induce the recruitment and expression of IL-10 in Tie-2 expressing monocytes78. Another member of fibrinogen superfamily FIBCD-1, was found to bind to chitin and reduce expression of IL-879. FGL2, a member of the fibrinogen family of proteins, has been shown to have potent immunosuppressive activity and has been implicated in the pathogenesis of a number of diseases.

1.2.2 Genomic localization and protein structure of FGL2

FGL2 also known as fibroleukin, was first cloned in 1987 from cytotoxic T cells and was identified as a member of the fibrinogen superfamily of proteins due to sharing 36% amino acid homology to the β and γ subunits of fibrinogen80. The fgl2 gene is a single copy gene in the 11 haploid genome and has been localized to chromosome 7 in humans81. In mice and pigs, the gene is located to chromosomes 5 and 9, respectively. The fgl2 gene contains two exons separated by a 2195 base-pair intron and is flanked by 5’ cis-elements including an AP1 site, an SP1 site, OCT sites, CEBP sites and Ets binding domains. In T regulatory cells (Treg), transgenic overexpression of CEBPα resulted in a significant increase in fgl2 expression82. Transcription of the gene results in two mRNA transcripts, 1.5kbp and 5kbp in size81. Transcripts can be found in heart, liver, lung, kidney, small intestine, and spleen. Translation results in a protein that is 439 amino acids in humans and 432 amino acids in mice. Under reducing conditions, the protein migrates with a molecular mass of 70 kDa, while under non-reducing conditions it migrates with a mass of 250-300 kDa, indicating that FGL2 exists as a tetramer held together by disulfide bonds83. FGL2 is divided into two domains: a linear domain containing α-helixes at the N- terminus and a globular portion at the C-terminus containing a FRED characteristically found on members of the fibrinogen family. Human and mouse FGL2 share about 70% homology overall, but homology in the FRED increases to 90%, indicating this portion is highly conserved84. FGL2 protein is expressed in two main forms: 1) a surface bound form on macrophages and endothelial cells and 2) a secreted form which has been found to be highly expressed by many types of Treg.

1.2.3 Membrane-bound FGL2

The membrane-bound form of FGL2 is a type II membrane protein and a serine protease, which has been shown to have prothrombinase activity, cleaving prothrombin to thrombin. Through site-directed mutagenesis, serine 89 was found to be critical for this prothrombinase activity85. Membrane-bound FGL2 has been implicated in the pathogenesis of several diseases. Membrane-bound form of FGL2 has been shown to have important physiological roles in the fetal development of mice86. FGL2 was found to be expressed in the maternal decidua as well as embryonic tissues during the early stages of pregnancy. Breeding of heterozygous fgl2+/-mice showed loss of both fgl2+/- and to an even greater extent, fgl2-/- embryos prior to day E11.5 due to hemorrhage. Interestingly, in the same study, injection of LPS at E6.5 which induces fetal loss was found to not impact fgl2-/- x fgl2-/- mating, demonstrating the dual roles of FGL2 as critical for normal fetal development, but also contributing to LPS-induced fetal loss.

During the course of viral hepatitis, fibrin deposition and thrombosis often result in hepatocellular injury and liver damage. In murine hepatitis strain 3 infection (MHV-3), an 12 experimental model of human fulminant hepatitis, it was found that activated macrophages and endothelial cells from BALB/cJ mice, which are highly susceptible to MHV-3 infection, expressed high levels of FGL2, indicating that the prothrombinase activity of FGL2 may be contributing to fibrin-mediated hepatic necrosis. C3H mice, which expressed lower levels of FGL2, did not die, and A/J mice, which did not produce FGL2 in response to infection, are resistant to MHV-387. FGL2 was further shown to be involved in the pathogenesis of MHV-3 hepatitis by the finding that MHV-3 infected fgl2-/-macrophages failed to induce a coagulation response and that fgl2-/- mice have improved survival with MHV-3 infection with little or no liver necrosis and fibrin deposition.

The prothrombinase activity of FGL2 has also been shown to contribute to the pathogenesis of graft loss in both xeno and allo transplantation. In acute vascular rejection after pig to primate kidney xenografts, FGL2 was found to be upregulated on the endothelium of the graft and contribute to thrombosis88. Targeted deletion of fgl2 in mouse to rat cardiac xenografts resulted in reduced fibrin, antibody and complement deposition compared to fgl2+/+ grafts consistent with lower acute vascular rejection89. Similarly, sinusoidal endothelial cell FGL2 expression was induced by TNFα and was found to mediate injury after hepatic reperfusion in mice90. In fgl2-/-mice, there was less hepatocyte necrosis and increased survival compared to fgl2+/+ mice. These studies suggest targeting the pro-coagulant activity of FGL2 may be beneficial in improving graft survival after transplantation.

1.2.4 Soluble FGL2

Soluble FGL2 has been shown to have potent immunoregulatory activity and directly or indirectly regulates the function of several immune cell types including B cells, dendritic cells and effector T cells. Recombinant FGL2 suppresses T cell proliferation in response to stimulation with alloantigens, anti-CD3/CD28 antibodies and concanavalin A in vitro in a dose- dependent manner91. In addition, FGL2 was able to suppress the production of IL-2 and IFNγ and increase the production of IL-4 and IL-10 in allogenic cultures, polarizing away from a Th1 response and towards a Th2 response. FGL2 was also found to induce apoptosis of B cells, illustrating a potent ability to regulate adaptive immune responses92. FGL2 been shown to have important regulatory effects on bone marrow-derived dendritic cells (BMDC). The addition of FGL2 to BMDC stimulated with LPS resulted in decreased maturation as measured by CD80 and 13

MHC II compared to LPS alone91. BMDC treated with FGL2 were also found to have a decreased ability to stimulate T cell responses. FGL2 was shown to inhibit translocation of NFκB from the cytoplasm to the nucleus in response to LPS indicating a possible mechanism by which FGL2 exerts its suppressive effect.

FGL2 has been identified as a key effector molecule of the TIGIT+ Foxp3+ subset of Treg. TIGIT is a co-inhibitory molecule that binds to CD15582. It was shown that ligation of TIGIT resulted in robust production of FGL2 and IL-10. These TIGIT+ Treg were able to selectively suppress Th1 and Th17 T cell responses, but did not suppress Th2 responses. Using fgl2-/- mice, it was observed that the selective suppression of Th1 and Th17 responses, but not Th2 was entirely dependent on FGL2. These findings serve to identify FGL2 as a novel immunosuppressive molecule with a wide variety of pleotropic effects on immune responses.

The cognate receptor for FGL2 has now been identified as FcγRIIB/RIII which is expressed primarily on the surface of a B cells, monocytes, macrophages, dendritic cells and endothelial cells, as well as activated T cells. The B cell line A20, which is known to only express FcγRIIB, was found to bind FGL2 as measured by flow cytometry, whereas the B cell line A20IIA1.6, which lacks FcγRIIB, failed to bind to FGL292. An antibody to FcγRIIB/FcγRIII completely prevented the binding of FGL2, further confirming that FcγRIIB is the receptor for FGL2. These findings were confirmed by plasmon resonance analysis. To identify the regions of FGL2 responsible for binding to FcγRs, synthetic oligomer peptides were generated93. It was found that peptides corresponding to regions in the β sheet plane in the FRED domain of FGL2 were able to block the biological activity of FGL2, indicating these regions in the FRED domain of FGL2 are responsible for mediating biding of FGL2 to FcγRIIB/RIII.

1.2.5 The role of FGL2 in disease

Mice genetically deficient in FGL2 exhibit a phenotype consistent with loss of an immunosuppressive molecule. Immunization of fgl2-/- mice with T-independent antigens resulted in increased IFNγ and decreased IL-4, indicating a Th1 polarization94. In addition, numbers of antibody producing B cells were also increased and dendritic cells expressed higher levels of co- stimulatory molecules following stimulation. Treg from fgl2-/- mice also showed significantly decreased suppressive activity compared to fgl2+/+ Treg. Interestingly fgl2-/- mice were prone to 14 developing autoimmune glomerulonephritis, which is consistent with defective immunoregulatory activity in these mice.

FGL2 has also been implicated in the pathogenesis of many important infectious diseases. In patients with chronic viral hepatitis, elevated levels of serum FGL2 have been observed when compared to healthy controls or patients with alcoholic cirrhosis95. FGL2 levels in these patients has also correlated with disease severity and returned to baseline levels in patients who responded to anti-viral therapy. Dr. Levy’s group has recently published on the role of FGL2 in acute viral hepatitis caused by LCMV WE and chronic hepatitis caused by LCMV Cl 13. In both LCMV WE and LCMV Cl 13 a rapid increase of plasma FGL2 was observed early in the course of infection96. Plasma levels of FGL2 remained elevated during the course of infection and returned to near baseline once the infection had resolved. Infection of fgl2-/- mice with LCMV WE resulted in increased dendritic cell maturation and serum TNFα levels. The virus-specific CD8+ T cell and neutralizing antibody response was also enhanced in these mice. Infection of fgl2-/- mice with LCMV Cl 13 resulted in clearance of virus by day 56 post-infection and undetectable virus in the lungs, liver and plasma, while wild-type mice remained persistently infected. This viral clearance was accompanied by increased APC activation, with dendritic cells and macrophages expressing higher levels of co-stimulatory molecules. Virus-specific T cells also expressed higher levels of TNFα and IFNγ upon restimulation as well as lower levels of PDL1, indicating restoration of anti-viral T cell responses. LCMV Cl 13 infected wild-type mice were giving anti-FGL2 and FcγRIIB/RIII antibody beginning at 25 days post-infection until day 45 post-infection. Interestingly anti-FGL2 therapy was able to restore anti-viral adaptive responses, and virus was cleared by day 56 post- infection. These results suggest that FGL2 is a major mediator of viral pathology in both experimental and human viral hepatitis and provide rational for the development of anti-FGL2 therapeutics for use in human patients.

Induction of FGL2 may be a mechanism by which parasites persist and protect against anti-parasitic immune responses. FGL2 was found to be highly elevated in the livers of mice infected with the helminth Echinococcus multilocularis97. Subsequent infection of fgl2-/- mice with this helminth resulted in lower parasitic load compared to wild-type mice98. IL-17A and FGL2 levels were found to be correlated, and treatment splenic mononuclear cells with IL-17A lead to increased FGL2 expression in a dose-dependent manner. Sorting on splenic Treg, CD4+, 15

CD8+, and APC cells demonstrated that Treg showed the highest increase of FGL2 mRNA after infection indicating that these cells were the major producer of FGL2. Interestingly infected fgl2- /- mice had fewer Treg than fgl2+/+ mice. Increased helminth control in fgl2-/- mice was also accompanied by increased IFNγ and IL-17A, and decreased IL-4 and IL-10 as well as increased APC expression of co-stimulatory molecules. These findings suggest that after Echinococcus multilocularis infection, IL-17A induces the expression of FGL2 by Treg. FGL2 has a suppressive effect on Th1 and Th17 immune responses and biases Th2 immune responses, which promotes Echinococcus multilocularis persistence and pathology.

FGL2 has also been implicated in cancer, and cancer cells may take advantage of this pathway to evade immune responses. Interestingly FGL2 has been shown to be produced by glioblastoma, colorectal carcinoma and hepatocellular carcinoma cells99-101. Glioblastoma, a highly malignant brain tumor, has been found to have high copy number mutations of the fgl2 gene. Lower-grade gliomas, on the other hand, retained 2 copies of the fgl2 gene99. FGL2 mRNA in the tumor also inversely correlated to patient outcome. In a glioblastoma mouse model, FGL2 overexpression was shown to enhance tumor growth and inhibition of FGL2 with anti-FGL2 antibodies slowed tumor growth and also increased median survival. These studies demonstrate the importance of FGL2 in tumor progression and suggest that blocking FGL2 may be a useful anti-cancer therapy.

FGL2 has been implicated in the induction of transplantation tolerance. In a murine heart transplant model, rapamycin-induced tolerance was shown to be dependent on Treg and the expression of FGL2. Treatment with anti-CD25 antibody which depletes Treg or anti-FGL2 antibody resulted in the prevention of tolerance and the loss of the allograft102. Addition of recombinant FGL2 also prevented rejection of mismatched cardiac allografts; however, these grafts were rejected once treatment with FGL2 was stopped103. Mice that ubiquitously overexpressing FGL2 (fgl2Tg) were also found to spontaneously accept 50% of allografts without the need for additional immunosuppression. These findings demonstrate that FGL2 may be a key molecule involved in transplantation tolerance.

1.3 FGL2 and macrophage M1/M2 polarization

While the immunoregulatory effects of FGL2 have been extensively studied on T cells, B cells and dendritic cells, the effects on macrophage phenotype and function are less well understood. M2 macrophage polarization has been reported in many diseases in which FGL2 has been found to have an immunoregulatory role; however, a direct link between FGL2 and M2 macrophage polarization has yet to be established. Using a mouse model, glioblastoma cells were stably transfected to over-express FGL2.These FGL2-overexpressing tumors had a higher growth rate than their non-transfected counterparts. Interestingly, the tumor environment of FGL2-overexpresssing tumors contained more myeloid-derived suppressor cells, Treg and M2 macrophages than non-transfected tumors99. This increase in suppressive cell subsets was abrogated in fcγriib-/-mice and after treatment with anti-FGL2 antibody, indicating the FGL2- FcγRIIB pathway may be supporting the accumulation of these suppressive cell types; however, it remains to be determined if FGL2 is directly responsible for the polarization or acts through a secondary molecule.

Fcγ receptor engagement on macrophages have been known to promote suppressive phenotypes. Macrophage M2b polarization characterized by high IL-10 and low IL-12 is induced by stimulation of immune complexes binding to FcγR on macrophages104. The selective suppression of IL-12 by FcγR ligation was found to occur at the transcriptional level and be dependent on intracellular calcium influx. These studies provide strong evidence for the role of FcγR in determining macrophage phenotype as well as a rationale for examining FGL2 as a novel determinant of macrophage M2-like polarization. 17

1.4 Hypothesis and Objectives

Hypothesis

We hypothesize that FGL2 directly polarizes macrophages to an M2-like phenotype through ligation of FcγRIIB/RIII receptors

Objectives

Dr. Levy’s lab has generated unique reagents including mice genetically deficient (fgl2-/-) or ubiquitous overexpressing FGL2 (fgl2Tg) as well as purified recombinant mouse and human FGL2 protein. We plan to use these reagents to test the hypothesis by:

1. Characterizing macrophage polarization in fgl2+/+, fgl2-/- and fgl2Tg mice, including M1/M2 cytokine profiles.

2. Examining the effect of recombinant FGL2 protein directly on macrophage M1/M2 polarization as determined by the ratio of production of IL12 /IL10.

3. Determining the biological significance of FGL2 on macrophages by studying phagocytosis.

Chapter 2 Materials and Methods Materials and Methods 2.1 Animal Care

Fgl2+/+ (C57BL/6J), fgl2-/- and fgl2Tg mice were housed in specific-pathogen free conditions at the Ontario Cancer Institute (OCI) Animal Resources Center. All animal work was conducted in accordance with the guidelines set by the Canadian Council for Animal Care.

2.2 Generation of fgl2-/- and fgl2Tg Mice

Mice were generated as reported previously87,103. Briefly, for fgl2-/- mice a targeting construct containing a LacZ reporter and a PKG-neo was inserted into the first coding exon of the fgl2 gene accompanied by mutation of the initiator methionine codon to a BamHI site. ES cell clones where injected into blastocysts derived from C57Bl/6 mice generating chimeric mice. Intercrossing chimeric mice produced fgl2-/- as confirmed by PCR and southern blot. For fgl2Tg mice, fgl2Loxp mice containing a β-geo reporter gene flanked by loxp sites under the control of a constitutive CMV enhancer/chicken beta actin (CAG) promoter were crossed with Ella-Cre mice which ubiquitously express Cre. After Cre-mediated excision of the β-geo reporter, the fgl2-egfp gene was brought under the control of the CAG promoter resulting in constitutive and widespread expression of FGL2 as confirmed by ELISA and western blot.

2.3 Peritoneal Exudate Cells

4% w/v Brewer’s thioglycollate (Fisher Cat # DF0430-17-1, Nepean, ON) was aged for 2 months in the dark at room temperature, after which it was stored at 4oC before use. 1ml of thioglycollate was injected intraperitoneally into mice. Cells were collected by peritoneal lavage 4 days post-injection with 5ml cold PBS and counted with a hemocytometer. Samples with blood in the lavage fluid were excluded from the study. Total cells were calculated by multiplying cells/ml by the lavage volume. Cells were spun at 300g and resuspended in 5ml media and counted. 1 x 105 peritoneal cells were plated per well of a 96 well flat-bottom suspension cell plate in DMEM supplemented with 10% fetal bovine serum, Lglutamine and penicillin + streptomycin and placed in incubator at 37oC and 5% CO2. After an overnight incubation, non- adherent cells were washed off with two washes with ice-cold PBS. To measure cell viability, 19

20µl AlamarBlue® (Thermo Fisher Cat # DAL1025) was added to 200µl cultures and incubated at 37oC for 2 hours. Fluorescence was read at 590nm.

2.4 Production and Purification of Recombinant FGL2 Protein

His-tagged FGL2 protein was produced and purified as previously described92. Briefly, a plasmid containing mouse fgl2 cDNA (pMPG2-HIS-FGL227–432) was used to stably transfect Chinese hamster ovary (CHO) cells. FGL2 was purified by nickel column chromatography. To assess purity, protein was run on SDS-PAGE and stained with Coomassie blue. Additionally, western blot using anti-His antibody conjugated to HRP was used to confirm the presence of His-tagged protein. FGL2 protein concentration was determined by NanoDrop™ 2000.

2.5 Stimulation of Peritoneal Macrophages

Thioglycollate –elicited macrophage cultures were stimulated with E. coli LPS (Sigma Cat# L3880, Oakville, ON), mouse rIFNγ (Biolegend Cat# 575304, San Diego, CA), rIL-4 (Biolegend Cat# 574304), anti-CD40 agonist antibody (Clone FGK45, generously provided by Dr. Tania Watts) or recombinant FGL2. Culture supernatants were collected and stored at -80oC until use.

2.6 Griesse and Arginase Assay

Griesse and Arginase assays were carried out as previously described. 1x105 peritoneal cells were plated and allowed to rest overnight. Non-adherent cells were washed off. To measure NO, cells were stimulated for 24 hours and culture supernatants were mixed 1:1 with Griesse reagent for 10 minutes in the dark. To measure arginase, cells were stimulated for 24 hours and lysed with 100µl 0.1% triton for 15 minutes. Cell lysates were mixed with 100µl 50mM Tris- HCl and 10µl 100mM MnCL2. 100µl was transferred to a 2ml safe-lock Eppendorf tube. Lysates were incubated for 7 minutes at 56oC to activate enzymes. 100µl of 0.5 M arginine substrate was added and incubated at 37oC for 2 hours. To stop the reaction, 800µl of an acid mix containing H3PO4, H2SO4 and H20 in a 1:3:7 ratio was added to each tube. Urea production was measured by adding 40µl 6% α-ISPP dissolved in ethanol and incubating at 95oC for 30 minutes. Samples were allowed to cool for 30 minutes at 4oC before being read. Both Griesse assay and arginase assay samples were read using an ELISA reader at 540nm. 20

2.7 ELISA

IL-12 (Biolegend Cat# 433604), IL-6 (Biolegend Cat # 431304) and TNFα (Biolegend Cat # 4309040) and IL-10 (R&D Cat # DY417-05, Minneapolis, MN) cytokine levels were quantified in the recovered culture supernatants by sandwich ELISA. To measure IL-12 and IL- 10, undiluted supernatants were used. For IL-6 and TNFα determinations, samples were diluted 50-fold and 10-fold respectively.

2.8 Flow Cytometry

Anti-F4/80 Pe/Cy7 (Biolegend Cat # 123113), anti- F4/80 APC (Biolegend Cat # 123115), anti-Ly6C Pe (Biolegend Cat # 128007), anti-TLR4 Pe (Biolegend Cat # 145403), anti- IFNγR β chain Pe (Biolegend Cat # 113603), anti-CD45 BV650 (Biolegend Cat # 103151), anti- MHCII I-A/I-E Alexa Fluor 700 (Biolegend Cat # 107622), anti-CD11b Pe/Cy7 (Biolegend Cat # 101215), anti-CD11b Alexa Fluor 700 (Biolegend Cat # 101222), anti-CD45 APC/Cy7 (Biolegend Cat # 103115), anti-B220 APC (Biolegend Cat # 103211), anti-CD86 APC/Cy7 (Biolegend Cat # 105029), anti-Siglec-F PerCP-Cy5.5 (BD Bioscience Cat # 565526, Franklin Lakes, NJ), anti-Ly6G APC/Cy7 (BD Bioscience Cat # 560660) where used for the studies. Cells were incubated with Fc block (TruStain fcX™ Biolegend Cat# 101319) for 15 minutes at 4oC before being stained. Dead cells were excluded using eBioscience Fixable Viability Dye eFluor 450 (Thermo Fisher Cat # 65-0863-14). For characterization of immune cell subsets, 1x106 peritoneal-exudate cells were stained in 100µl FACs buffer for 30 minutes at 4oC and analyzed. For characterization of co-stimulatory molecule expression, 2x106 total peritoneal cells were plated on bacteriological plates and allowed to rest overnight. Non-adherent cells were washed off with cold PBS and cells were stimulated for 24 hours. Adherent cells were removed by adding 1ml of Versene solution (Thermo Fisher Cat # 15040066), incubating at 4oC for 15 minutes and pipetting vigorously. Cells were visualized by using an LSRII analyzer (BD Biosciences) and data were analyzed using FlowJo software version 10 (Tree Star Inc., Ashland, OR)

2.9 Phagocytosis Assay

1x106 peritoneal exudate cells were incubated with 10µg of pHrodo Red E. coli Bioparticles (Thermo Fisher Cat # P35361) in 2ml DMEM supplemented with 10% fetal bovine serum, L- 21 glutamine and penicillin + streptomycin for 2 hours at 37oC. Cells were washed and stained for F4/80 and CD11b and analyzed by flow cytometry.

2.10 Statistics

Statistics were calculated using Graphpad Prism version 6 (Graphpad Inc., La Jolla, CA). Parametric Anova with Tuckey’s post hoc test or Student’s t test were used for analysis. 22

Chapter 3 Results Results 3.1 Fewer cells are collected from the peritoneal cavity of fgl2Tg mice after injection of thioglycollate

Injection of thioglycollate into the peritoneal cavity induces sterile inflammation and is a well described method to induce infiltration of macrophages, which are collected 4 days post- injection. The immune response to thioglycollate in fgl2-/- and fgl2Tg mice, however, has not been characterized. Four days post-thioglycollate injection, significantly fewer total cells accumulated in the peritoneal cavity of fgl2Tg mice (2.1 x 107 ± 6.1 x 105) compared to fgl2+/+ (2.5 x 107 ± 7.3 x 105) or fgl2-/- mice (2.8 x 107 ± 1.5 x 106) (p ≤ 0.05 and p ≤ 0.0001 respectively) (Fig 1A.). Analysis of total cell numbers in the peritoneal cavity of fgl2+/+, fgl2-/- and fgl2Tg mice at D0 before injection revealed that all 3 groups of mice had similar numbers of resident cells (Fig 1B.). At D1 post-injection there was a rapid increase in total cells. Total cell numbers peaked at D3 post injection and began to decline to near baseline levels by D7

To identify which cell type was responsible for the difference in total cell numbers, flow cytometry was used to characterize different cell subsets in the peritoneal cavity during the course of thioglycollate-induced inflammation. Gating on CD45+ hematopoietic cells, the frequencies of macrophages (F4/80+ CD11b+), neutrophils (Ly6G+), monocytes (Ly6C+ CD11b+) and eosinophils (Siglec-F+) and B cells (B220+) were determined in the peritoneal cavity (Fig 2A.). Total cell numbers of cell subsets were determined by multiplying the frequency of each subset by the total count derived by counting with a hemocytometer (Fig 2B-F.). At steady state, the peritoneal cavity in the 3 groups of mice contained mostly macrophages (Fig 2E.) and B cells (Fig 2D.). After injection of thioglycollate there was a rapid influx of neutrophils (Fig 2B.) and monocytes (Fig 2C.) into the peritoneal cavity, which is consistent with an acute inflammatory response. There was also infiltration of eosinophils (Fig 2F.) and a decrease in total resident macrophage and B cells. By D3 post-injection, the numbers of monocytes and neutrophils had decreased, while B cells and monocyte-derived macrophages began to increase. Interestingly, at D3 post-injection eosinophils peaked and there was a significant difference between the total numbers of eosinophils in fgl2-/- (1.76 x 107 ± 2.87 x 106) and fgl2Tg (9.87 x 106 ± 1.84 x 106)

23 mice (p≤ 0.05) (Fig 2F.). At D4, all three groups had diminished neutrophils and monocytes, while macrophage numbers remained similar to D3. At D4, total eosinophils were fewer than at D3 in all three groups with a trend towards fewer eosinophils in fgl2Tg mice; however, this did not reach significance. At later time points (D5 and D7) the number of eosinophils continued to decrease, and macrophage numbers increased slightly. From this thioglycollate time course, eosinophils accounted for the significant difference seen in total numbers of cells recovered from peritoneal cavity in fgl2Tg mice.

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Figure 1. Total cell numbers recovered from the peritoneal cavity in response to thioglycollate in fgl2+/+, fgl2-/- and fgl2Tg mice. A) Total cells in the peritoneal cavity 4 days post-thioglycollate injection. Fgl2+/+, fgl2-/-, and fgl2Tg mice were injected with thioglycollate and lavaged with PBS 4 days post-injection. Cells were counted by hemocytometer and concentration of cells/ml were calculated. Total cells were calculated by multiplying cells/ml by total volume used to lavage. N = 21-18 per group from 5 independent experiments. B) Time course of total cells recovered from the peritoneal cavity following injection of thioglycollate. Mice were sacrificed at the indicated time points following injection of thioglycollate. The peritoneal cavity was lavaged with 5ml PBS and total cells recovered were determined by multiplying the concentration of cells/ml by lavage volume. N = 3-4 mice/group at each time point. Mean ± SD. * P ≤ 0.05, **** P ≤ 0.0001. 25

H A

- -

FSC

SSC SSC SSC

FSC-A FSC-W SSC-W CD45-BV650

SSC

FSC

SSC

Cy7

CD11b A700

F480 PE

CD11b-A700 Ly6G-APC-Cy7 Siglec F- B220-APC Ly6C-PE PercpCy5.5

Figure 2. Flow cytometric analysis and total numbers of cell subsets. A) Flow cytometry gating scheme used to identify cell subsets of cells in the peritoneal cavity. Single cells were gated on CD45+ hematopoietic cells. The percentage of macrophages (F4/80+ CD11b+), neutrophils (Ly6G+), eosinophils (Siglec-F+), B cells (B220+) and monocytes (Ly6C+ CD11b+) of total CD45+ cells were determined using flow cytometry. Total numbers of B) Neutrophils (Ly6G+) C) monocytes (Ly6C+ CD11b+) D) B cells (B220+) E) macrophages (F4/80+ CD11b+) and F) eosinophils (Siglec-F+) in the peritoneal cavity were calculated using the following formula (% of total CD45+ x total cell number) during the course of thioglycollate induced inflammation in fgl2+/+, fgl2-/- and fgl2Tg mice at the indicated time points. N = 3-4 mice/group at each timepoint. Mean ± SD. * P ≤ 0.05. 26

3.2 Macrophages from fgl2-/- and fgl2Tg mice express similar levels of iNOS and arginase to macrophages from fgl2+/+ mice.

To examine M1 polarization of peritoneal macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice, macrophages were recovered from the peritoneal cavity and examined for nitric oxide expression at the steady state and in response to stimulation. Macrophages from all 3 groups produced low levels of nitric oxide at steady state. After stimulation with 100ng/ml LPS and 300U/ml IFNγ for 24 hours there was a significant (~15 fold) increase in nitric oxide; however, there was no significant differences between fgl2+/+, fgl2-/- and fgl2Tg macrophages in nitric oxide produced post-stimulation (Fig 3A.). M2 polarization was assessed by measuring expression of arginase in macrophage cells. Cells were left unstimulated or stimulated with 10U/ml IL-4 for 24 hours and lysed. Lysates were incubated with arginine and urea production was measured. Unstimulated macrophages from all 3 groups expressed similar levels of arginase (Fig 3B.). After stimulation with IL-4, which induces M2 polarization, there was a significant (~ 4fold) increase in arginase expression from baseline; however, there was no significant difference between groups.

B C

Figure 3. Nitric Oxide and Arginase expression by cultured peritoneal macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice. 1 x 105 cells were plated and allowed to rest overnight. Non- adhered cells were washed off and adhered cells were stimulated for 24 hours. A) Nitric oxide was measured by adding Griesse reagent. OD values were measured and compared to a standard curve. B) Cells were lysed and cell lysates were incubated with arginine at 37oC for 2 hours, after which urea production was measured. 1 unit of arginase is defined as the amount of enzyme able to hydrolyze 1 µM arginine per minute. N = 5 mice/group. Mean ± SD. 28

3.3 Fgl2Tg macrophages produce significantly less IL-12 in response to stimulation

As discussed in the introduction, the differential expression of IL-12 (M1) and IL-10 (M2) are markers of macrophage M1/M2 polarization. To assess if FGL2 has a role in macrophage polarization, peritoneal macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice were stimulated with 100ng/ml LPS or a combination of 100ng/ml LPS and 300U/ml IFNγ for 24 hours and the production of IL-12 and IL-10 was measured. IL-12 was undetectable in cultures of unstimulated macrophages; however, following stimulation, IL-12 increased in cultures from the three groups of mice (Fig 4A.). Interestingly, IL-12 expression was significantly reduced in fgl2Tg macrophages (40 ± 6.5 pg/ml) compared to fgl2-/- macrophages (68 ± 11 pg/ml) or fgl2+/+ macrophages (59 ± 5.7 pg/ml) after stimulation with LPS (p≤ 0.05). After stimulation with LPS and IFNγ, there was a significant increase in total IL-12 production by all 3 groups compared to LPS alone. Again, macrophages from fgl2Tg mice produced significantly less IL-12 than either fgl2+/+ or fgl2-/- macrophages. IL-10 levels were also measured after stimulation with 100ng/ml LPS for 24 hours (Fig 4B.). IL-10 was undetectable in unstimulated macrophages. After stimulation, there was an increase in total IL-10, but there were no significant differences between the 3 groups.

29 A C

Figure 4. IL-12 and IL-10 production by macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice. 1 x 105 macrophages were plated and allowed to rest overnight. Non-adhered cells were washed off and adhered cells were stimulated for 24 hours. A) Macrophages were stimulated with LPS (100ng/ml) or the combination of LPS (100ng/ml) and IFNγ (300U). IL-12 in culture supernatants was measured by ELISA. N = 9 per group from 2 independent experiments. Mean ± SEM. B) Macrophages were stimulated with 100ng/ml LPS. IL-10 in culture supernatants was measured by ELISA. N = 4 mice/group. Mean ± SD. * P ≤ 0.05, ** P ≤ 0.01.

3.4 Fgl2Tg macrophages express significantly lower TLR4 than fgl2+/+ or fgl2-/- macrophages

Engagement of FGL2’s cognate receptors FcγRs, have shown to downregulate expression of macrophage cell surface receptors such as IFNγR105. To examine if downregulation of cell surface receptors on macrophages from fgl2Tg mice could account for the decreased IL-12 expression in response to stimulation, flow cytometry was performed. In these experiments, the expression of the IFNyR β subunit, the signaling chain of the receptor for IFNγ, and TLR4, the receptor for LPS on freshly isolated cells, was measured. Gating on F480+ CD11b+ macrophages, comparable expression of IFNγR was found from all 3 groups on mice as measured by Median Fluorescence Intensity (MFI) (Fig 5A.). In contrast, the MFI of TLR4 on fgl2Tg macrophages (625 ± 23) was found to be significantly lower compared with fgl2+/+ (709 ± 19) or fgl2-/-(751 ± 52) macrophages (Fig 5B.) (p≤ 0.05). These data suggest that the reduced production of IL-12 (after LPS stimulation) by fgl2Tg macrophages is related to lower expression of surface TLR4.

Figure 5. Expression of IFNγR and TLR4 on macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice. 1 x 106 thioglycollate-elicited peritoneal cells were isolated and stained. Gating on F4/80+ CD11b+ macrophages, the MFI of A) IFNγR and B) TLR4 were measured by flow cytometry. Representative histograms for fgl2Tg (blue), fgl2-/- (orange) and fgl2+/+(red) compared to FMO (green) are illustrated. N = 10 mice/group from 2 independent experiments. Mean ± SEM. * P ≤ 0.05.

3.5 FGL2 protein suppresses IL-12 by macrophages in a dose- dependent manner.

In the previous studies, IL-12 production was found to be suppressed in macrophages that were genetically engineered to overexpress FGL2 (fgl2Tg). We next investigated if recombinant FGL2 (rFGL2) could suppress IL-12 in fgl2+/+macrophages stimulated with LPS. IL-12 in culture supernatants was measured after macrophages were stimulated for 24 hours with 100ng/ml LPS alone or in combination with 10ng/ml, 100ng/ml, 200ng/ml, 500ng/ml or 1µg/ml rFGL2 (Fig 6A.). A dose-dependent suppression of IL-12 production was observed compared to LPS alone (143 ± 6.6 pg/ml) starting at 100ng/ml (97 ± 10 pg/ml) rFGL2, reaching maximum suppression at 1µg/ml of FGL2 (69 ± 4.9 pg/ml, p≤ 0.05). Expressed as a percent inhibition compared to LPS alone, 100ng/ml FGL2 inhibited IL-12 production by 28 ± 4.5%. Suppression was increased to 51 ± 2.1% with 1µg/ml FGL2 (Fig 6A.).

As FGL2 protein has been shown to induce apoptosis in some cell types, cell viability was assessed to exclude the possibility of cell death as the cause for the decrease in IL-12 production. AlamarBlue was used to measure cell viability (Fig 6B.), which becomes fluorescent in the reducing environment of a viable cell. AlamarBlue fluorescence was not reduced in FGL2 treated samples compared to LPS alone, suggesting that the suppression of IL-12 was not due to macrophage apoptosis.

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Figure 6. IL-12 production is suppressed by rFGL2 in fgl2+/+ macrophages. A) 1 x 105 cells were plated and stimulated with 100ng/ml LPS alone or in combination with different concentrations of FGL2 protein. IL-12 levels were measured by ELISA. Percent inhibition was calculated by dividing LPS and FGL2 treated IL-12 values by LPS alone values. N = 9 from 2 independent experiments. Mean ± SEM. B) Cell viability was assessed by AlamarBlue. 1 x 105 cells were treated as indicated. After 24 hours AlamarBlue was added and fluorescence was measured by ELISA reader. N = 5. mean ± SD. * P ≤ 0.05. 34

3.6 FGL2 protein suppresses IL-12 by macrophages to a variety of stimuli

We next investigated whether FGL2 could suppress IL-12 production in response to stimuli other than LPS. CD40 is known to be able to induce IL-12 production by myeloid cells in vivo. An anti-CD40 agonist antibody (clone FGK45) was used to stimulate cells to produce IL- 12, and FGL2 was added to determine whether it could also suppress this stimulation. As has been reported in the literature, anti-CD40 alone was insufficient to induce IL-12 in vitro106,107. However, in combination with 100ng/ml LPS, anti-CD40 could increase IL-12 production to higher levels compared to LPS alone (207 ± 24 pg/ml vs 275 ± 48 pg/ml, p≤ 0.05) (Fig 7.). Addition of 1µg/ml FGL2 protein was able to completely suppress the synergistic effect of LPS and anti-CD40, and suppressed IL-12 production to levels similar to LPS and FGL2 alone (133 ± 15 pg/ml vs 104 ± 30 pg/ml). These results indicate that FGL2 inhibits IL-12 production in response to stimuli other than LPS alone.

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Figure 7. IL-12 expression in fgl2+/+ macrophages in response to stimulation with CD40 agonist, LPS and rFGL2 protein. 1 x 105 cells were plated and stimulated with LPS alone, or in combination with rFGL2 and anti-CD40. IL-12 levels were measured by ELISA. N = 3. Mean ± SD. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

3.7 FGL2 inhibits IL-12 at early and late timepoints after stimulation and with low and high doses of LPS.

To examine whether FGL2 could completely inhibit LPS-induced IL-12 production, lower concentrations of LPS were used. IL-12 production was also measured at both 6 and 24 hours of stimulation. IL-12 production was measured at 6 hours post-stimulation with (Fig 8A.) 10ng/ml LPS or (Fig 8B.) 100ng/ml LPS and increasing concentrations of rFGL2. Using 10ng/ml LPS in combination with FGL2, there was a trend towards suppression of IL-12 at 6 hours, however this did not reach statistical significance (Fig 8A.). At a higher dose of 100ng/ml LPS there was a significant suppression of IL-12 at 6 hours post stimulation (Fig 8B.). At 24 hours post-stimulation, FGL2 suppressed IL-12 production with both a low dose of 10ng/ml LPS (Fig 8C.) and a higher dose of 100ng/ml LPS (Fig 8D.) These experiments suggest that FGL2 is able to suppress IL-12 at early time points post-stimulation, however is insufficient to completely inhibit the production of IL-12 even after stimulation with a low dose of LPS.

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Figure 8. IL-12 production by fgl2+/+ macrophages at early and late timepoints in response to FGL2 and lower and higher doses of LPS. 1 x 105 cells were plated and stimulated as indicated. IL-12 cytokine levels were measured by ELISA 6 hours post-stimulation with A) 10ng/ml LPS B) 100ng/ml LPS or 24 hours post-stimulation with C) 10ng/ml LPS and D) 100ng/ml LPS. N = 3. Mean ± SD. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

3.8 FGL2 inhibits IL-6 but not TNFα in response to LPS simulation

As rFGL2 was able to suppress the inflammatory cytokine IL-12, we next determined whether rFGL2 was able to suppress the production of other inflammatory cytokines, such as TNFα and IL-6, which are important inflammatory cytokines produced by activated M1 macrophages. Macrophages were stimulated with 10ng/ml LPS in combination with 10ng/ml, 100ng/ml or 1µg/ml rFGL2 and expression of TNFα and IL-6 at 6 hours and 24 hours post- stimulation was measured. TNFα production was unaffected after stimulation with 10ng/ml LPS at all concentrations of rFGL2 used at 6 hours (Fig 9A.) and 24 hours post-stimulation (Fig 9B). Although IL-6 production was unaffected at 6 hours post stimulation (Fig 9C.), there was a significant suppression of IL-6 production by FGL2 at 24 hours post-stimulation (Fig 9D.).

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Figure 9. TNFα and IL-6 production by fgl2+/+ macrophages at early and late timepoints in response to FGL2 and LPS. 1 x 105 cells were plated and stimulated as indicated. TNFα cytokine levels were measured by ELISA at A) 6 hours and B) 24 hours after stimulation. IL-6 levels were also measured at C) 6 hours and D) 24 hours after stimulation. N = 3. Mean ± SD. * P ≤ 0.05, ** P ≤ 0.01.

3.9 Fgl2+/+, fgl2-/- and fgl2Tg macrophages express similar levels of MHCII and CD86 in response to LPS or IFNγ

Macrophages have an important role in antigen presentation beyond cytokine production. To examine if FGL2 influenced the ability of macrophages to present antigen, macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice were plated and stimulated with either 100ng/ml LPS or 300U IFNγ for 24 hours and analyzed for expression of MHCII and CD86 by flow cytometry. Percentage of CD86 expression was similar on unstimulated cells from all 3 groups. After stimulation with LPS or IFNγ, there was a profound upregulation of CD86 on macrophages from all 3 groups of mice; however, there were no significant differences between groups (Fig 10A.). Similarly, MCHII expression was comparable on unstimulated macrophages of all 3 groups, and after LPS or IFNγ stimulation, the percent of MHCII positive cells increased; however, there were no differences between the three groups (Fig 10B.). The MFI of CD86 (Fig 10C.) and MHCII (Fig 10D.) was also not different before or after stimulation on macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice.

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Figure 10. Expression of co-stimulatory molecules on macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice. 2 x 106 peritoneal cells were plated and non-adherent cells were washed off. Cells were stimulated for 24 hours with 100 ng/ml LPS or 300 U/ml IFNγ. Cells were stained and analyzed by flow cytometry. Percentages of cells positive for A) CD86 and B) MHCII of total were determined. MFI of C) CD86 and D) MHCII were also determined on macrophages post- stimulation. N = 5. Mean ± SD.

3.10 Phagocytosis of Bacteria is impaired in fgl2Tg macrophages

Macrophages are important in the clearance of bacteria as they are primarily responsible for phagocytosing bacterial cells. M1 polarized macrophages have an increased ability to phagocytosis and destroy bacteria, while M2 macrophages have a decreased capacity for phagocytosis. To examine if FGL2 could modulate macrophage phagocytosis, the ability of thioglycollate-elicited peritoneal macrophages from fgl2+/+, fgl2-/- and fgl2Tg to phagocytose labelled E. coli bioparticles was determined. After 2 hours of incubation with the E. coli particles, the percentage of macrophages that had taken up the E. coli (stained positively) was similar in all 3 groups of mice (Fig 11A-B.) However, fgl2Tg macrophages had a significantly lower MFI compared with fgl2+/+ and fgl2-/- macrophages (Fig 11C.). This suggests that although the number of macrophages that had phagocytosed bacteria was similar, fewer bacteria were phagocytosed by fgl2Tg macrophages.

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Chapter 4 Discussion Discussion

Macrophages are critical cells for the maintenance of homeostasis and for the initiation, progression and resolution of inflammation. Due to the expression of many different PRRs, they act as immune sentinels. Macrophages detect bacterial, viral or fungal pathogens and help to initiate the appropriate immune responses through the expression of cytokines such as IL-1β, TNFα IL-6, IL-12 and IL-10. Macrophages are also key in modulating adaptive T cell and B cell responses as changes in the expression of cytokines and surface molecules during the course of inflammation can either promote or inhibit adaptive responses. Macrophages have thus become attractive targets for therapeutic intervention in diseases including atherosclerosis, cancer, autoimmune disease, metabolic disease and infectious disease and there is great interest in therapeutic agents that polarize macrophage phenotypes. FGL2 has been identified as a potent immunoregulatory molecule and recently has been implicated in the pathology of viral and parasitic infections as well as cancer. We hypothesized that FGL2 promotes an immunoregulatory M2-like phenotype in macrophages. The availability of mice that lack expression of FGL2 (fgl2-/-) or overexpress FGL2 (fgl2Tg) as well as the availability of recombinant purified FGL2 provided a unique opportunity to conduct these studies.

4.1 FGL has suppressive roles in eosinophil accumulation in the peritoneal cavity in response to thioglycollate.

We first investigated the response to thioglycollate in fgl2-/- and fgl2Tg mice compared to control fgl2+/+ mice. Thioglycollate-induced peritonitis is a well described method for the induction of large numbers of monocyte-derived macrophages in mice. Aging thioglycollate results in the nonenzymatic formation of aged glycation end products from proteins and sugars, which increases its ability to induce inflammation and to recruit large numbers of cells into the peritoneal cavity108. Macrophages were collected 4 days after administration of thioglycollate. Initially, significant differences in total cell numbers recovered at this time point were observed with fgl2-/- and fgl2+/+ mice having significantly more total cells compared to fgl2Tg mice. The thioglycollate induced inflammatory response triggers the influx of multiple immune cell types into the peritoneal cavity and to identify the cell type(s) responsible for the observed differences 45 in cell number, a time course was conducted with flow cytometry to identify immune cell populations in the peritoneal cavity on days 0,1,3,4,5,7 post-injection. During steady-state conditions, the peritoneal cavity of all 3 groups of mice had comparable numbers of total cells. The peritoneal cavity is known to be home to large numbers of resident macrophages as well as the B1 subset of B lymphocytes. The majority of cells were found to be macrophages and B cells in the peritoneal cavity of all 3 groups of mice at the steady state. At D1 post-thioglycollate injection, a rapid influx of Ly6G+ neutrophils and Ly6C+ monocytes were observed, which is characteristic of acute inflammation. Interestingly at this early time point, the influx of eosinophils was also observed into the peritoneal cavity, which are known to make up a large portion of total cells post-thioglycollate. A decrease in the total numbers of resident macrophages recovered was also observed. This phenomenon is well described and is known as the macrophage disappearance reaction, in which resident macrophages in the peritoneal cavity rapidly disappear from the peritoneal lavage fluid after acute inflammatory stimuli such as LPS or thioglycollate and are gradually replaced by monocyte-derived macrophages over time109. By D3 post-injection, the neutrophil and monocyte numbers began to decline and were almost undetectable at later time points; however, macrophage numbers increased as monocytes began to differentiate into macrophages. At this time point, eosinophil numbers also reached a maximum. Significant differences in eosinophil numbers were found, with fgl2-/- having significantly more eosinophils than fgl2Tg at D3. This trend persisted at D4 but did not reach statistical significance. These results indicate that the differences seen in total cell numbers in the 3 strains of mice post-thioglycollate can be predominantly attributed to eosinophils.

The mechanism by which FGL2 may inhibit accumulation of eosinophils into peritoneum is currently unclear. This finding could be attributed to decreased eosinophil chemotaxis into the peritoneal cavity or defects in eosinophil survival in fgl2Tg mice. As eosinophils are known to express both FcγRIIB and FcγRIII, FGL2 may be able to directly act on these cells. One study found that engagement of FcγRIIB/RIII on eosinophils using the monoclonal antibody 2.4G2, which recognizes both FcγRIIB and FcγRIII resulted in chromatin condensation and Annexin V binding, ultimately resulting in apoptosis within 24 hours after antibody addition110. This apoptosis was found to be dependent on FcγRIIB and independent of FcγRIII. FGL2 may thus be directly acting on eosinophils to induce apoptosis in fgl2Tg mice, leading to lower numbers of cells after thioglycollate. However hematological analysis of fgl2Tg mice found eosinophil 46 numbers in the blood to be comparable to fgl2+/+ and fgl2-/- at birth until 6 months of age103. By 1 year after birth the numbers of eosinophils in the blood of fgl2Tg mice were significantly higher than fgl2+/+ or fgl2-/- mice suggesting apoptosis may not be the mechanism responsible for fewer eosinophils recovered in the peritoneal cavity of fgl2Tg mice after injection of thioglycollate. Another possibility may be that eosinophil survival is reduced in fgl2Tg mice. Eosinophil survival is dependent on autocrine or exogenous IL-5, IL-3 and GMCSF and these cytokines have been found to prolong survival of eosinophils in vitro111. Inhibition of IL-5 by FcγRIIB complexed with galectin-3 has been previously described; however, the role of FGL2 in this interaction remains unknown112. Future studies may also look at the production of these cytokines in the peritoneal cavity, as well as the ability of eosinophils to respond to these survival factors in fgl2Tg mice. Monocyte recruitment into the peritoneal cavity in response to thioglycollate was found to be heavily dependent on the chemokine CCL2. These results did not find significant differences in monocyte numbers in the peritoneal cavity, suggesting that CCL2 levels are similar in all 3 strains. However, thioglycollate also induces the expression of CCL7, also known as MCP-3, which is known to be a potent chemoattractant of eosinophils113,114. There is some evidence that FcγRIIB deletion increases CCL7 expression indicating that the FcγRIIB-FGL2 pathway may important for the regulation of CCL7115. Future studies may focus on measuring the levels of CCL7 in the peritoneal cavity or examining the receptors of CCL7, CCR1, CCR2 and CCR3 expression on eosinophils from fgl2Tg mice. Other chemokines are also known to act as chemoattractants to eosinophils, (e.g., RANTES (CCL5) Eotaxin (CCL11) and Eotaxin-2 (CCL24)) may also be downregulated in fgl2Tg mice. It is also important to note that these experiments did not control for differences in microbiota that may contribute to the observed response to thioglycollate. Future studies should include fgl2+/+, fgl2-/- and fgl2Tg littermate controls to ensure that the observed phenotypes are microbiota independent.

Eosinophilia is a hallmark of a number of pathologies including allergy and asthma as well as parasitic and fungal infections. In asthma, eosinophil accumulation has a causal role in airway hyper-sensitivity. In patients exhibiting late asthmatic responses, eosinophils were found to be in a “primed” state and expressed increased FcγRIIB116. In mouse models of allergic asthma, FcγRIIB was found to downregulate inflammation and disruption of fcyriib lead to increased inflammation compared to controls, demonstrating the critical role of FcγRIIB in regulating inflammation in this model117. FGL2 may therefor also be critical in the resolution of 47 airway inflammation and because engagement of these FcγRIIB has been shown to induce apoptosis in eosinophils, FGL2 may be a viable therapeutic option for the elimination of Eosinophils during asthma or other airway inflammatory diseases. Eosinophils have also been shown to provide survival factors for antibody producing plasma cells, which have also been implicated in auto-antibody production in autoimmune disorders. Eosinophils therefore have been the interest of therapeutic intervention in these disorders and the development of drugs to prevent eosinophil activation and accumulation in organs is ongoing118. Currently glucocorticoids are the most effective therapy; however, these drugs have potentially severe side effects and other therapies including targeting chemokines and adhesion molecules have shown promise in animal models. These findings provide rational for future studies examining the ability of FGL2 to inhibit eosinophil accumulation and for the development of FGL2-based anti- eosinophilia therapeutics.

4.2 FGL2 is a novel regulator of IL-12 expression by macrophages.

The next series of studies focused on identifying M1-like or M2-like phenotypes of macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice in response to stimulation in vitro. Macrophage M1/M2 polarization was first historically defined based on differential arginine metabolism and therefore the expression of iNOS and arginase were first measured. Macrophages from all 3 groups of mice expressed low levels of nitric oxide at baseline, indicating that deletion or overexpression of FGL2 was not directly polarizing to an M1-like phenotype. To examine if FGL2 had synergistic effects in enhancing or suppressing M1 polarization, macrophages were stimulated with IFNγ and LPS for 24 hours to polarize to an M1 phenotype. This treatment resulted in approximately a 15-fold increase in NO. However, there were no significant differences between the 3 groups of mice. Likewise, macrophages from the 3 groups did not express significantly different levels of arginase at baseline or after stimulation with IL-4, which increased arginase expression approximately 3-fold. These studies indicate that the absence or overexpression of FGL2 did not directly induce the expression of arginase or iNOS and that macrophages from fgl2-/- and fgl2Tg mice are able to respond to stimuli and upregulate these enzymes similar to fgl2+/+ mice. 48

While arginase and iNOS are good indicators of macrophage polarization, these markers do not translate to human macrophages and thus IL-12 and IL-10 production are also commonly used to identify macrophage polarization. Macrophages were stimulated with LPS alone or in combination with IFNγ and IL-12 production was measured as a marker of M1-like polarization. Interestingly there was a marked decrease in IL-12 produced in response to both LPS alone or in combination of IFNγ by fgl2Tg macrophages compared to fgl2+/+ or fgl2-/- macrophages. IL-10 was however not significantly increased in fgl2Tg macrophages following stimulation. However, based on these results, the ratio of IL-12 to IL-10 has shifted towards IL-10 in macrophages treated with FGL2. These results may have implications on adaptive immune response and Th1- Th2 polarization of T cell.

To confirm these findings were the direct result of FGL2 acting on macrophages, a dose titration of recombinant FGL2 protein and LPS was used to stimulate fgl2+/+ macrophages. There was a clear dose-dependent suppression of IL-12 in response to increasing concentrations of protein. As FGL2 is known to cause apoptosis in some cell types, it was confirmed this was not the case in macrophages. The expression of IL-12 was also examined at earlier time points (6 hours) and using lower doses of LPS (10ng/ml). FGL2 was able to suppress IL-12 as early as 6 hours post-stimulation, but it did not completely suppress IL-12 even at lower doses of LPS. These results suggest that FGL2 suppresses M1-like polarization in macrophages. The transcription of IL-12 in response to LPS involves TLR4-induced MYD88 signaling, ultimately resulting in the degradation of IκB and the translocation of NFκB into the nucleus. IL-12 is a heterodimeric cytokine and the transcription of the two genes, Il12a (IL-12p35) and Il12b (IL- 12p40) are regulated in different ways119. Transcription factors promoting the expression of Il12a include NFκB and IRF-1. Transcription of Il12b is regulated by transcription factors including NFκB, PU.1, IRF-1, AP1, NFAT and C/EBPβ and requires nucleosome remodeling. This study examined the production of IL-12p70; however, it did not identify if the decrease in IL-12p70 was due to decreased IL-12p35 and/or IL-12p40. Previous work from Dr. Levy’s lab has shown that FGL2 has the ability to prevent NFκB translocation in dendritic cells, which may also be contributing to lower levels of IL-12 observed in this study91. Future studies will examine the expression of the two subunits of IL-12. Examining the transcription of the IL-12 genes to identify if the suppression of IL-12 is at the level of transcription may also delineate which transcription factors may be involved. Future studies will also examine the expression of 49 other IL-12 family cytokines including IL-23 and IL-35, which are heterodimeric and share common subunits to IL-12. Others have also shown the suppression of IL-12 in macrophages by Fc receptor ligation and this was determined to be dependent on extracellular calcium influx104. It would thus be important to examine of FGL2 causes an influx of calcium in macrophages and if the suppression of IL-12 by FGL2 can be reversed by the addition of calcium chelators.

The ability of FGL2 to suppress the expression of IL-12 in response to CD40 was also examined. In vitro, CD40 agonistic antibodies require a second stimuli to fully activate macrophages120. Macrophages were stimulated with the anti-CD40 agonist mAb FGK45 in combination with LPS and found to significant increase IL-12 compared to LPS alone. Addition of FGL2 was able to suppress the production of IL-12 in response to LPS and anti-CD40. Interestingly, although FGL2 is able to suppress IL-12 in response to LPS + IFNγ, the levels of IL-12 produced are still significantly higher than LPS alone, but FGL2 was able to suppress IL- 12 in response to LPS + anti-CD40 to levels comparable to LPS alone. These results suggest that FGL2 can inhibit IL-12 production in response to multiple stimuli.

Next, we determined if FGL2 was specifically suppressing the production of IL-12, or if FGL2 was suppressing a wide range of inflammatory cytokines. The expression of TNFα, IL-6 after LPS stimulation was measured. There was a modest suppression of IL-6; however, TNFα was not affected. Cytokines produced in response to LPS can broadly be divided into two categories: primary response genes and secondary response genes121. Primary response genes are under the control of transcription factors that are expressed but are latent in the cell. Once TLR signaling is triggered, these transcription factors (e.g., NFκB and IRF3) translocate to the nucleus and initiate transcription. Secondary response genes are dependent on de novo synthesis of transcription factors and nucleosome remodeling and thus are slower to be expressed than primary response genes. TNFα is a primary response gene, while IL-6 and IL-12 are examples of secondary response genes. It is possible that FGL2 is suppressing the synthesis of the transcription factors that are needed for the expression of IL-12 and IL-6; however, this remains to be determined.

IL-12 is an important cytokine for Th1 polarized T cell responses. IL-12 can induce IFNγ from NK cells and T cells, which then acts on macrophages to increase IL-12 expression in a positive feedback loop. IL-12 has long been studied in the context of infectious disease, 50 including chronic viral infections. Hepatitis B (HBV) is a global health concern, with over 250 million people persistently infected. Chronic viral infection results in a state termed “T cell exhaustion”, in which anti-viral T cell are unable to effectively respond to and clear viral antigens, leading to the persistence of virus. IL-12 has been explored as a means to restore T cell function. A prior study found that IL-12, acting as a third signal after T cell receptor activation and co-stimulation, was able to rescue exhausted HBV-specific CD8+ effector T cells122. IL-12 stimulation resulted in increased T-bet transcription and effector cytokine production, as well as decreased expression of PD1 on the effector T cells. IL-12 has also been observed to directly inhibit viral replication in transgenic mice through induction of IFNγ, leading to dysfunctional viral particle assembly123. IL-12 has also been explored as an adjuvant for vaccines. In a HBV- carrier mouse model, in which the mice had become tolerized and unable to respond to HBV antigen, vaccination in conjunction with IL-12 was able to break HBV tolerance and restore anti- HBV antigen immune responses124. IL-12 may also be an important for vaccines against HIV. In a primate model, a SIV-DNA vaccine co-administered with a IL-12 expression plasmid enhanced SIV-specific CD8+ T cell responses demonstrating IL-12-mediated enhancement of vaccines as a potential strategy to be explored in humans125. It is also interesting to note that virus such as HBV and HIV increase expression of FGL2, which may contribute to viral persistence through suppression of IL-12126,127.

In addition to treating chronic viral infections, IL-12 may also have promise as a treatment in cancer. IL-12 alone or in combination with chemotherapy has been explored as a treatment option for cancer with promising results in animal models and clinical trials. In a murine model, IL-12 alone was found to have anti-tumor effects on various cancers including, Lewis lung carcinoma and renal cell carcinoma and B16 melanoma and this effect was further synergized by radiation therapy128. In another study, administration of the chemotherapy drug paclitaxel in conjunction with IL-12 coding adenoviral expression vectors resulted in decreased tumor size and increased survival compared to drug alone129. Similarly, another study found that treatment of tumor bearing mice with the drug 5-aza-2′-deoxycitydine in conjunction with IL-12 greatly increased the efficacy compared to drug alone130. This enhancement of anti-tumor effects was found to be dependent on CD4+ and CD8+ T cells. Remarkably, several studies have also reported complete elimination of tumor cells after IL-12 transduction131,132. Due to the promising results with animal models, several clinical trials have also tested the efficacy of IL-12 in 51 patients. Although the results from these trials have been mixed, several trials have shown positive results. In a study of patients with recurrent or refractory non-Hodgkin’s B cell lymphoma (NHL), administration of IL-12 for 5 days every 3 weeks, or twice weekly resulted in ~21% of patients exhibiting partial responses or complete remission and ~34% with stable disease133. Another trial examining patients with cutaneous T cell lymphoma (CTCL), 10 patients with CTCL were treated with IL-12 twice a week for 24 weeks134. Only one patient out of the 9 evaluated did not respond. Treatment was accompanied by increased CD8+ T cells in the lesions and adverse effects to treatment were mild. These trials demonstrate that IL-12 can be a promising treatment option for various types of cancer.

As a mechanism to evade the immune response, tumor cells are known to secrete cytokines (e.g., IL-10 and IL-11) that inhibit the expression of IL-12135-137. Tumor cells have also been shown to express FGL2 and one study found that 83% of glioblastoma tumors had copy number gains or gene amplification of the FGL2 gene, while 72% of lower grade gliomas retained 2 copies of the FGL2 gene, indicating that increased FGL2 expression by tumor cells may be contributing to pathogenesis99.One mechanism by which tumor-derived FGL2 may be contributing to tumor progression is by the downregulation of IL-12. The tumor microenvironment also contains immunoregulatory cells including Treg and TAMs which contribute to tumor maintenance. Although macrophages are an important source of IL-12 normally, TAMs in the tumor microenvironment suppress IL-12 production endogenously as well as by other cell types such as dendritic cells through secretion of IL-10138. TAMs themselves may also be an important source of FGL2, as FGL2 mRNA was also found to be highly upregulated in human M2 polarized macrophages, suggesting FGL2 is also an important M2 effector molecule139. It remains currently unclear if the predominate source of FGL2 in the tumor microenvironment is tumor cell derived, TAM and Treg derived, or both. Future studies using conditional knockouts of fgl2 may allow for the identification of the primary source of FGL2 in tumors and determine if FGL2 is suppressing the production of IL-12, leading to weakened anti-tumor immune responses. Suppression of IL-12 by FGL2s may thus be a novel mechanism by which tumor cells inhibit anti-tumor immune responses, and targeting FGL2 with monoclonal antibodies alone or in combination with other chemotherapies may restore IL-12 production and promote anti-tumor immune responses. 52

High levels of IL-12 may play a role in promoting autoimmune diseases. One study examining patients who developed de novo autoimmune hepatitis after receiving liver transplants were found to have impaired Treg and increased expression of IL-12140. This increased IL-12 was found to predominantly derived from macrophages and responsible for promoting Treg dysfunction. Another study found that patients with progressive multiple sclerosis but not relapsing-remitting, there was increased IL-12 induced by CD40-CD40L interactions, indicating IL-12 may be contributing to disease progression seen in multiple sclerosis141. As FGL2 has been shown to regulate the expression of IL-12 by macrophages, it would therefore be of interest to examine if recombinant FGL2 can prevent the initiation or progression of this or other autoimmune disorders. One possible advantage FGL2 has above other immunosuppressive therapies is that FGL2 was found to downregulate but not completely inhibit IL-12 expression by macrophages. Patients given potent immunosuppressive drug treatments for prolonged periods may have adverse side effects, such as being more susceptible to infection or to the development of cancer. As FGL2 does not completely suppress IL-12 production, it may be a safer alternative for long term patient use than some currently available immunosuppressive therapies.

4.3 FGL2 suppresses the anti-bacterial response in macrophages

Besides cytokine production, macrophages have important roles in the detection and clearance of pathogens and in antigen presentation. We determined if FGL2 can modify these key macrophage functions. The expression of MHCII and CD86 on macrophages from fgl2+/+, fgl2-/- and fgl2Tg mice at baseline and after stimulation was examined and found that all 3 groups expressed similar levels of thee markers a baseline. After stimulation with LPS or IFNγ, there was large increase in CD86 expression in all 3 groups; however, there were no significant differences in percentage positive or MFI. Likewise, MHCII was significantly upregulated after stimulation, but there were no significant differences between the 3 groups of macrophages. This is in contrast to earlier studies that demonstrated that recombinant FGL2 protein was able to suppress dendritic cell maturation and lead to decreased expression of MHCII on bone marrow- derived dendritic cells in response to LPS compared to untreated. These findings may be explained by the fact that MHCII is regulated differently in macrophages and dendritic cells. Class II transactivator (CIITA) is thought of as a master regulator of MHCII expression. The expression of CIITA is regulated by three separate promoters, PI, PIII and PIV, which encode 53 type I, III, and IV CIITA respectively. It has been reported that that after activation, macrophages predominantly expressed type I and type IV CIITA, while dendritic cells express predominantly type I CIITA142. These differences in regulation of MHCII expression between dendritic cells and macrophages may explain why there was not a decreased MHCII expression on macrophages in response to FGL2.

Macrophages also detect and respond to PAMPs to initiate immune responses. Due to the findings that macrophages make less IL-12 in response to stimulation, the expression of the surface receptors TLR4 and IFNγR were next tested. Comparable levels of IFNγR on macrophages from all 3 groups were found, however TLR4 MFI was significantly lower on macrophages from fgl2Tg mice. One method by which TLR4 signaling was found to be regulated was by the translocation of TLR4 into lysosomes for degradation, which decreased inflammatory cytokine production by macrophages and this finding may also partially explain why fgl2Tg macrophages make less IL-12 in response to LPS, however this remains to be determined and can be the focus of subsequent studies143. Although TLR4 levels were found to be lower when comparing fgl2Tg macrophages to fgl2+/+macrophages, it remains to be determined if this also occurs after treatment of fgl2+/+ macrophages with rFGL2 and if this the main mechanism for the suppression of IL-12.

As macrophages also have critical roles in the phagocytosis and degradation of bacteria, we next examined if FGL2 altered these functions. Using labeled bacteria, fgl2Tg macrophages had lower MFI of labeled bacteria, indicating that they took up less bacteria per macrophage. These results are in line with the finding that these macrophages express less TLR4, as TLR4 expression has been found to be important for macrophage phagocytosis. Bone-marrow derived macrophages from tlr4-/- mice had reduced ability to phagocytose bacteria; however, phagocytosis of apoptotic cells was similar to wild type macrophages, indicating specific defects in bacterial phagocytosis144. Another study examining peritoneal sepsis in mice found that macrophages from tlr4-/- phagocytosed significantly fewer bacteria than their wild type counterparts in vivo145. Although TL4 downregulation may be the mechanism by which fgl2Tg macrophages phagocytose fewer bacteria, another explanation may be that fgl2Tg macrophages have defects in their ability to acidify the phagosome. The pHrodo E. coli only become fluorescent in an acidic environment, such as a lysosome. Differences in lysosome acidity may lead to the decreased fluorescence seen in fgl2Tg macrophages. 54

Phagocytosis of bacteria is also enhanced in M1-like polarized macrophages and has been found to be suppressed in M2-like polarized macrophages146,147. Likewise, the FGL2- FcγRIIB pathway may be a currently unexplored pathway for the regulation of phagocytosis. A prior study found that macrophages deficient in FcγRIIB had increased phagocytic capacity compared to wild-type macrophages, indicating FcγRIIB can suppress phagocytosis and that FGL2 signaling may mediate this suppression148. To further explore these findings, future work can examine the role of FGL2 on macrophage phenotypes in vivo using sepsis models such as cecal puncture to examine the ability of macrophages from fgl2Tg and fgl2-/- mice to control and clear bacteria.

4.4 Future Directions

Future work on this project can explore the mechanisms by which FGL2 regulates eosinophils, IL-12 and phagocytosis, and identify clinically relevant models to assess the importance of FGL2 in vivo. The next series of experiments should determine if FGL2 can cause apoptosis in eosinophils directly in vitro, as seen with other FcγRIIB agonists. Afterwards, in vivo analysis of Annexin V staining of eosinophils may explain the decrease in eosinophils recovered from fgl2Tg mice. Finally, mouse models of chronic allergen exposure have been able to recapitulate some of the hallmarks of human asthma, such as airway remodeling and eosinophilia. It would thus be of interest to examine differences in the response to chronic allergen exposure in fgl2+/+, fgl2-/- and fgl2Tg mice, as well as explore treating wild type mice with exogenous FGL2 to ameliorate symptoms.

This study demonstrated a clear ability of FGL2 to regulate the expression of IL-12 from macrophages; however, the mechanisms by which it does so remain unclear. It would be of interest to determine if FGL2 is regulating IL-12 at the level of transcription, post-transcriptionally or post- translationally and if 12p40, 12p35 or both are affected. Quantitative PCR to measure mRNA can be used to determine which stage the regulatory effect of FGL2 is occurring and to determine if other IL-12 family cytokines are similarly inhibited by FGL2. FGL2 has been shown to regulate NFκB in dendritic cells; however, if this occurs in macrophages has not been determined. Using western blot to examine the degradation kinetics of IκB as well as the phosphorylation of NFκB subunits will allow for more detailed molecular analysis of the transcription networks FGL2 signaling may be regulating. IL-12 has been shown to be important in the control of many clinically 55 relevant mouse models including various cancer models, Listeria monocytogenes and leishmania donovani. It would thus be useful to determine if FGL2 is upregulated in these models and if the deletion of FGL2 (fgl2-/- mice) results in improved outcomes, after which IL-12 expression by macrophages can be assessed and determined to contribute to these improved outcomes. Finally, it would also be of interest to examine the role of FGL2 on regulating IL-12 produced by dendritic cells. Transcription of IL-12 by macrophages and dendritic cells differs and FGL2 may affect these two cell types differently.

In this study, we also demonstrate deficiencies in phagocytosis of bacteria by fgl2Tg macrophages. Although this was determined in vitro with non-viable bacteria, it would next be of interest to test phagocytosis in the three groups of mice in vivo. Viable bacteria can be injected into the peritoneal cavity and recovered and quantified at later time points to determine how effectively they are cleared. These findings can be translated into mouse models of sepsis, including cecal puncture, to determine if deletion of FGL2 can improve outcomes to bacterial sepsis, or if FGL2 over-expression inhibits bacterial clearance.

4.5 Conclusions

In these series of experiments, the ability of FGL2 to modulate macrophage activation and to polarize macrophages to either M1-like or M2-like phenotypes was examined. This study is the first to explore the effect of FGL2 on macrophage polarization directly and has revealed important information on the biology of FGL2. As the role of FGL2 in a number of relevant diseases including chronic viral infection and cancer is becoming more apparent, understanding the effects of FGL2 on macrophages, which are also heavily implicated in these pathologies, will allow for the development of novel therapeutic approaches. Based on the literature and previous work from Dr. Levy’s lab, we hypothesized that FGL2 would act to polarize macrophages to an M2-like phenotype. In this study, increases in M2 macrophage markers such as arginase or IL-10 were not observed. However, M1 macrophage markers such as IL-12 and IL-6 were suppressed and phagocytosis was decreased in macrophages in response to FGL2. These results indicate that FGL2, although not directly polarizing to an M2-like phenotype, is suppressing the ability of macrophages to be polarized to an M1-like phenotype and respond to bacterial stimuli (Fig 12.) These results may have implications in cancer, in which FGL2 has been shown to be highly 56 upregulated. Future studies may examine the role of tumor-derived FGL2 in preventing macrophage activation and tumoricidal activity, contributing to the TAM phenotype.

Figure 12. Model of the effect of FGL2 on macrophage phenotype. Treg or tumor-derived FGL2 signaling in macrophages results in suppression of IL-12, IL-6, TLR4 and macrophage bacterial phagocytosis ultimately polarizing macrophages toward an M2 phenotype and away from an M1 phenotype. Lower IL-12 production may result in diminished IFNγ production by Th1 CD4 T cells, inhibiting the IL-12-IFNγ positive feedback loop and an increase in the IL- 10/IL-12 ratio, potentially suppressing Th1 T cell responses.

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