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STAT6 and STAT4 in murine atherosclerosis Tai, Daven C H 2013

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STAT6 and STAT4 in Murine Atherosclerosis by Daven C H Tai B.M.L.Sc., The University of British Columbia, 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  Doctor of Philosophy  in  The Faculty of Graduate and Postdoctoral Studies  (Pathology and Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)  December 2013  ? Daven C H Tai, 2013 ii Abstract Atherosclerosis is a chronic inflammatory condition and the major underlying cause of heart attacks and strokes. Since the immune system is paramount in all stages of atherosclerosis, modulating the immune response is an attractive therapeutic strategy for atherosclerotic disease. Signal transducers and activators of transcription (STAT) 6 and STAT4 are essential orchestrators of the anti-inflammatory Th2 response and the pro-inflammatory Th1 response, respectively. Using bone marrow transplantation to deplete STAT6 and STAT4 expression in specific immune compartments in low density lipoprotein receptor knockout (Ldlr-/-) mice, we investigated the validity of modulating the immune response through STAT6 and STAT4 as a prospective treatment strategy for atherosclerosis. We found that myeloid-specific STAT6 depletion did not significantly impact atherosclerotic lesion area or stability in Ldlr-/- mice fed an atherogenic diet for 8 or 14 weeks. In addition, hematopoiesis in the myeloid and lymphoid lineages was not significantly affected by the absence of myeloid STAT6. In contrast, total hematopoietic system STAT4 depletion profoundly exacerbated atherosclerotic lesion area and vulnerability in Ldlr-/- mice following 8 weeks of atherogenic diet. Hematopoietic perturbations in mice transplanted with STAT4-deficient bone marrow are highly reminiscent of interferon (IFN)-?-dependent effects on immune cell development in interleukin-12-treated mice, suggesting that IFN-? levels may be elevated in response to hypercholesterolemia in the absence of STAT4. Myeloid-specific STAT4-deficient mice also developed larger atherosclerotic lesions in the aortic root, providing evidence that even partial STAT4 insufficiency can potentially accelerate atherosclerosis. This thesis provides novel insights into the functions of STAT6 and STAT4 in atherosclerosis.  iii Preface Chapter 2: Dr. John S. Hill, Dr. Kenneth W. Harder, and I were responsible for the experimental design of the studies. I was responsible for all experiments and data analyses with the exception of the following:  Figure 2.3: Jennifer L. Beer performed the bone marrow transplantation, flow cytometry, and RT-PCR for this experiment.  Figure 2.4: Both Jennifer L. Beer and I were responsible for weighing the mice for the duration of the atherogenic diet.  Figure 2.19 and Figure 2.20: Wax-IT Histology Services, Inc. was responsible for the embedding, sectioning, and staining of aortic root samples.  All bone marrow transplantation procedures were performed by Jennifer L. Beer.  The sacrifice and tissue collection for all murine atherosclerosis studies in this chapter involved the coordinated efforts of Eugene M. Chu, Jennifer L. Beer, and I.  Chapter 3: Dr. John S. Hill, Dr. Kenneth W. Harder and I were responsible for the experimental design of the studies. I was responsible for all experiments and data analyses with the exception of the following:  Figure 3.2 and Figure 3.9: Both Jennifer L. Beer and I were responsible for weighing the mice for the duration of the atherogenic diet.  Figure 3.8 and Figure 3.18: Wax-IT Histology Services, Inc. was responsible for the embedding, sectioning, and staining of aortic root samples.  All bone marrow transplantation procedures were performed by Jennifer L. Beer.  The sacrifice and tissue collection for total hematopoietic system STAT4 depletion studies involved the coordinated efforts of Eugene M. Chu, Jennifer L. Beer, Gabriel Dorighello, and I. Eugene M. Chu, Gabriel Dorighello, and I were responsible for the sacrifice and tissue collection for myeloid-specific STAT4 depletion studies.  Ethics Approval: All studies involving animals in Chapter 2 and Chapter 3 have been approved by the UBC Animal Care Committee under the project title Macrophage Polarization and Atherosclerosis (ID number : A08-0786).  iv Table of Contents Abstract .......................................................................................................................... ii  Preface .......................................................................................................................... iii  Table of Contents ......................................................................................................... iv  List of Tables ................................................................................................................ ix  List of Figures ............................................................................................................... x  List of Symbols and Abbreviations .......................................................................... xiii  Acknowledgements ..................................................................................................... xv  Dedication ................................................................................................................... xvi  CHAPTER 1: Introduction ............................................................................................. 1  1.1 Atherosclerosis ? Background ................................................................................. 1  1.2 Oxidized Low Density Lipoprotein ........................................................................... 2  1.3 The Innate Immune System .................................................................................... 3  1.3.1 Monocytes and Atherosclerosis ......................................................................... 4  1.3.2 Macrophages in Atherosclerosis ........................................................................ 7  1.3.3 Macrophage Polarization and Heterogeneity ..................................................... 8  1.3.4 Macrophage Heterogeneity in Atherosclerosis ................................................. 10  1.3.5 Introduction to Dendritic Cells .......................................................................... 11  1.3.6 Dendritic Cells in Atherosclerosis ..................................................................... 12  1.3.7 Activation of T Cells by Dendritic Cells ............................................................. 13  1.3.8 Neutrophils in Atherosclerosis .......................................................................... 13  1.3.9 Natural Killer Cells in Atherosclerosis .............................................................. 14  1.4 The Adaptive Immune System .............................................................................. 14  v 1.4.1 Introduction to T Cells ...................................................................................... 15  1.4.2 CD4+ T Cells in Atherosclerosis ....................................................................... 16  1.4.3 The Th1 Response ........................................................................................... 16  1.4.4 The Th2 Response ........................................................................................... 17  1.4.5 CD8+ Cytotoxic T Cells in Atherosclerosis ....................................................... 18  1.4.6 B Cells in Atherosclerosis ................................................................................ 19  1.5 Janus Kinase-Signal Transducers and Activators of Transcription ....................... 20  1.5.1 STAT Structure and Signaling .......................................................................... 20  1.5.2 STAT Proteins in Atherosclerosis ..................................................................... 22  1.6 STAT6 ................................................................................................................... 23  1.6.1 STAT6 Signaling .............................................................................................. 24  1.6.2 Biological Significance of STAT6 ..................................................................... 25  1.6.3 IL-4, IL-13 and STAT6 in Atherosclerosis ........................................................ 26  1.7 STAT4 ................................................................................................................... 27  1.7.1 STAT4 Signaling .............................................................................................. 28  1.7.2 Biological Significance of STAT4 ..................................................................... 30  1.7.3 IL-12 Family of Cytokines and STAT4 in Atherosclerosis ................................ 31  1.8 The Mouse Model of Atherosclerosis .................................................................... 32  1.9 Research Objectives and Hypotheses .................................................................. 34  CHAPTER 2: Investigation of STAT6 in a Mouse Model of Atherosclerosis .......... 36  2.1 Background ........................................................................................................... 36  2.2 Materials and Methods .......................................................................................... 39  2.2.1 Animals ............................................................................................................ 39  vi 2.2.2 Isolation and Culturing of BMDMs .................................................................... 39  2.2.3 LDL Preparation and Oxidation ........................................................................ 40  2.2.4 Cholesterol Accumulation Assay ...................................................................... 40  2.2.5 Bone Marrow Transplantation .......................................................................... 41  2.2.6 Induction of Atherosclerosis ............................................................................. 41  2.2.7 Sacrifice and Tissue Collection ........................................................................ 42  2.2.8 Plasma Cholesterol Content ............................................................................ 43  2.2.9 Embedding and Sectioning of Aortic Roots ...................................................... 43  2.2.10 Staining and Scanning Aortic Root Atherosclerotic Lesions ........................... 43  2.2.11 Blinding of the Observer ................................................................................. 44  2.2.12 Quantification of Aortic Root Lesion Area ...................................................... 44  2.2.13 Quantification of Aortic Root Lesion Necrosis ................................................ 44  2.2.14 Quantification of Aortic Root Lesion Lipid Content ......................................... 45  2.2.15 En Face Analysis of Atherosclerosis .............................................................. 45  2.2.16 Preparation of Tissues for Flow Cytometry .................................................... 45  2.2.17 Staining of Blood, Bone Marrow, and Spleen Cells for Flow Cytometry ......... 46  2.2.18 Statistical Analysis ......................................................................................... 47  2.3 Results .................................................................................................................. 48  2.3.1 STAT6 Deficiency Abolishes IL-4-Induced Increases in Cholesterol Accumulation in Murine BMDMs ............................................................................... 48  2.3.2 Validation of the Irradiation and Bone Marrow Transplantation Procedures for the Generation of M-Stat6-/- and M-Stat6+/+ Mice ...................................................... 49  2.3.3 Body Weight and Total Plasma Cholesterol Measurements in M-Stat6-/- Mice Fed an Atherogenic Diet for 8 or 14 Weeks .............................................................. 51  2.3.4 Assessment of Bone Marrow Reconstitution in M-Stat6+/+ and M-Stat6-/- Mice 53  vii 2.3.5 The Effects of Myeloid-Specific STAT6 Depletion on Monocyte Populations in the Blood, Bone Marrow, and Spleens of Ldlr-/- Mice ................................................ 66  2.3.6 Myeloid-Specific STAT6 Depletion Does Not Alter Splenic DC Populations in Ldlr-/- Mice Following 8 Weeks of Atherogenic Diet ................................................... 67  2.3.7 Myeloid-Specific STAT6 Depletion Does Not Affect NK Cell and Neutrophil Populations in the Spleen of Ldlr-/- Mice .................................................................... 75  2.3.8 The Effects of Myeloid-Specific STAT6 Depletion on Lymphopoiesis in the Blood, Bone Marrow, and Spleens of Ldlr-/- Mice ...................................................... 78  2.3.9 Myeloid-Specific STAT6 Depletion Does Not Affect Atherosclerosis in Ldlr-/- Mice .......................................................................................................................... 85  2.4 Discussion ............................................................................................................. 89  CHAPTER 3: Investigation of STAT4 in a Mouse Model of Atherosclerosis .......... 97  3.1 Background ........................................................................................................... 97  3.2 Materials and Methods ........................................................................................ 100  3.2.1 Animals .......................................................................................................... 100  3.2.2 Bone Marrow Transplantation ........................................................................ 100  3.2.3 Induction of Atherosclerosis ........................................................................... 101  3.2.4 Atherosclerosis Measurements and Flow Cytometry ..................................... 102  3.3 Results ................................................................................................................ 102  3.3.1 Body Weight and Total Plasma Cholesterol Measurements in Total Hematopoietic System STAT4 Deficient Mice ......................................................... 102  3.3.2 Changes in Monocyte Populations in Hematopoietic System STAT4-Deficient Mice Following 8 Weeks of Atherogenic Diet .......................................................... 102  3.3.3 Total Hematopoietic System STAT4 Deficiency Reduces DC Frequency in the Spleens of Ldlr-/- Mice ............................................................................................. 107  3.3.4 Total Hematopoietic System STAT4 Deficiency Enhances NK Cell and Neutrophil Populations in the Spleens of Ldlr-/- Mice ............................................... 109   viii 3.3.5 Lymphocyte Development is Impaired in Total Hematopoietic System STAT4-Deficient Mice ......................................................................................................... 111  3.3.6 Total Hematopoietic System STAT4 Deficiency Exacerbates Atherosclerosis in Ldlr-/- Mice ............................................................................................................... 115  3.3.7 Body Weight and Total Plasma Cholesterol Measurements M-Stat4-/- Mice .. 118  3.3.8 Assessment of Bone Marrow Reconstitution in M-Stat4+/+ and M-Stat4-/- Mice ................................................................................................................................ 120  3.3.9 Myeloid STAT4 Deficiency Increases Absolute Monocyte Counts in the Spleen of Ldlr-/- Mice ........................................................................................................... 127  3.3.10 Myeloid STAT4 Deficiency Increases Absolute DC Counts in the Spleens of Ldlr-/- Mice ............................................................................................................... 130  3.3.11 Splenic NK Cell Frequency and Absolute Numbers are Elevated in M-Stat4-/- Mice ........................................................................................................................ 132  3.3.12 Myeloid STAT4 Deficiency Does Not Affect Lymphocyte Populations in Ldlr-/- Mice ........................................................................................................................ 134  3.3.13 Myeloid STAT4 Deficiency Does Not Significantly Affect Aortic Atherosclerosis in Ldlr-/- Mice ........................................................................................................... 137  3.3.14 Myeloid STAT4 Deficiency Increases Aortic Root Lesion Area but Does Not Affect Lesion Lipid Content or Necrosis in Ldlr-/- Mice ............................................. 139  3.4 Discussion ........................................................................................................... 142  CHAPTER 4: Conclusions and Future Directions .................................................. 149  4.1 Overall Summary ................................................................................................ 149  4.2 Limitations and Future Directions ........................................................................ 152  4.3 Concluding Statement ......................................................................................... 158  Bibliography .............................................................................................................. 159  ix List of Tables Table 2.1 Combination of Antibodies Used to Stain Each Tissue for Flow Cytometry .. 47   x List of Figures Figure 1.1 The Immune Response in Atherosclerosis ..................................................... 5  Figure 1.2 Organization and Function of STAT Protein Domains .................................. 21  Figure 1.3 The STAT6 Signaling Pathway .................................................................... 25  Figure 1.4 The STAT4 Signaling Pathway .................................................................... 29  Figure 2.1 Experimental Design for in vivo STAT6 Studies ........................................... 42  Figure 2.2 STAT6 Deficiency Abolishes IL-4-Induced Increases in Cholesterol Accumulation in Murine BMDMs ................................................................................... 48  Figure 2.3 Validation of the Irradiation and Bone Marrow Transplantation Procedures for the Generation of M-Stat6-/- and M-Stat6+/+ Mice .......................................................... 50  Figure 2.4 Body Weight and Total Plasma Cholesterol Measurements in M-Stat6-/- Mice Fed an Atherogenic Diet for 8 or 14 Weeks .................................................................. 52  Figure 2.5 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Blood of M-Stat6+/+ and M-Stat6-/- Mice Following 8 Weeks of Atherogenic Diet .......... 56  Figure 2.6 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Bone Marrow of M-Stat6+/+ and M-Stat6-/- Mice Following 8 Weeks of Atherogenic Diet ...................................................................................................................................... 57  Figure 2.7 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Spleens of M-Stat6+/+ and M-Stat6-/- Mice Following 8 Weeks of Atherogenic Diet ....... 60  Figure 2.8 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Blood of M-Stat6+/+ and M-Stat6-/- Mice Following 14 Weeks of Atherogenic Diet ........ 62  Figure 2.9 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Bone Marrow of M-Stat6+/+ and M-Stat6-/- Mice Following 14 Weeks of Atherogenic Diet ...................................................................................................................................... 63  Figure 2.10 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Spleens of M-Stat6+/+ and M-Stat6-/- Mice Following 14 Weeks of Atherogenic Diet ..... 66  Figure 2.11 Monocyte Populations in the Blood, Bone Marrow, and Spleens of  M-Stat6+/+ and M-Stat6-/- Mice Following 8 Weeks of Atherogenic Diet ......................... 70  Figure 2.12 Monocyte Populations in the Blood, Bone Marrow, and Spleens of  M-Stat6+/+ and M-Stat6-/- Mice Following 14 Weeks of Atherogenic Diet ....................... 73  xi  Figure 2.13 Myeloid-Specific STAT6 Depletion Does Not Alter Splenic DC Populations in Ldlr-/- Mice Following 8 Weeks of Atherogenic Diet ....................................................... 75  Figure 2.14 Myeloid-Specific STAT6 Depletion Does Not Affect Splenic NK Cell and Neutrophil Populations in Ldlr-/- Mice Following 8 Weeks of Atherogenic Diet .............. 76  Figure 2.15 Myeloid-Specific STAT6 Depletion Does Not Affect Splenic NK Cell and Neutrophil Populations in Ldlr-/- Mice Following 14 Weeks of Atherogenic Diet ............ 77  Figure 2.16 Lymphocyte Populations in the Blood and Spleens of M-Stat6+/+ and  M-Stat6-/- Mice Following 8 Weeks of Atherogenic Diet ................................................ 81  Figure 2.17 Lymphocyte Populations in the Blood and Spleens of M-Stat6+/+ and  M-Stat6-/- Mice Following 14 Weeks of Atherogenic Diet .............................................. 84  Figure 2.18 Myeloid-Specific STAT6 Depletion in Ldlr-/- Mice Does Not Influence Aortic Atherosclerosis in Mice Fed an Atherogenic Diet for 8 or 14 Weeks ............................. 86  Figure 2.19 Myeloid-Specific STAT6 Depletion in Ldlr-/- Mice Does Not Significantly Influence Aortic Root Lesion Area or Lipid Content in Mice Fed an Atherogenic Diet for 8 or 14 Weeks ............................................................................................................... 87  Figure 2.20 Myeloid-Specific STAT6 Depletion in Ldlr-/- Mice Does Not Significantly Influence Aortic Root Necrosis in Mice Fed an Atherogenic Diet for 8 or 14 Weeks ..... 88  Figure 3.1 Experimental Design for in vivo STAT4 Studies. ........................................ 102  Figure 3.2 Body Weight and Total Plasma Cholesterol Measurements in Total Hematopoietic System STAT4 Deficient Mice ............................................................. 104  Figure 3.3 Changes in Monocyte Populations in Total Hematopoietic System STAT4-Deficient Mice Following 8 Weeks of Atherogenic Diet ............................................... 107  Figure 3.4 Total Hematopoietic System STAT4 Deficiency Reduces DC Frequency in the Spleens of Ldlr-/- Mice............................................................................................ 109  Figure 3.5 Total Hematopoietic System STAT4 Deficiency Enhances NK Cell and Neutrophil Populations in the Spleen .......................................................................... 110  Figure 3.6 Lymphocyte Development is Impaired in Total Hematopoietic System STAT4-Deficient Mice ................................................................................................. 114  Figure 3.7 Total Hematopoietic System STAT4 Deficiency in Ldlr-/- Mice Increases Aortic Atherosclerosis Lesion Area Following 8 Weeks of Atherogenic Diet. .............. 116   xii Figure 3.8 Total Hematopoietic System STAT4 Deficiency in Ldlr-/- Mice Increases Aortic Root Lesion Area and Necrosis......................................................................... 118  Figure 3.9 Body Weight and Total Plasma Cholesterol Measurements M-Stat4-/- Mice .................................................................................................................................... 119  Figure 3.10 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Blood of M-Stat4+/+ and M-Stat4-/- Mice ....................................................................... 123  Figure 3.11 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Bone Marrow of M-Stat4+/+ and M-Stat4-/- Mice ........................................................... 124  Figure 3.12 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Spleens of M-Stat4+/+ and M-Stat4-/- Mice Following 8 Weeks of Atherogenic Diet ..... 127  Figure 3.13 Myeloid STAT4 Deficiency Increases Absolute Monocyte Counts in the Spleens of Ldlr-/- Mice ................................................................................................. 130  Figure 3.14 Myeloid STAT4 Deficiency Increases Absolute DC Counts in the Spleens of Ldlr-/- Mice ................................................................................................................... 132  Figure 3.15 Splenic NK Cell Frequency and Absolute Numbers are Elevated in  M-Stat4-/- Mice Following 8 Weeks of Atherogenic Diet .............................................. 133  Figure 3.16 Myeloid STAT4 Deficiency Does Not Affect Lymphocyte Populations in  Ldlr-/- Mice ................................................................................................................... 137  Figure 3.17 Myeloid STAT4 Deficiency Does Not Affect Aortic Atherosclerosis in Ldlr-/- Mice Fed an Atherogenic Diet for 8 Weeks ................................................................. 138  Figure 3.18 Myeloid STAT4 Deficiency Increases Aortic Root Lesion Area but Does Not Affect Lesion Lipid Content or Necrosis in Ldlr-/- Mice ................................................. 141    xiii List of Symbols and Abbreviations APC:  Antigen Presenting Cell ApoB100:  Apolipoprotein B 100 ApoE:  Apolipoprotein E BAFF:  B Cell Activating Factor BMDM:  Bone Marrow-Derived Macrophage CCR:  C-C Motif Chemokine Receptor CD:  Cluster of Differentiation Cre:  Cre Recombinase CTL:  Cytotoxic Lymphocytes CX3CR:  C-X3-C Motif Chemokine Receptor DAMPs: Danger-Associated Molecular Patterns DBD:  DNA Binding Domain DC:  Dendritic Cell DTR:  Diphtheria Toxin Receptor ER:  Endoplasmic Reticulum FACS:  Fluorescence-Activated Cell Sorting FBS:  Fetal Bovine Serum Fc:  Fragment, Crystallizable FSC:  Forward Scatter GAPDH:  Glyceraldehyde 3-Phosphate Dehydrogenase GAS:  Gamma-Activated Sequence Gy:  Gray H&E:  Hematoxylin and Eosin HDL:  High Density Lipoprotein HSP:  Heat Shock Protein ICAM-1:  Intercellular Adhesion Molecule-1 IFN:  Interferon Ig:  Immunoglobulin IL:  Interleukin iTreg Induced T Regulatory Cell JAK:  Janus Kinase JAM-A:  Junctional Adhesion Molecule-A LCR:  Locus Control Region LDL:  Low Density Lipoprotein LDLR:  Low Density Lipoprotein Receptor LPS:  Lipopolysaccharide LXR:  Liver X Receptor LysM:  Lysozyme M M-CSF:  Macrophage Colony-Stimulating Factor MHC:  Major Histocompatibility Complex MMP:  Matrix Metalloproteinase M-STAT4:  Myeloid-STAT4 M-STAT6:  Myeloid-STAT6 NK:  Natural Killer  xiv Nur77:  Nerve Growth Factor IB OxLDL:  Oxidized Low Density Lipoprotein PAMPS: Pathogen-Associated Molecular Patterns PBS:  Phosphate Buffered Saline PD-1:  Programed Cell Death-1 PI:  Propidium Iodide PPAR:  Peroxisome Proliferator-Activated Receptor RA:  Rheumatoid Arthritis RAG:  Recombination Activation Gene RBCs: Red Blood Cells ROR?t:  Retinoic Acid Receptor-Related Orphan Nuclear Receptor SA:  Streptavidin SH2:  Src Homology 2 SLE:  Systemic Lupus Erythematosus SMC:  Smooth Muscle Cell SOCS:  Suppressors of Cytokine Signaling SR-A:  Scavenger Receptor Class A SSC:  Side Scatter STAT:  Signal Transducers and Activators of Transcription TAD:  Transcriptional Activation Domain T-bet:  T-box Transcription Factor TCR:  T Cell Receptor TGF:  Transforming Growth Factor Th:  T helper TNF:  Tumor Necrosis Factor Treg:  Regulatory T Cells TYK:  Tyrosine Kinase VCAM-1:  Vascular Cell Adhesion Molecule-1 VLDL:  Very Low Density Lipoprotein WT:  Wild Type ?c:  Common ? Chain  xv Acknowledgements I would like to express my sincerest appreciation for all of the individuals who made it possible for me to complete this thesis. I offer my deepest gratitude to my supervisor Dr. John Hill, who taught me the importance of passion, persistence, and integrity in the pursuit for scientific knowledge. I am eternally grateful for the wonderful opportunity to study under his supervision.  I am especially thankful for my co-supervisor Dr. Ken Harder, who constantly provided invaluable advice and encouragement during my studies. I am extremely fortunate and forever indebted to him for providing me with the opportunity to learn in his lab.  I would like to thank my committee chair, Dr. David Granville, and my committee members Dr. Haydn Pritchard, Dr. Pauline Johnson, and Dr. Ken Harder for their scientific expertise and guidance during my research and thesis preparation.  I would like to acknowledge my friend and colleague Eugene Chu for his support and companionship, both in science and in life.  I extend my thanks to Jennifer Beer for her valuable technical expertise in animal studies and her constant assistance during various experiments.  I thank all members of the Harder Lab for their assistance and their friendship.  I am extremely grateful for my wife, Sarah Kam, my mother and father-in-law, Regina Leung and M.C. Kam, and my parents, Jason and Jodie Tai, for their unconditional love and support in my studies and, most of all, in my life.  xvi Dedication   To my friends and family For holding on together 1 CHAPTER 1: Introduction 1.1 Atherosclerosis ? Background Advances in science and medicine over the past few decades have led to a steady decline in fatal cardiovascular events.1 Despite these efforts, however, cardiovascular disease, including myocardial infarction and stroke, continue to reign as the top two leading causes of mortality worldwide, accounting for nearly a quarter of all deaths in the human population.2 In Canada, heart disease and stroke claimed 26.6% of Canadian lives in 2009, second only to cancer at 29.8%.3  Atherosclerosis, the hardening of arteries due to a buildup of plaque, is the major underlying condition leading to myocardial infarctions and strokes. Perceived as a passive cholesterol storage disease as recently as the last quarter century, atherosclerosis is now widely recognized as a chronic inflammatory condition resulting from the body?s response to lipids, in particular low density lipoproteins (LDL).4  The retention and subsequent oxidative modification of LDL within the intima in areas of disturbed blood flow is an instigator of atherosclerosis.5 The presence of modified LDL promotes endothelial cell expression of leukocyte adhesion molecules, cytokines, and chemokines. The ensuing recruitment and infiltration of professional inflammatory cells into the intima initiates a life-long process of atherosclerotic plaque generation, progression, and destabilization. The acute clinical events of atherosclerosis emerge when the plaque ruptures, exposing thrombogenic material to the blood followed by the rapid formation of a thrombus or embolus. The occlusion of a vessel in the heart or the brain by the blood clot is often the cause of heart attacks and strokes.6  2 The immune response plays fundamental roles in all stages of atherosclerosis. Pro-inflammatory T helper (Th) 1 responses are atherogenic while the antagonizing Th2 responses have been proposed to be atheroprotective, although the latter remains controversial.7 Cytokines provide the major driving force behind T cell-mediated immunity; modulating the immune response through the manipulation of cytokine signaling pathways is therefore an attractive therapeutic strategy for atherosclerosis.8 Indeed, treatment of rheumatoid arthritis, an inflammatory condition, by blocking the Th1 cytokine tumor necrosis factor (TNF)-? reduced the incidence of myocardial infarction in a patient cohort.9 The aforementioned study provides a glimmer of hope for targeting cytokines as a means of treatment for cardiovascular diseases in the foreseeable future. Signal Transducers and Activators of Transcription (STAT) proteins are transcription factors essential for the signaling of certain cytokines, such as interleukins (IL)-12 and IL-4/13. IL-12, signaling through STAT4, promotes Th1-mediated immunity whereas IL-4/13, signaling through STAT6, is a hallmark of the Th2 response.10, 11 Studies in mice have revealed a predominantly atherogenic role of IL-12, as will be discussed in detail in later sections.12,13 The influences of IL-4/13, however, are much more ambiguous.12, 14, 15 Importantly, the consequences of a loss of STAT4 or STAT6 in atherosclerosis have not yet been explored in detail. This thesis documents the work performed to elucidate the functional significance of STAT4 and STAT6 in atherosclerosis using a LDL receptor knockout (Ldlr-/-) mouse model.  1.2 Oxidized Low Density Lipoprotein LDL is the major particle involved in the transportation of plasma cholesterol to peripheral tissues. The LDL particle is composed of a single apolipoprotein B100  3 (ApoB100) particle, an outer layer of phospholipids and free cholesterol, and an inner core of cholesterol esters.16 Elevated plasma LDL promotes their accumulation in the intima, where they bind to proteoglycans in the extracellular matrix.17 Once retained in the subendothelium, both the lipid and ApoB100 components of LDL particles are vulnerable to oxidation, enzymatically and non-enzymatically, to become oxidized LDL (oxLDL).18 Multiple studies have shown that oxLDL is crucial to atherogenesis and can serve as a potential biomarker for future cardiovascular events.19, 20, 21  1.3 The Innate Immune System OxLDL triggers the endothelial cell expression of adhesion molecules such as vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, P-selectin, and junctional adhesion molecule (JAM)-A, resulting in monocyte recruitment, adhesion, and transmigration across the endothelium.22?25 Recruited monocytes differentiate into macrophages and dendritic cells (DCs) and phagocytose oxLDL through scavenger receptors, most notably scavenger receptor A (SR-A) and CD36, to become lipid-laden foam cells.26, 27 Intracellular accumulation of oxLDL can trigger endoplasmic reticulum stress, leading to the apoptosis of phagocytic cells.28 During early atherosclerosis, the clearance of apoptotic cells, termed efferocytosis, by neighboring phagocytes remains effective.29 Successful efferocytosis not only removes inflammatory components from the intima but also induces the production of anti-inflammatory mediators such as IL-10 and transforming growth factor (TGF)-?.30 With disease progression, however, defective efferocytosis, possibly due in part to macrophage class switching (discussed below), contributes to secondary necrosis, a characteristic of advanced and vulnerable atherosclerotic lesions.31, 32 Monocyte-derived  4 macrophages continue their attack on vulnerable lesions by secreting matrix-degrading metalloproteinases.33 The resulting thinning and weakening of the fibrous cap may cause plaque rupture and ultimately lead to acute cardiovascular events.34 In addition to initiating the primary response to atherosclerosis-relevant antigens, an important role of the innate immune system is to activate the adaptive immune response (Figure 1.1).6 DCs, and to a lesser extent, macrophages, are efficient antigen presenting cells (APCs) capable of processing and presenting antigens to T cells, leading to T cell activation and proliferation.35, 36 Activated T cells secret a host of inflammatory mediators which may then feed back into the innate immune system to perpetuate the immune response and propel atherosclerosis development.37  1.3.1 Monocytes and Atherosclerosis As alluded to previously, monocyte recruitment and differentiation into macrophages and DCs is one of the initial events in atherogenesis. The importance of this early event is emphasized by the attenuation of atherosclerosis in animals in which monocyte recruitment is perturbed via genetic manipulation.38, 39 Although the bone marrow was originally thought to be the exclusive source of monocytes, it has recently been shown that the spleen can also serve as a major source of monocytes during atherosclerosis.40 In humans and mice, monocytes can be recognized by their expression of macrophage-colony stimulating factor receptor (CD115) and membrane-activating complex 1 (CD11b).41, 42 At least two monocyte subsets have been identified based on their differential expression of CD14/CD16 in humans and Ly6C in mice.43 In humans, CD14hiCD16- cells are termed classical, or inflammatory, monocytes and are analogous to murine Ly6Chi cells. On the other hand, human CD14+CD16+ cells are 5  LumenIntimaEndothelial CellsIFN-?DCMonocyteTh1 cellPro-inflammatory MacrophageMHC II B7 TCR CD28 oxLDLAnti-inflammatory MacrophageIL-12IL-4/13T cellMacrophageTh2 cellIL-4/13(Source unclear)T cellIFN-?IL-12  Figure 1.1 The Immune Response in Atherosclerosis OxLDL triggers monocyte recruitment into the intima. Recruited monocytes differentiate into macrophages and DCs which phagocytize oxLDL. DCs and macrophages present oxLDL-derived antigens on MHCII molecules to T cells. The simultaneous engagement of the T cell receptor (TCR) and costimulatory molecules CD28/B7 leads to T cell activation. In the presence of IL-12, T cells differentiate into the IFN-?-producing Th1 subset, creating positive feedback to perpetuate inflammation. When exposed to IL-4/13, T cells develop into Th2 cells, which further secrete IL-4/13 and dampen inflammation.   6 termed non-classical, or patrolling, monocytes and share similar properties with murine Ly6Clow cells.44 There is evidence to suggest that Ly6Chi monocytes lose their Ly6C expression during maturation and serve as precursors to Ly6Clow monocytes.42 This conversion, however, may not be the only source of Ly6Clow monocytes, as mice lacking the nuclear receptor Nur77 possess a Ly6Chi monocyte population but are devoid of Ly6Clow monocytes.45 Additionally, mice deficient in Krupple-like factor 4 lack Ly6Chi monocytes yet still contain detectable amounts of Ly6Clow monocytes.46 These studies support the current belief that Ly6Clow monocytes can arise from both Ly6Chi-dependent and independent pathways. A third subset, referred to as intermediate monocytes (CD14hiCD16+), has recently been described in humans and non-human primates.47 However, the proper identification of this monocyte subset in mice, as well as their functional significance, have yet to be explored.47 The mouse has served as an invaluable model organism for studying the functions of monocyte subsets in atherosclerosis. During normal conditions, circulating monocytes consist of approximately equal proportions of pro-inflammatory versus patrolling subsets.48 Under conditions of hypercholesterolemia, however, Ly6Chi monocyte numbers are increased in the blood, bone marrow, and spleen, resulting in cholesterol-induced monocytosis.48 These pro-inflammatory monocytes readily accumulate in atherosclerotic lesions through the C-C motif chemokine receptor (CCR) 2 and the C-X3-C motif chemokine receptor (CX3CR) 1.49 Ly6Clow monocytes can also enter lesions via CCR5, but they do so significantly less frequently than their Ly6Chi counterparts.49 Indeed, it has been suggested that greater than 90% of monocyte-derived cells in the lesion originate from the Ly6Chi subset.48 Ly6Chi monocytes are  7 sources of inflammatory cytokines, proteolytic enzymes, reactive oxygen species, and contribute to lesional foam cells?all of which exacerbate atherosclerosis.40 The importance of Ly6Clow monocytes has also been demonstrated in vivo: mice lacking this anti-inflammatory subset following Nur77 deletion develop increased atherosclerosis.50 A recent study, however, found that bone marrow Nur77 deficiency decreases Ly6Clow monocytes but does not alter atherosclerosis, suggesting that monocyte subsets, though important, are not the sole factor in determining atherosclerotic disease outcome.51  1.3.2 Macrophages in Atherosclerosis Once in the intima, monocytes differentiate into macrophages under the influence of endothelial cell-derived macrophage colony stimulating factor (M-CSF).52 Macrophages are the most abundant leukocytes in atherosclerotic lesions and constitute a significant portion of total cellular bulk within the plaque.53 Macrophage proliferation is positively correlated with atherosclerosis progression; inhibition of growth suppressors such as cyclin-dependent kinase inhibitor 1B, retinoblastoma, and p53 increases macrophage proliferation and exacerbates atherosclerosis in ApoE-/- and Ldlr-/- mice.54, 55, 56 It has also been shown that the number of macrophages in human atherosclerotic lesions, determined by immunohistochemical staining of macrophage markers, increases with lesion advancement.57 As professional phagocytes, macrophages employ pattern recognition receptors such as SR-A and CD36 to recognize and ingest modified lipoproteins, eventually becoming lipid-laden foam cells?the hallmarks of atherosclerotic plaques.58 An important question that remains is how excessive lipid accumulation affects  8 macrophage function, especially in relation to atherosclerosis. Several lines of evidence suggest that macrophage foam cells exhibit enhanced pro-inflammatory signaling due to increases in their membrane free cholesterol and lipid raft content.59, 60, 61 Moreover, oxLDL can induce ER stress and trigger apoptosis in foam cells, which promotes atherosclerotic lesion growth, advancement, and destabilization.62 Advanced and vulnerable atherosclerotic plaques are characterized by a large necrotic core and a thin fibrous cap: a dangerous combination that precedes plaque rupture.63 The formation of the necrotic core is a consequence of the non-resolving inflammation brought about by macrophage death and defective efferocytosis.64 Furthermore, the degradation of the fibrous cap results from the actions of macrophage-derived matrix metalloproteinases (MMPs), a class of proteases involved in the degradation and remodeling of the extracellular matrix.65 The accumulation of macrophages proximal to rupture-prone shoulder regions of the atherosclerotic plaque further strengthens the hypothesis that these cells are intimately involved in the final stages of atherosclerosis leading up to acute cardiovascular events.57  1.3.3 Macrophage Polarization and Heterogeneity  Macrophage heterogeneity in atherosclerotic lesions was observed two decades ago and continues to be a central focus of atherosclerosis research today.66 In response to stimuli in their microenvironment, macrophages can be polarized to display a range of inflammatory states. Stimulation of macrophages with the Th1 cytokine interferon (IFN)-? followed by lipopolysaccharide (LPS) results in classically-activated, or M1, macrophages, characterized by their production of inflammatory cytokines, such as TNF-?; IL-12; and IL-1?, and reactive oxygen and nitrogen species.67 Conversely,  9 alternatively-activated (M2) macrophages differentiate in response to treatment with Th2 cytokines such as IL-4 or IL-13. M2 macrophages are important in the resolution of inflammation through their production of anti-inflammatory mediators such as IL-10 and TGF- ?.67,68 Alternatively-activated macrophages can be further divided into M2a (IL-4 or IL13), M2b (immune complexes plus IL-1? or LPS), and M2c (IL-10, TGF-?, or glucocorticoids) subtypes.32  The number of macrophage subsets is constantly expanding, with phenotypes such as M4, Mox, Mhem, and Mres recently being described.69?72 Although the classification of macrophages into distinct subsets is a convenient method for studying macrophage heterogeneity in vitro, this simplified approach may not reflect the true complexity of the atherosclerotic lesion environment in vivo. It has been hypothesized that, instead of falling into discrete polarization states, macrophages within atherosclerotic plaques display a continuous spectrum of inflammatory potentials with classically- and alternatively-activated macrophages representing the phenotypic extremes.73 It may therefore be relevant, at least in the context of atherosclerosis, to direct our research based on the functional differences of macrophages (especially with regards to inflammation) instead of focusing entirely on the traditional macrophage subsets. Nonetheless, the M1 and M2 nomenclature provides an efficient way to distinguish macrophages of differing inflammatory potentials. The subsequent section will therefore use M1 and M2 to represent pro-inflammatory and anti-inflammatory macrophages, respectively.      10 1.3.4 Macrophage Heterogeneity in Atherosclerosis Despite considerable advances in our understanding of macrophage heterogeneity in vitro, the relative contributions of different macrophage sub-phenotypes to various stages of atherosclerosis in vivo are only beginning to be appreciated. Since M1 macrophages have a high propensity to secrete pro-inflammatory cytokines and produce reactive oxygen/nitrogen species, M1 macrophages have been hypothesized to contribute more positively to atherosclerosis compared to their more anti-inflammatory M2 counterparts.74 Nonetheless, both classically and alternatively-activated macrophages have been observed in human and murine atherosclerotic plaques.75,76 In humans, M1 and M2 macrophages increase concomitantly with advancing atherosclerosis.57 Interestingly, the ruptured plaque does not show any preference for expressing M1 versus M2 markers.57 The same authors did, however, note a differential spatial distribution of polarized macrophage within the plaque; M1 macrophages preferentially accumulate around the shoulder regions of the plaque while M2 macrophages reside within the adventitia.57 The proximity of M1 macrophages to the rupture-prone regions of the plaque prompts the hypothesis that these cells are central to plaque rupture. Indeed, macrophage-derived MMPs have been shown to accumulate at sites of potential rupture and can greatly influence plaque stability and disruption.77, 78, 79 In contrast to human lesions, early atherosclerotic lesions in ApoE-/- mice (at 20 weeks of age) are dominated by anti-inflammatory macrophages, which express the M2-specific marker, arginase I.80 As the lesion advances, there is a dramatic shift towards the M1 sub-phenotype, as demonstrated by an increase in the M1-associated  11 marker, arginase II, in the plaque at 55 weeks of age.80 It was also shown that fully-polarized macrophages retain the capacity to undergo a phenotypic switch and convert to the opposite phenotype in vitro.80 The nuclear receptors peroxisome proliferator-activated receptor (PPAR)-? and liver X receptor (LXR)-? are involved in the control of macrophage alternative activation.76, 81, 82 PPAR-? activation is associated with reduced production of inflammatory cytokines, such as IL-12, TNF-?, IL-1?, and IL-6, by human and murine macrophages.83, 84  Pharmacological activation of PPAR-? using troglitazone inhibits atherosclerosis in ApoE-/- mice.85 In addition, conditional knockout of macrophage PPAR-? exacerbates atherosclerosis in Ldlr-/- as well as C57BL/6 mice.86 Similarly, macrophage-specific overexpression of LXR-? promotes cholesterol efflux, inhibits inflammatory response by macrophages, and significantly reduces atherosclerosis in Ldlr-/- mice.87 Taken together, these studies support the hypothesis that alternative macrophage activation plays protective roles in atherosclerosis.  1.3.5 Introduction to Dendritic Cells In addition to differentiating into macrophages, monocytes possess the capacity to develop into DCs in vivo.88, 89 DCs are identified by their expression of CD11c in mice; however, it has recently been shown that monocytes can express these markers as well.90 DCs are professional APCs that, upon antigen uptake, migrate to secondary lymphoid organs, such as the lymph nodes or the spleen, and present antigens on MHC molecules to antigen-specific T lymphocytes.91 In doing so, DCs act as the bridge between the innate and the adaptive immune systems. In addition to activating T cells through antigen presentation, DCs also produce a variety of inflammatory cytokines  12 (e.g. IL-12, TNF-?, IL-6) and chemokines (e.g. CCL2, CCL17, and CCL22) to promote the recruitment and activation of monocytes and lymphocytes during inflammation.92  1.3.6 Dendritic Cells in Atherosclerosis DCs can be found in healthy aortas of humans and mice and are capable of accumulating lipids and developing into foam cells.26, 93, 94 Similar to macrophages, the expansion of intimal DCs is associated with increased atherosclerosis.95 Since most of the efforts put into deciphering the roles of myeloid cells in atherosclerosis have been focused on macrophages, the functional significance of DCs in atherogenesis are only beginning to be understood. Deficiency in CX3CR1 decreases DC accumulation in the intima of ApoE-/- mice and reduces aortic lesion area.95 In addition, inhibition of other molecules involved in DC recruitment and antigen presentation (e.g. CD74, CD80, and CD86) protects Ldlr-/- mice from atherosclerosis.96, 97 Furthermore, targeted depletion of DCs using transgenic mice expressing the diphtheria toxin receptor under the CD11c promoter (CD11c-DTR) reduces intimal lipid and foam cell accumulation during initial stages of lesions formation.94  The above studies invite the speculation that DCs play a primarily pro-atherogenic role. However, it was found that prolonging DC survival, by overexpressing the anti-apoptotic gene hBcl-2, enhances the Th1 response (increased IFN-? and IL-12, for example) but also decreases plasma cholesterol levels, resulting in no net change in atherosclerosis.98 More recently, it was shown that a subset of DCs, characterized by their expression of CD103 and dependence on Fms-like tyrosine kinase 3 signaling for development, protects against atherosclerosis by stimulating regulatory T cell (Treg) differentiation.99 A wide variety of DC subsets have been identified in mice; additional  13 studies are required to further our knowledge of the functions of these cells in atherosclerosis.92  1.3.7 Activation of T Cells by Dendritic Cells DCs are well-known for their antigen presenting to, and activation of, T cells to initiate the adaptive immune response. Potential atherosclerosis-related antigens for DC uptake and antigen presentation include oxLDL, heat shock protein (HSP) 60, and ApoB100.100, 101, 102 Antigen-loaded DCs emigrate from the intima to para-aortic lymph nodes where they present antigens on MHCII molecules to na?ve T cells.92 The recognition of antigen in conjunction with the engagement of costimulatory molecules (B7/CD28) on T cells leads to T cell activation and clonal expansion.103 Interestingly, as atherosclerosis progresses, DC emigration from lesions becomes impaired.104,105 The retention of DCs within the plaque may yield additional opportunities for local activation of T cells. Indeed, immunohistochemical staining of atherosclerotic plaques found that activated DCs are always in close proximity to activated T cells, suggesting that the DC-T cell interaction is an active process in atherosclerosis.106 The consequences of such an interaction will be visited in the following sections.  1.3.8 Neutrophils in Atherosclerosis Neutrophils are innate immune cells that act as the first responders to sites of infection and tissue damage.107 Neutrophils phagocytize foreign material and produce reactive oxygen species, myeloperoxidase, and proteolytic enzymes that eliminate infection but also injure the surrounding tissue.107 Due to their low abundance in lesions, the functions of neutrophils in atherosclerosis have attracted little attention until  14 recently.108 Current insights into neutrophil biology in atherosclerosis are either correlative or inferred from their roles in inflammation. In mice, neutrophils can be detected in the atherosclerotic lesion, near the fibrous cap, or in the adventitia.109 Neutrophilia is induced shortly after the initiation of a high fat diet and correlates with lesion size.110 Furthermore, depletion of neutrophils leads to a reduction in atherosclerotic lesion area.110, 111, 112 Clearly, neutrophils represent a significant cell type in atherosclerosis; the mechanisms by which they act in lesion formation warrant further investigation.  1.3.9 Natural Killer Cells in Atherosclerosis Natural killer (NK) cells are bone marrow-derived lymphocytes that are critical in the defense against pathogens, especially viruses.113 Activated NK cells can secrete inflammatory cytokines including IFN-? and TNF-?, which may have potential implications for atherosclerosis.114 Even though NK cells have been detected in human and mouse atherosclerotic lesions, direct evidence for the effects of NK cells in atherosclerosis are still lacking.111, 115 To date, only a single study has clearly demonstrated that mice deficient in functional NK cells are protected from atherosclerotic lesion development.115   1.4 The Adaptive Immune System The adaptive immune response is initiated upon antigen recognition by membrane-bound immunoglobulins on B cells or the T cell receptor on T cells.116 Engagement of costimulatory molecules (e.g. CD40 on B cells and CD28 on T cells) following antigen binding is required for the full activation of lymphocytes.116 The  15 adaptive immune response has a constant presence in atherosclerosis. Antibodies against oxLDL and HSPs have been detected in human serum and within atherosclerotic plaques.117, 118 Additionally, antibody titers against HSP65 (a member of the HSP60 family) are positively correlated with atherosclerosis and have predictive value for cardiovascular mortality.119, 120 Animal models have shown that mice with severe combined immunodeficiency or mice lacking mature lymphocytes develop significantly smaller lesions compared to immunocompetent animals, providing further evidence for the pro-atherogenic qualities of lymphocytes during lesion development. 121, 122 On the other hand, certain subsets of adaptive immune cells, such as Tregs and B cells, may exhibit atheroprotective properties.7, 123 The ultimate outcome of atherosclerosis is therefore influenced by the cumulative effects of the complex interplay between pro- and anti-atherogenic forces exerted by lymphocytes in vivo.  1.4.1 Introduction to T Cells T lymphocytes originate from hematopoietic stem cells in the bone marrow and undergo maturation in the thymus to become either CD4+ or CD8+ T cells;124, 125 T cells can also be detected in the spleen and the peripheral blood.126 CD4+ T lymphocytes, including T helper (Th) cells and Tregs, perform critical roles in the immune system. Some of these tasks include helping B cells produce antibody,127 optimizing the response of CD8+ cells to pathogens,128 and driving macrophage polarization, among others.129 Activated CD8+ T cells, or cytotoxic T lymphocytes (CTLs), are involved in the direct killing of infected and damaged cells.130 The presence of T cells within human atherosclerotic plaques was first confirmed in the mid-1980s.131 Since then, tremendous  16 efforts have been devoted to deducing the functional significance of these cells in atherosclerosis.6, 7, 37   1.4.2 CD4+ T Cells in Atherosclerosis CD4+ T lymphocytes play central roles during atherosclerosis. Indeed, transfer of CD4+ T cells into athero-resistant immunodeficient mice leads to a dramatic increase in atherosclerosis, accompanied by increases in T cell-homing to lesions and elevated circulating IFN-? concentrations.122 Under the collective influences of the antigen, costimulatory molecules, and cytokines, na?ve CD4+ T cells can differentiate into at least 4 well-characterized subsets: Th1, Th2, Th17, and induced T regulatory (iTreg) cells.132 These subsets are separated based on their cytokine profiles; the defining cytokines are IFN-? for Th1 cells; IL-4, IL-5, and IL-13 for Th2 cells; IL-17 and IL-22 for Th17 cells; and TGF-? for iTregs.37, 132   1.4.3 The Th1 Response The Th1 response is initiated by IFN-? and IL-12 signaling through STAT1 and STAT4, respectively; interestingly, IFN-? is also the major effector cytokine of Th1 immunity.133 There is now an abundance of observational and experimental evidence to suggest that atherosclerosis is driven by the Th1 response. Activated T cells and IFN-? can be detected in human atherosclerotic lesions.134, 135 Furthermore, genetic ablation of IFN-? and its receptor significantly reduces lesion size in both ApoE-/- and Ldlr-/- mouse models.136, 137, 138 Similarly, postnatal blocking of IFN-? signaling, by overexpressing a soluble mutant of the IFN-? receptor, not only decreases disease burden in ApoE-/- mice but also stabilizes the lesions by reducing lipid and macrophage  17 content and increasing fibrotic and smooth muscle cell (SMC) areas.139, 140 Intraperitoneal injections of IFN-?, on the other hand, accelerates the development of atherosclerosis.141 Studies have also found pro-atherogenic roles for IL-12 and IL-18, which drive Th1 differentiation and stimulate IFN-? production by T cells.12, 142  Mechanistic studies on the atherogenicity of IFN-? have revealed that IFN-? induces endothelial VCAM-1 and ICAM-1 expression;143, 144 prevents SMC proliferation and production of plaque-stabilizing collagen;145, 146 promotes foam cell formation and apoptosis;147, 148 and augments the inflammatory response by enhancing antigen presentation to T cells and inflammatory cytokine production by macrophages.53, 149 It is interesting to note that IFN-? can also exert anti-atherogenic functions: IFN-? increases the expression of inducible nitric oxide synthase in endothelial cells, SMCs, and macrophages, thereby inhibiting LDL oxidation.150, 151, 152 Nonetheless, current experimental evidence strongly supports an overall pro-atherogenic role for IFN-? and the Th1 response.  1.4.4 The Th2 Response The Th2 response, characterized by the production of IL-4, IL-5, and IL-13 and the inhibition of the Th1 response, is initiated upon activation of the T cell antigen receptor and the binding of IL-4 to IL-4 receptors.153, 154 Signal transduction from IL-4 receptors is performed by STAT6, which up-regulates the transcription factor GATA3, the master regulator of Th2 differentiation.155, 156 Unlike the clear roles of the Th1 response, the data surrounding the effects of Th2 immunity in atherosclerosis are limited and ambiguous. Th2 cytokines such as IL-4, IL-5, and IL-13 are rarely detected in human atherosclerotic lesions.157 Animal studies have also yielded confusing and  18 contradictory results. Some studies show that inhibiting the Th2 response reduces atherosclerosis lesion area,12, 15 while others have found that Th2 immunity is either atheroprotective or has no effect on atherosclerosis.14, 158, 159 Additional research by targeted, and perhaps cell type-specific, deletion of proteins in the Th2 cytokine signaling pathway may be required to fully understand the function, or the lack thereof, of the Th2 response during atherosclerosis.  1.4.5 CD8+ Cytotoxic T Cells in Atherosclerosis Although less abundant than CD4+ cells in the plaque, CD8+ T cells can nonetheless represent a significant portion of the T cell population in the lesion.160, 161 CD8+ T cells proliferate in response to hypercholesterolemia in ApoE-/- mice;162 however, the increase in CD8+ T cells in this instance was neither pro- nor anti-atherogenic.163 Treatment of ApoE-/- mice with an agonist against CD137, a costimulator of T cells, increases lesion size and promotes CD8+ T cell infiltration into the lesions.164 In the same study, the authors also noted that CD137 signaling may potentially act on multiple other cell types including monocytes, B cells, DCs, SMCs, and endothelial cells. 164 Therefore, it becomes challenging to attribute the observed biological effects to a specific cell type. Genetic ablation of programed cell death-1(PD-1) or its ligand PD-L1/2, which negatively regulate T cell activation, exacerbates atherosclerosis and increases CD8+ T cell, CD4+ T cell, and macrophage accumulation the lesions in Ldlr-/- mice.165, 166 Once again, the importance, if any, of the CD8+ lineage of T cells cannot be confirmed due to potential influences from other cell types.    19 1.4.6 B Cells in Atherosclerosis In humans and mice, B lymphocytes originate from common lymphoid progenitors in the bone marrow, mature in the spleen, and can be identified based on their surface expression of the CD19 molecule.167, 168 B cells may be divided into two distinct developmental subsets, termed B-1 and B-2.167 B-1 B cells are perceived as part of the innate immune system for their roles in T cell-independent secretion of natural IgM antibodies.169 B-2 B cells populate secondary lymphoid organs and mediate both innate-like and adaptive immune responses.170 Several lines of evidence point toward a predominantly atheroprotective role for B cells. Adoptive transfer of B cells into splenectomized ApoE-/- mice can counteract the atherogenic effects of splenectomy.171 In the same study, the authors showed that transfer of B cells confers atheroprotection even in non-splenectomized mice.171 In addition, irradiated Ldlr-/- mice reconstituted with B cell-deficient bone marrow develop more atherosclerosis compared to mice transplanted with wild type bone marrow.123 Consistent with these findings, Ldlr-/- mice deficient in IgM develop larger and more complex lesions compared to IgM wild type mice, demonstrating a protective role for B-1 B cell-derived IgM in atherosclerosis.172 The binding of IgM to oxLDL, which prevents macrophage uptake of oxLDL and inhibits foam cell formation, is thought to contribute importantly to the suppression of atherosclerosis by B cells.173, 174 Contrary to the findings described above, it was recently shown, by two independent groups, that B cell depletion using an anti-CD20 antibody decreases atherosclerosis in ApoE-/- mice.175, 176 Interestingly, the CD20 antibody preferentially depletes B-2 B cells over B-1 B cells, hinting at potential discrepancies between the  20 atherogenicity of distinct B cell lineages.177 Indeed, two recent studies found that deleting the B-2 cell population by knocking out the B-cell activating factor (BAFF) receptor protects ApoE-/- and Ldlr-/- mice from atherosclerosis.178, 179 The pro-atherogenic roles of B-2 B cells was further supported by an increase in atherosclerosis following the adoptive transfer of B-2 B cells into B cell-deficient ApoE-/- mice.176  1.5 Janus Kinase-Signal Transducers and Activators of Transcription The Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway represents an evolutionary-conserved signaling mechanism for various extracellular cytokines and growth factors.180 Since the initial discovery of STAT1 and STAT2 in 1992, the STAT family rapidly grew to incorporate a total of 7 family members designated STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6.181, 182, 183 The JAKs, sequenced at approximately the same time as the STATs, include JAK1, JAK2, JAK3, and TYK2.183 Although STAT molecules share many similarities in their structure and signaling mechanism, each STAT family member senses and responds to a specific set of extracellular stimuli.  1.5.1 STAT Structure and Signaling STAT proteins range from ~750 to ~850 amino acids in size and share 5 structurally and functionally similar domains: an amino-terminal domain, a coiled-coil domain, a DNA binding domain (DBD), a linker-domain, and the SH2/tyrosine activation domain.184, 185 Each STAT protein also contains a unique transcriptional activation domain (TAD) that contributes to signaling specificity (Figure 1.2).185 The amino-terminal domain is involved in STAT nuclear translocation and deactivation;186 the  21 coiled-coil domain is responsible for receptor binding, tyrosine phosphorylation, and nuclear export;187, 188 the DBD facilitates the recognition of binding of STATs to the consensus sequence TTCN2-4GAA on the promoter of target genes;189 the linker domain bridges the DBD and the SH2/tyrosine activation domains and also regulates transcription;190 finally, the SH2 domain, the most highly conserved of the STAT domains, is essential for the recruitment of STATs to cytokine receptors, the binding of STATs to JAKs, and the homo- or heterodimerization of activated STAT proteins.191, 192, 193   750 ? 850 amino acidsAmi o T rminalCoiled-CoilDNA BindingLinkerSH2/Tyrosine ActivationTranscriptional Activation? Nuclear translocation? STAT Deactivation? Tyrosine phosphorylation? Receptor binding? Nuclear export? Bind target DNA? Receptor binding? JAK binding? Dimerization? Activate transcription? Signaling specificity  Figure 1.2 Organization and Function of STAT Protein Domains STAT proteins range from 750 to 850 amino acids in length and possess 5 conserved domains: amino-terminal, coiled-coil, DNA binding, linker, and SH2/tyrosine activation. The characteristic tyrosine phosphorylation site required for efficient STAT dimerization occurs in the SH2 domain and is indicated by the circled P. The transcriptional activation domain is poorly conserved amongst STAT family members and confers signaling specificity.   22 The canonical JAK-STAT signaling pathway begins with the binding of cytokines to corresponding cell surface receptor dimers, which causes a conformational change in the cytoplasmic portion of the receptors; this allows receptor-associated JAKs to transphosphorylate and gain the ability to phosphorylate specific tyrosine residues on the receptor tails. STAT proteins bind to the phosphotyrosine on the receptors via their SH2 domain and are in turn phosphorylated, also on a tyrosine residue, by the JAKs. Phosphorylated STATs dissociate from the receptor, dimerize, and translocate into the nucleus where they bind to the enhancers of target genes and activate transcription.184, 185, 194 The activity of STATs is regulated by dephosphorylation of JAKs and STATs by tyrosine phosphatases;195 nuclear export of dephosphorylated STATs;196 and negative feedback by suppressors of cytokine signaling (SOCS) family proteins.197  1.5.2 STAT Proteins in Atherosclerosis Although STATs are essential for the signaling of various cytokines, many of which play critical roles in atherosclerosis, literature describing the roles of STATs in atherosclerotic disease is scarce; indeed, at the present time, only STAT1 and STAT3 have been explored in detail in the context of atherosclerosis. STAT1 is responsible for the signaling of IFN-?/? and IFN-?, which have pro-atherogenic properties.136?141, 198, 199 Transplantation of Stat1-/- bone marrow into Ldlr-/- mice inhibits macrophage apoptosis and significantly reduces lesion area and necrosis.200 STAT3 is a versatile protein with roles in embryogenesis, skin maintenance, nervous system health, mammary development, and cytokine signaling.201 STAT3 is activated in response to the pleotropic cytokine IL-6 (and other IL-6 family members) and the anti-inflammatory cytokine IL-10.202, 203 Activated STAT3 can be detected in human atherosclerotic lesions and  23 endothelial cell-specific knockout of STAT3 reduces lesion area and macrophage content in mice.204 Consistent with the above findings, antisense oligodeoxynucleotide inhibition of SOCS3, which negatively regulates STAT1 and STAT3 signaling, exacerbates atherosclerosis in ApoE-/- mice.205  1.6 STAT6 The story of STAT6 began in the year 1988, when a group led by Laurie Glimcher described a novel DNA-binding protein in B cells that was up-regulated in response to IL-4.206 A number of years later, between 1994 and 1995, both human and murine homologs of this protein, initially designated IL-4 Stat, were purified and the encoding genes were cloned.207, 208 In humans and mice, the STAT6 gene is located on chromosome 12 and chromosome 10, respectively.209, 210 Although STAT6 is expressed ubiquitously, it has been shown that its expression can be augmented in active T and B cells.211 Splice variants of STAT6 (STAT6a, STAT6b, and STAT6c), potentially acting as dominant negatives, have been observed; however, the full functions of different STAT6 isoforms have yet to be investigated.209, 212 The STAT6 protein is composed of 847 amino acids (837 in mice) with a total size of 94 kD.207, 208 The organization of protein domains in STAT6 is consistent with that of other STAT family members. The amino-terminal domain is followed by the coiled-coil domain, the DBD, the linker-domain, the SH2/ tyrosine activation domain, and finally the TAD at the carboxy terminus.213 The DBD, SH2/tyrosine, and TAD are essential for the transcriptional activity of STAT6, as mutations within any one of these domains abolishes DNA binding and transcriptional regulation by STAT6.213   24 1.6.1 STAT6 Signaling STAT6 is required for IL-4 and IL-13 signaling, which occurs through a receptor dimer consisting of IL-4R? combined with either the common ? chain (?c) or IL-13R?.11, 214 The major IL-4 receptor, also known as the type I IL-4 receptor, is composed of the IL-4R? and the ?c subunits.215 Since the ?c chain is involved in the signaling of various other cytokines, including IL-2, IL-7, IL-9, IL-15, and IL-21,209, 210 the signaling specificity of IL-4, at the receptor level, is achieved through the preferential binding of IL-4 to IL-4R? prior to the dimerization of IL-4R? with the ?c chain.218 The type II IL-4 receptor, a heterodimer of IL-4R? with IL-13R?1, is able bind to both IL-4 and IL-13.218, 219 The formation of the type II receptor occurs through the initial binding of IL-4 or IL-13 to IL-4R? or IL-13R?1, respectively, followed by the dimerization with the other component of the receptor complex.218 Engagement of the cytokine receptor by IL-4/13 leads to the tyrosine-phosphorylation of STAT6 at position 641 (Y641) by receptor-associated JAKs;213 all JAK family members (JAK1, JAK2, JAK3, and TYK2) have been shown to phosphorylate STAT6.220?223 Notably, STAT6 phosphorylation occurs extremely rapidly, reaching a plateau in as little as 3 minutes after IL-4 stimulation.208 Phosphorylated STAT6 dimerizes through the reciprocal interaction of the SH2 domain of one STAT6 molecule with the phosphotyrosine of another STAT6 molecule. The STAT6 homodimer then translocates to the nucleus and binds to the highly conserved gamma-activated sequence (GAS) DNA motif to activate or repress gene transcription (Figure 1.3).224, 225   25 PIL-4IL-13IL-4R?JAK JAKSTAT6PPSTAT6STAT6PPSTAT6STAT6PPGATA3P P? IL-4, IL-5, IL-13 Production? Enhance Th2 Response? Antagonize Th1 ResponseGAS MotifCytoplasmNucleus Figure 1.3 The STAT6 Signaling Pathway IL-4 and IL-13 bind to a receptor dimer consisting of IL-4R? and either the ?c or IL-13R?. Receptor-associated JAKs phosphorylate STAT6, which then dimerizes and translocates to the nucleus where it binds to the GAS motif to regulate transcription. STAT6 induces the expression of the master regulator of Th2 differentiation, GATA3, which enhances the Th2 response and antagonizes the Th1 response.  1.6.2 Biological Significance of STAT6 The most prominent role of STAT6 in physiology is orchestrating the Th2 response by regulating the expression of GATA3, the transcription factor necessary for Th2 cell proliferation and cytokine production (Figure 1.3).154, 156 Indeed, in Stat6-/- mice, the production of characteristic Th2 cytokines IL-4, IL-5, and IL-13 is dramatically reduced compared to Stat6+/+ mice.226, 227 In B lymphocytes, IL-4/STAT6 promotes immunoglobulin class switching to IgE and IgG1?antibodies that have strong implications in host defense against helminthes and in allergic airway inflammation.227,  26 228, 229 Understandably, STAT6 knockout mice are more susceptible to parasitic nematodes and have a blunted response against allergen challenge.229, 230, 231 In addition to lymphocytes, macrophages and DCs also employ STAT6 for IL-4 and IL-13-mediated responses. STAT6 signaling promotes the differentiation of macrophages into the alternatively activated sub-phenotype with increased MHCII expression, 232 reduced IL-12 and TNF-? production,233 decreased STAT1 activation,234 and the ability to inhibit T cell proliferation.235 The IL-4/STAT6 signaling pathway has also been shown to facilitate the transcription of PPAR-?-regulated genes in macrophages and dendritic cells.236 Unexpectedly, IL-4 and STAT6 can also induce the Th1 response by inhibiting IL-10 and stimulating IL-12 production by DCs.237 Although STAT6 is important in orchestrating immune cell function and differentiation, the roles of STAT6 in hematopoiesis are limited; mice deficient in STAT6 exhibit normal 233hematopoiesis despite having an increased number of myeloid progenitors in the bone marrow.238, 239, 240  1.6.3 IL-4, IL-13 and STAT6 in Atherosclerosis Literatures surrounding IL-4 have yet to reach a consensus on the roles of this cytokine in atherosclerosis. One study found that, at 30 weeks of age, Il-4-/-ApoE-/- mice have reduced aortic root atherosclerosis compared to ApoE-/- mice; however, at 45 weeks of age, the significant difference in aortic root lesion area is lost and replaced by a reduction in aortic arch atherosclerosis in Il-4-/-ApoE-/- mice.12 Consistent with the above findings, Alan Daugherty?s group showed that Ldlr-/- mice transplanted with Il-4-/- bone marrow develop smaller lesions in the aortic arch and the thoracic aorta but not the aortic root compared to mice transplanted with Il-4+/+ bone marrow.15 The same  27 group, however, later showed that neither IL-4 deficiency nor intraperitoneal injections of exogenous IL-4 affect atherosclerosis in Ldlr-/- and ApoE-/- mice.14 In contrast, another study found that administration of IL-4 into C57BL/6 mice abolishes the Th1 response and leads to an extremely significant 90% reduction in atherosclerotic lesion area.158 To complicate matters further, depending on the type of atherogenic stimulus, IL-4 can either be essential or dispensable in fatty streak formation.241, 242 Compared to IL-4, very few studies have explored the functional significance of IL-13 and STAT6 in atherosclerosis. Although it has long been known that IL-4 and IL-13 share redundant features, a report showing direct evidence that IL-13 affects atherosclerosis in vivo was unavailable until as recently as late 2012. The study found that administration of IL-13 reduces monocyte recruitment, decreases plaque macrophage content, and increases collagen content in established plaques without affecting lesion area.243 Furthermore, it was shown, in the same study, that atherosclerosis development is accelerated in Ldlr-/- mice transplanted with Il-13-/- bone marrow compared to mice transplanted with Il-13+/+ bone marrow.243 To date, only a single study, published in 2001, has reported a direct link between STAT6 and atherosclerosis. Deletion of STAT6 in the normally athero-resistant, Th2-prone, BALB/c mouse strain renders the mice susceptible to atherosclerosis.158 The increase in atherosclerosis is associated with an enhanced Th1 and impaired Th2 response based on the production of IL-4 and IFN-? by the lymphocytes of Stat6-/- mice.158  1.7 STAT4 STAT4 was first cloned in 1994 by two independent research groups using degenerate PCR based on sequence homology to STAT1.244, 245 Unlike STAT6, which  28 is expressed ubiquitously, STAT4 expression is limited to lymphoid and myeloid cells, spleen, thymus, and testes.244, 246, 247 The gene encoding STAT4 is mapped to chromosome 2 and chromosome 1 in human and mice, respectively.248, 210 The monomeric STAT4 protein is composed of 748 amino acids (749 in mice) and has a molecular weight of 89 kDa.249 An isoform of STAT4, termed STAT4?, lacks the C-terminal transactivation domain, making the protein 44 amino acids shorter than full length STAT4.249 The domain organization of STAT4 is identical to that of the other STAT family members.249  1.7.1 STAT4 Signaling STAT4 is phosphorylated and activated in response to IL-12, which is mainly secreted by monocytes/macrophages, neutrophils, and DCs when exposed to danger- or pathogen-associated molecular patterns (DAMPs and PAMPs).250, 251 IL-12 is a heterodimeric cytokine composed of two subunits: the 35-kDa IL-12? chain and the 40-kDa IL-12? chain.252 In a similar fashion, the IL-12 receptor complex is made up of two chains designated IL-12R?1 and IL-12R?2.253 The binding of IL-12 to IL-12 receptor triggers the tyrosine phosphorylation of JAK2 and TYK2, which then phosphorylate STAT4 on tyrosine 693.254, 255 The phosphorylation of serine at position 721, located in the TAD, is also critical for the transcriptional activity of STAT4.256 Like other STAT family members, phosphorylated STAT4 dimerizes, translocates to the nucleus, and binds to the GAS DNA sequence to regulate transcription (Figure 1.4).257    29 PIL-12IL-12R?1JAK2 TYK2STAT4PPSTAT4STAT4PPSTAT4STAT4PPT-betP P? IFN-? Production? IL-12R? Expression? Enhance Th1 Response? Antagonize Th2 ResponseCytoplasmNucleusGAS Motif  Figure 1.4 The STAT4 Signaling Pathway The signaling events of the IL-12/STAT4 pathway are similar to those of the other STATs. Activated STAT4 operates in conjunction with T-bet, the master regulator of Th1 differentiation, to drive the production of IFN-?, induce IL-12R? expression, and enhance the Th1 response while inhibiting Th2 differentiation.  In addition to IL-12, STAT4 is also phosphorylated in response to other cytokines in the IL-12 family, including IL-23; IL-27; and, more recently, IL-35.258, 259, 260 IL-23 signals through a heterodimer of STAT3 and STAT4; however, compared to IL-12, activation of STAT4 by IL-23 is considerably weaker.258 IL-27 is an inhibitory cytokine that preferentially activates STAT1, STAT3, and STAT5 but can also phosphorylate STAT4.259 IL-35, which also appears to be an inhibitory cytokine, employs STAT1 and STAT4 for its functions.260 In human cells, STAT4 is also activated by type I IFNs; the extent to which this pathway is conserved in the murine model, however, remains controversial.261?264  30 1.7.2 Biological Significance of STAT4 The main biological functions of IL-12 and STAT4 include mediating the differentiation of CD4+ T cells into Th1 cells and stimulating IFN-? production by NK cells, T cells, B cells, and APCs.265?268 Optimal Th1 polarization and IFN-? production requires STAT4 working in conjunction with the T-box transcription factor (T-bet), often considered to be the master regulator of Th1 differentiation.266, 269 T-bet is absent in na?ve CD4+ T cells but its expression can be induced by IFN-? and IL-12. 269, 270 T-bet subsequently amplifies IFN-? production and enhances the expression of the IL-12R?2 subunit of the IL-12 receptor, creating a positive feedback loop to drive Th1 differentiation (Figure 1.4).269, 270 Furthermore, T-bet antagonizes Th2 and Th17 lineage development by suppressing the expression of GATA3 and retinoic acid receptor-related orphan nuclear receptor (ROR?t), respectively.271, 272   STAT4 knockout mice display an impaired Th1 response but are otherwise identical to wild type mice with regards to physical development and hematopoiesis, with the exception of decreases in myeloid progenitors in the bone marrow of Stat4-/- mice.240, 265, 273 Since Th1 immunity is important for the clearance of intracellular parasites and bacteria, STAT4-deficient mice are, as predicted, more susceptible to infection by these microorganisms. Indeed, STAT4 knockout mice have increased disease burdens following infection with intracellular parasites, such as Leishmania major, Leishmania mexicana, Trypanosoma cruzi and Toxoplasma gondii, and the intracellular bacterium Mycobacterium tuberculosis .274?278  Interestingly, in animal models, STAT4 deficiency can confer resistance against several experimentally-induced, T cell-driven autoimmune conditions, including  31 encephalomyelis, arthritis, colitis, myocarditis, and diabetes.279?283 In humans, STAT4 has been identified as a risk factor for rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and Sj?gren's syndrome, all of which are autoimmune conditions.284, 285 Furthermore, single nucleotide polymorphisms in the third intron of STAT4 are associated with increased susceptibility to RA, SLE, and Sj?gren's, further strengthening the involvement of STAT4 in these diseases.286 Intriguingly, patients with RA or SLE develop accelerated atherosclerosis, inviting the speculation that STAT4 may also play important roles in atherosclerotic disease.287, 288  1.7.3 IL-12 Family of Cytokines and STAT4 in Atherosclerosis The importance of IL-12 in atherosclerosis has been explored using mouse models. In studies described so far, IL-12 possesses a mainly pro-atherogenic role. ApoE-/- mice receiving daily intraperitoneal injections of IL-12 for 30 days develop lesions that are more than twice as large as those found in PBS-injected controls.289 Moreover, lesions from IL-12-treated mice are more advanced as evidenced by higher numbers of Th1 cells and increased IFN-? mRNA expression in the plaque.289 On the other hand, blocking IL-12 signaling by protein vaccination leads to smaller and more stable lesions in Ldlr-/- mice.13 In addition, aortic root atherosclerosis is reduced in Il-12-/-ApoE-/- mice at 30 weeks of age compared to ApoE-/- and Il-4-/-ApoE-/- mice.12 Interestingly, from 30 weeks to 45 weeks of age, there is a dramatic 142% increase in aortic root atherosclerosis in Il-12-/-ApoE-/- mice, which abrogates previous differences in lesion area seen between Il-12-/-ApoE-/- and the other genotypes.12 The rapid acceleration of atherosclerosis between 30 and 45 weeks of age in Il-12-/-ApoE-/- is also observed in the aortic arch, thoracic aorta, and abdominal aorta.12 These findings  32 suggest that IL-12 is an important cytokine in early lesion formation; its roles in advanced atherosclerosis, however, remain to be elucidated. It is important to note that the studies listed above do not reveal whether IL-12-mediated plaque development is dependent upon STAT4. The influences of IL-23 and IL-35 deficiency on murine atherosclerosis have not been studied. On the other hand, IL-27 has been shown to inhibit atherosclerosis by two independent laboratories.290, 291 Ldlr-/- mice deficient in IL-27, IL-27 receptor, and mice transplanted with Il-27ra-/- bone marrow all develop increased atherosclerosis.290, 291 Again, the relevance of STAT4 in these studies was not addressed. Despite evidence that the Th1 response, which is enhanced by STAT4, drives atherosclerotic plaque formation, STAT4 itself has received little attention in atherosclerosis; nonetheless, some studies might suggest a possible functional significance of STAT4 in this disease. In a murine lupus model, simvastatin causes a cholesterol-independent reduction of atherosclerotic lesion area that was associated with decreases in STAT4 phosphorylation and reductions in serum TNF-? and IFN-?.292 Furthermore, administration of suppressive oligodeoxynucleotides inhibits STAT1/STAT4 phosphorylation, T-bet expression, and atherosclerosis in ApoE-/- mice.293 Although these findings are exciting, direct evidence for the functional significance of STAT4 in atherosclerosis still needs to be generated with the help of animal models.   1.8 The Mouse Model of Atherosclerosis Over the past two decades, the mouse has gained tremendous popularity as a model organism for human atherosclerosis due to their low cost of maintenance, rapid  33 breeding, and ease of genetic manipulation. Wild type mice, however, are resistant to atherosclerosis since mice transport the majority of their plasma cholesterol using HDL particles, which have been shown to be atheroprotective in humans.294, 295 The generation of ApoE and LDL receptor knockout mice in 1992 and 1993, respectively, proved invaluable for in vivo atherosclerosis research.294, 296 Both ApoE-/- and Ldlr-/- mice develop hypercholesterolemia when given a high fat diet and, like humans, are susceptible to diet-induced atherosclerosis.294, 297?299 Interestingly, transplantation of wild type bone marrow, which express ApoE and LDLR, can normalize the lipid profile and attenuate atherosclerosis in ApoE-/- mice but not in Ldlr-/- mice.297?300 This quality makes the Ldlr-/- mouse the primary choice for studying the roles of bone marrow-derived cells in atherosclerosis. The development of atherosclerotic lesions in ApoE-/- and Ldlr-/- mice shares many similarities as well as differences with the human version of the disease. The process of atherosclerosis occurs much more rapidly in mice, typically spanning weeks instead of the decades required for humans.301 Immune cells are abundant in the plaque of both species but the SMC content is much higher in human compared to murine lesions.301 The preferential sites of lesion formation, in areas of low shear stress (the aortic root; lesser curvature of the aorta; brachiocephalic artery; and carotid and subclavian branch points), are common between the two species.302 The most notable difference, however, is perhaps the rarity of spontaneous plaque rupture and thrombosis in mice, which severely limits their usefulness as a model for the major clinical consequence of human atherosclerotic disease.303 Nonetheless, the murine  34 model remains a tremendously important tool for studying the process of atherogenesis and progression leading up to plaque rupture.  1.9 Research Objectives and Hypotheses The importance of the immune system during all stages of atherosclerosis is no longer controversial. Research efforts in the past few decades have revealed that certain immune responses are atherogenic while others may protect against atherosclerosis.6 One might then speculate that augmenting anti-atherogenic immunity while inhibiting the pro-atherogenic immune response is an effective treatment strategy for atherosclerotic diseases. However, the complexity of the immune system and its indispensable roles in maintaining tissue homeostasis warrant extreme caution when modulating certain aspects of immunity for disease treatment, especially for chronic conditions like atherosclerosis. Therefore, we must continue to broaden our understanding of atherosclerosis-related immunity in order to evaluate whether current, or novel, pathways are suitable potential targets for pharmacological intervention in the treatment of atherosclerosis. STAT6 and STAT4 are essential mediators of two antagonizing arms of the immune response, yet very little is known about their functional significance in atherosclerosis.304 Moreover, as mentioned in previous sections, a number of cytokines and receptors in the STAT6/STAT4 pathway have important influences on murine atherosclerosis; this prompted me to explore the consequences of STAT6 and STAT4 deficiency on atherogenesis in mice. My research aim was to examine how the absence of STAT6 or STAT4 in bone marrow-derived cells would affect atherosclerosis in Ldlr-/- mice fed an atherogenic diet. I hypothesized that STAT6 deficiency would dampen the  35 Th2 response, leading to an enhanced Th1 response and therefore increased atherosclerosis. On the other hand, I predicted that bone marrow STAT4 knockout mice would be protected from atherosclerosis due to a suppression of their atherogenic Th1 response. In the following chapters, I outline the experiments performed to test these hypotheses as well as the major findings. I also discuss the potential impacts of my discoveries on the current understanding of atherosclerosis, address possible limitations of my research, and propose important future studies while speculating on the expected outcomes of these experiments.  36 CHAPTER 2: Investigation of STAT6 in a Mouse Model of Atherosclerosis 2.1 Background STAT6 is critical for the signaling of IL-4/13 and the initiation and maintenance of the Th2 response.154 Despite previous research efforts, the roles of the STAT6 pathway in atherosclerotic disease remain ambiguous. Studies in mice have demonstrated that IL-4 can exacerbate, attenuate, or be a bystander in atherosclerosis.12, 14, 15, 158 Furthermore, IL-4-dependent effects on lesion formation can differ based on anatomical location and the animal?s age. In Il-4-/-ApoE-/- mice, for example, atherosclerosis is reduced only in the aortic root at 30 weeks of age and only in the aortic arch at 45 weeks of age compared to ApoE-/- mice.12 In addition, Ldlr-/- mice transplanted with Il-4-/- bone marrow develop smaller lesions in the aortic arch and the thoracic aorta but not in the aortic root compared to mice transplanted with Il-4+/+ bone marrow.15 Compared to IL-4, IL-13 has received considerably less attention; nevertheless, Cardilo-Reis et al. recently published a pioneering study showing that IL-13 confers resistance against atherosclerosis by reducing monocyte and macrophage content while increasing collagen content in the lesions of IL-13-treated Ldlr-/- mice.243  It was shown more than a decade ago that deletion of STAT6 in the normally athero-resistant BALB/c mouse strain impaired the Th2 response and allowed these mice to develop atherosclerotic lesions similar to those found in wild type C57BL/6 mice.158 However, the ubiquitous expression of STAT6 introduces challenges when attributing phenotypic changes in Stat6-/- BALB/c mice to particular cellular lineages. We have therefore chosen to focus our research efforts on the effects of myeloid-specific  37 STAT6 on murine atherosclerosis. Understanding the impacts of lineage-specific STAT6 deficiency on atherosclerosis is a step towards developing targeted therapeutics to treat atherosclerotic disease without severely compromising the immune system.  In this chapter, I describe the research undertaken to examine the effects of myeloid-specific STAT6 depletion on atherosclerosis in Ldlr-/- mice at two time points: 8 and 14 weeks after the initiation of atherogenic diet. These time points were chosen, based on previous literature, to represent early and intermediate stages of atherosclerosis.305, 306, 307 The specific aims of this chapter are 1. To evaluate whether STAT6 affects cholesterol accumulation in murine bone marrow-derived macrophages (BMDMs) following IL-4 stimulation. 2. To assess the effects of myeloid-specific STAT6 depletion on atherosclerotic lesion size and complexity in Ldlr-/- mice fed an atherogenic diet for 8 or 14 weeks. 3. To determine whether myeloid STAT6 affects the immune cell populations in the blood, bone marrow, and spleen of Ldlr-/- mice fed an atherogenic diet for 8 or 14 weeks. Since IL-4 induces the expression of CD36, a scavenger receptor involved in oxLDL uptake, in the murine RAW264.7 macrophage cell line,308 we hypothesized that IL-4 would increase cholesterol accumulation in murine BMDMs. Furthermore, because STAT6 is involved in IL-4 signaling, we proposed that STAT6-deficient BMDMs would have reduced cholesterol accumulation following IL-4 treatment compared to IL-4-treated wild type BMDMs.  38 In myeloid cells, activation of the STAT6 signaling pathway triggers an array of potentially anti-atherogenic qualities such as reducing IL-12 and TNF-? production, decreasing IFN-?-induced STAT1 activation, and inhibiting T cell proliferation through the STAT6-dependent expression of PD-L2.233?235 Although IL-4 has been shown to exacerbate, attenuate, and have no effect on atherosclerosis in mouse models, germline deletion of the Stat6 gene enhances atherosclerosis in BALB/c mice.158 Based on the aforementioned observations, we hypothesized that Ldlr-/- mice transplanted with Stat6-/-Rag1-/- bone marrow (myeloid STAT6-deficient mice, designated M-Stat6-/-) would be more susceptible to atherosclerosis compared to Ldlr-/- mice transplanted with Stat6+/+Rag1-/- bone marrow (M-Stat6+/+ mice). We speculated that the loss of STAT6 in myeloid cells would lead to increased inflammation within atherosclerotic lesions, resulting in greater lesion area due to increased innate and adaptive immune cell recruitment and proliferation. Enhanced inflammatory cytokine production brought about by the loss of myeloid STAT6 may stimulate the preferential conversion of macrophages into the M1 subset, thus increasing the ratio of M1 to M2 macrophages. Therefore, we also expected to observe increases in lesion necrosis in M-Stat6-/- mice due to the defective efferocytosis of apoptotic cells in the plaque, a process mainly orchestrated by M2 macrophages.29 In STAT6 knockout mice, the numbers and cycling status of granulocyte-macrophage, erythroid, and multipotential progenitor cells in the bone marrow and spleen are significantly elevated compared to wild type mice. Despite the changes in progenitor cell homeostasis, the development of mature myeloid cells is unaffected in STAT6 knockout mice.238?240 However, chronic unresolved inflammation, such as that  39 observed during atherosclerosis, leads to the continuous recruitment of immune cells to sites of inflammation.309 An important question to answer is whether increased numbers of progenitor cells in STAT6 knockout mice enhances inflammatory cell recruitment to atherosclerotic lesions by accelerating the replenishment of the pool of available cells. If so, this is another potential mechanism by which STAT6-depletion can exacerbate atherosclerosis.   2.2 Materials and Methods 2.2.1 Animals Stat6-/-, Ldlr-/-, Rag1-/-, BoyJ, and C57BL/6 animals were purchased from The Jackson Laboratory. Stat6-/- mice were crossed with Rag1-/- mice to generate the Stat6-/-Rag1-/- genotype. The animals were housed at the Centre for Disease Modeling at the University of British Columbia under standard pathogen-free conditions. All experimental protocols were performed in compliance with the University of British Columbia Animal Care Committee.  2.2.2 Isolation and Culturing of BMDMs Two femurs were removed from wild type C57BL/6 or Stat6-/- mice after euthanasia and flushed with a syringe filled with RPMI1640 media (Hyclone) to isolate bone marrow. Bone marrow cells were cultured in RPMI1640 media supplemented with 10 ng/mL recombinant murine M-CSF (R&D Systems), 10% heat-inactivated FBS (Hyclone), 100 U/mL penicillin, 100 ?g/mL streptomycin, 2% sodium bicarbonate, 1% sodium pyruvate, and 0.2% beta-mercaptoethanol (Invitrogen) in T-175 flasks (Sarstedt)  40 at 37oC in a 5% CO2 atmosphere. Media changes were performed on days 3 and 6 of culture.  2.2.3 LDL Preparation and Oxidation Pooled normal human plasma (Innovative Research) was adjusted to a density of 1.019 g/mL using sodium bromide (Sigma Aldrich) and spun at 50,000 rpm and 8 oC for 24 hours in the L8-55 M Ultracentrifuge (Beckman Coulter). After the spin, the top layer of liquid containing chylomicrons and VLDL was removed using a pipette. The remaining solution was readjusted to a density of 1.063 g/mL using sodium bromide and centrifuged for another 24 hours at 50,000 rpm and 8 oC. The top layer of native LDL was transferred into 7 K MWCO Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific) and dialyzed in 4L of PBS at 4 oC for 48 hours. PBS was changed a total of 4 times during dialysis. Following dialysis, LDL was oxidized using 200 ?g/mL Cu2+ (from Cu2SO4) per 5 ?M of LDL for 20 hours. The successful oxidation of LDL was verified by detecting the formation of fluorescent Schiff base imine products at Ex: 360 nm Em: 430 nm using the Tecan Safire Plate Reader (TECAN).    2.2.4 Cholesterol Accumulation Assay On day 8 of culture, murine BMDMs were detached from the flasks using pre-warmed Accutase (Innovative Cell Technologies) and re-plated in 48-well plates at a density of 1 x 105 cells/well. Cells were treated with 10 ?g/mL recombinant murine IL-4 (R&D Systems) for 24 hours and then incubated with 50 ?g/mL of Cu2+ oxLDL for 24 hours. Culture media was then removed and the number of cells per well was estimated through DNA content measured using the CyQuant Kit (Invitrogen). Cells were then  41 lysed and endogenous peroxides were eliminated using 1U of bovine catalase. Cellular cholesterol content was measured using the Amplex Red Kit and normalized to DNA content.  2.2.5 Bone Marrow Transplantation To induce bone marrow aplasia, 8 week-old female Ldlr-/- mice on a C57BL/6 background were exposed to two doses of 6.5 Gy total body irradiation 4 hours apart (total of 13 Gy). On the day following lethal irradiation, mice were transplanted, via the tail vein, with 1 x 107 total bone marrow cells containing 90% of Stat6-/-Rag1-/- bone marrow, which cannot generate mature T or B cells,310 plus 10% of wild type bone marrow from the BoyJ mouse strain, which expresses the congenic leukocyte marker CD45.1, to reduce STAT6 expression in myeloid cells. Mice transplanted with 90% of Stat6+/+Rag1-/- and 10% wild type BoyJ bone marrow were used as controls (Figure 2.1). The validity of this procedure in generating M-Stat6-/- mice was confirmed in a proof of principle experiment using flow cytometry and qRT-PCR. Following bone marrow transplantation, animals were given normal chow diet and fresh water ad libitum for 6 weeks to allow for the full reconstitution of the immune compartments by donor bone marrow.  2.2.6 Induction of Atherosclerosis Following the recovery period, the chow diet was substituted with an atherogenic diet (TD.94059, Harlan Teklad) containing 15.8% fat (w/w) and 1.25% cholesterol (w/w) for 8 weeks or 14 weeks (Figure 2.1). Mice were monitored twice per day and weighed twice per week for the duration of the diet.  42 2.2.7 Sacrifice and Tissue Collection Mice were anesthetized with isoflurane and placed onto a dissection pad. Blood was collected via cardiac puncture and death was confirmed by cervical dislocation.  The chest cavity was exposed and a syringe filled with 10 mL of PBS was inserted into the left ventricle to flush remaining blood from the vasculature. The femurs and spleen were removed and saved for flow cytometry. The heart and the aorta were then perfused with 10 mL of 10% formalin. Under a dissecting microscope, the fat and adventitial tissue surrounding the heart and the aorta were carefully removed using surgical forceps and scissors. The heart and the entire aorta, from the top of the heart to ~3 mm after the iliac bifurcation, were removed and stored in 10% formalin.   Stat6-/-Rag1-/-orStat6+/+Rag1-/-(CD45.2)Wild Type BoyJ (CD45.1)Lethally-Irradiated Ldlr-/-(CD45.2)10%90%SacrificeAndAnalysisAtherogenicDiet8 or 14 weeksRecovery6 weeks Figure 2.1 Experimental Design for in vivo STAT6 Studies Ldlr-/- mice were lethally irradiated and reconstituted with 90% Stat6-/-Rag1-/- or Stat6+/+Rag1-/- bone marrow plus 10% wild type BoyJ bone marrow to create M-Stat6-/- or M-Stat6+/+ mice, respectively. Following a 6-week recovery period, the mice were fed an atherogenic diet for 8 weeks or 14 weeks prior to sacrifice.   43 2.2.8 Plasma Cholesterol Content  Blood was centrifuged at 1500 rpm for 10 minutes to isolate plasma. Plasma total cholesterol content was measured using the enzymatic colorimetric Cholesterol E kit (Wako Diagnostics) following manufacturer?s instructions.  2.2.9 Embedding and Sectioning of Aortic Roots  Sectioning and staining of the aortic roots was performed by Wax-it Histology Services Inc. at the University of British Columbia. Hearts were washed in tris-buffered saline and then embedded in NEG 50 (Richard-Allen Scientific) using liquid nitrogen-cooled isopropanol. Sectioning was done using the Leica CM 1900 cryomicrotome (Leica Microsystems) at a thickness setting of 10 ?m and a temperature setting of -18 ?C. Sections were placed on Superfrost?Plus Microscope Slides (Fischer Scientific) for staining.  2.2.10 Staining and Scanning Aortic Root Atherosclerotic Lesions  Slides were incubated in a 37 ?C oven for 1.5 hours and then fixed in a fixative solution consisting of 100% ethanol, 10% neutral buffered formalin, glacial acetic acid, and distilled water. For H&E staining, slides were stained, in order, with Gill?s 2 hematoxylin, 1% acid alcohol, lithium carbonate, and 2% eosin Y followed by dehydration and mounting procedures. For Oil Red O staining, slides were first counterstained in Mayer?s Hematoxylin, washed in distilled water, and dipped in lithium carbonate. The slides were then equilibrated in 70% ethanol prior to staining with Oil Red O. After staining, slides were rinsed with 70% ethanol and distilled water and subsequently mounted with DAKO Faramount. Stained sections were scanned using  44 the ScanScope XT (Aperio Technologies) for quantitative analysis of atherosclerotic lesions.  2.2.11 Blinding of the Observer  All procedures involving image analysis of atherosclerosis, including en face, aortic root lesion area, aortic root necrosis, and aortic root lipid content, were performed by Daven Tai after all of the information used to identify the treatment and control groups were concealed by Eugene Chu.  2.2.12 Quantification of Aortic Root Lesion Area  Atherosclerotic lesion area was quantified using the Aperio ImageScope software (Aperio Technologies) by manually tracing around the Oil Red O-positive areas. The total lesion areas of 3 to 4 tissue sections were averaged to represent the aortic root lesions area of a single animal.  2.2.13 Quantification of Aortic Root Lesion Necrosis  Necrotic areas were defined as acellular empty spaces within an atherosclerotic lesion.311 On H&E-stained slides, lesion area and necrotic core areas were traced manually using the Aperio ImageScope software. The extent of necrosis within a lesion was then calculated by dividing the necrotic core area by the total lesion area and multiplying by 100%. The percentage of necrosis of 3 to 4 tissue sections was averaged to represent the aortic root necrosis of a single animal.       45 2.2.14 Quantification of Aortic Root Lesion Lipid Content  The Positive Pixel Count algorithm in Aperio ImageScope was used to measure aortic root lesion lipid content on Oil Red O-stained slides. The total number of ?positive? and ?strong positive? pixels were multiplied by the area per pixel and then divided by lesion area to obtain the lesion lipid content. The results of the algorithm were verified using the manual color segmentation method in the Image-Pro Plus software (Media Cybernetics).   2.2.15 En Face Analysis of Atherosclerosis Entire aortas were sliced open longitudinally using surgical scissors and then pinned flat onto a black dissection pad to expose the intimal surface. The aortas were then stained with Sudan IV to highlight atherosclerotic areas. Images of the aorta were taken using the Infinity2 digital CCD camera (Lumenera) connected to a dissection microscope. The lesion area as a percentage of total aortic surface area was quantified using the ImagePro Plus software.  2.2.16 Preparation of Tissues for Flow Cytometry  Blood was added drop-wise to 10 mL of pre-warmed red cell lysis buffer (90% 0.16 M NH4Cl and 10% 0.17 M Tris, pH 7.65) and incubated at 37 ?C for 5 minutes. The blood was then centrifuged for 5 minutes at 1500 rpm and 4 ?C. The supernatant was removed and the blood was incubated in Fc block for 15 minutes prior to staining.  Bone marrow was flushed from femurs into 10 mL of PBS using a 21 G needle attached to a 1 mL syringe. Bone marrow was then centrifuged for 5 minutes at 1500 rpm and 4 ?C. The supernatant was removed and cells were resuspended in 10 mL of  46 PBS. The cells were counted using a hemocytometer and 5 x 106 cells were used for each flow cytometry stain. The cells were incubated in Fc block for 15 minutes prior to staining.  Spleens were homogenized with scalpel blades and passed through a 70 ?m filter to achieve single cells suspensions. The cells were centrifuged for 5 minutes at 1500 rpm and 4 ?C. The supernatant was removed and splenocytes were resuspended in 10 mL of FACS buffer (2% FBS, 2.5 mM EDTA, 0.05% NaN3 in MT-PBS). The cells were counted using a hemocytometer and 5 x 106 cells were used for each flow cytometry stain. The cells were incubated in Fc block for 15 minutes prior to staining.  2.2.17 Staining of Blood, Bone Marrow, and Spleen Cells for Flow Cytometry  Please refer to Table 2.1 for the combination of antibodies used in each stain (A, B, C, and D) as well as the type of stain used for each tissue. Following Fc block, cells were incubated in the appropriate primary antibody mixture for 45 minutes followed by the corresponding secondary antibody (SA-PB for stains A and B; SA-APC for stain C; SA-APC/Cy7 for stain D) for 30 minutes. Cells were then washed in FACS buffer, centrifuged for 5 minutes at 1500 rpm and 4 ?C, and resuspended in 250 ?L of propidium iodide. Flow cytometry was performed using the BD LSRII flow cytometer and data was collected using the BD FACSDiva Software. The analysis of flow cytometry data was performed using the FlowJo Software (Tree Star Inc.).         47 2.2.18 Statistical Analysis Statistical significance was tested using the two-tailed Student?s t-test in GraphPad Prism 5 or 6 (GraphPad Software). A p-value of 0.05 or smaller was considered statistically significant.   Stain A B C D Primary Antibody (Clone) Ly6C (AL-21) CD11b (M1/70) CD3 (145-2C11) CD49b (DX5) CD62L (MEL-14) F4/80 (BM8) CD4 (RM4-5) NK1.1 (PK136) CD11b (M1/70) MHCII (M5/114.15.2) CD19 (1D3) GR-1 (RB6.8C5) CD115 (AFS98) CD11c (N418) CD8a (53-6.7) CD11b (M1/70) MHCII (M5/114.15.2) CD45.1 (A20) CD45.1 (A20) CD45.1 (A20) CD45.1 (A20) CD45.2 (104) CD45.2 (104) CD45.2 (104) Tissues Stained ? Blood ? Bone Marrow ? Spleen ? Spleen ? Blood ? Spleen ? Spleen  Table 2.1 Combination of Antibodies Used to Stain Each Tissue for Flow Cytometry      48 2.3 Results 2.3.1 STAT6 Deficiency Abolishes IL-4-Induced Increases in Cholesterol Accumulation in Murine BMDMs  To determine whether STAT6 is involved in cholesterol homeostasis in murine BMDMs, murine Stat6+/+ and Stat6-/- BMDMs were stimulated with IL-4 and incubated with Cu2+ oxLDL for 24 hours. Cellular cholesterol content was then measured enzymatically and normalized to DNA content. There was a 33% increase in cholesterol accumulation in IL-4-treated Stat6+/+ BMDMs compared to untreated Stat6+/+ BMDMs. IL-4 treatment did not increase cholesterol accumulation in Stat6-/- BMDMs (Figure 2.2). There were no statistically-significant differences in cholesterol accumulation between Stat6+/+ and Stat6-/- BMDMs in either untreated or IL-4-treated conditions.  B)A)Stat6+/+IL-4Stat6+/+Untreated Stat6-/-IL-4Stat6-/-Untreated Figure 2.2 STAT6 Deficiency Abolishes IL-4-Induced Increases in Cholesterol Accumulation in Murine BMDMs On day 8 of culture, murine BMDMs were treated with 10 ng/mL of IL-4 for 24 hours. Cells were then loaded with 50 ?g/mL of Cu2+oxLDL for 24 hours and cholesterol content was measured enzymatically using the Amplex Red kit. Cholesterol content was normalized by DNA content using the CyQuant Kit. A) Cholesterol accumulation in Stat6+/+ BMDMs. B) Cholesterol accumulation in Stat6-/- BMDMs. Error bars represent SD. n = 6 mice for Stat6+/+ and n = 4 mice for Stat6-/-.* p < 0.05 by two-tailed t-test.  49 2.3.2 Validation of the Irradiation and Bone Marrow Transplantation Procedures for the Generation of M-Stat6-/- and M-Stat6+/+ Mice Prior to initiating our in vivo studies, we performed a proof of principle experiment to demonstrate that our bone marrow transplantation procedure successfully generates mice in which the lymphoid compartment is derived from wild type BoyJ (CD45.1+) bone marrow while the myeloid compartment comes from Rag1-/- (CD45.2+) donors, assuming complete ablation of the recipient?s myeloid compartment (which is also CD45.2+). 8 week-old Ldlr-/- mice were lethally irradiated and transplanted with 90% Stat6+/+Rag1-/- or Stat6-/-Rag1-/- plus 10% wild type BoyJ bone marrow. Following a 9 week-recovery period, the expression of CD45.1 on CD115+CD11b+ myeloid cells and CD19+ B cells was measured by flow cytometry (Figure 2.3 A). We found that greater than 90% of myeloid cells from the blood, bone marrow, and spleen of M-Stat6-/- mice were negative for CD45.1, suggesting that <10% of recipient myeloid cells were derived from the BoyJ donor (Figure 2.3 B). On the other hand, nearly all B cells from the blood were positive for CD45.1, confirming that the lymphoid compartment was successfully repopulated by BoyJ-derived cells (Figure 2.3 C). We next evaluated the expression of Stat6 mRNA in the bone marrow cells of recipient mice using RT-qPCR. We found that Stat6 expression was dramatically reduced in the bone marrow of M-Stat6-/- mice compared to M-Stat6+/+ mice. Moreover, the expression level of Stat6 in M-Stat6-/- mice was ~10% of that in M-Stat6+/+ mice, which correlated with the ~10% of CD45.1+ wild type cells found in the M-Stat6-/- mice, suggesting that CD45.1 expression can be used as an indirect marker for Stat6 expression in recipient mice. (Figure 2.3 D).   50 A)Stat6+/+Rag1-/-orStat6-/-Rag1-/-(CD45.2)WT BoyJ(CD45.1)90%10%Lethally-irradiatedLdlr-/-mice (CD45.2)9 week RecoveryANALYSISD)M-Stat6+/+M-Stat6-/-CD115CD11bCD45.1FSC8.1  3.692.2  3.4B) C)CD45.1-CD45.1+CD19+B CellsMonocytes Figure 2.3 Validation of the Irradiation and Bone Marrow Transplantation Procedures for the Generation of M-Stat6-/- and M-Stat6+/+ Mice Lethally irradiated Ldlr-/- mice, reconstituted with 90% Stat6+/+Rag1-/- or Stat6-/-Rag1-/- and 10% wild type BoyJ bone marrow, were euthanized 9 weeks post irradiation. A) Schematic of the experimental design. B) Gating scheme showing ~10% of CD45.1+ to ~90% CD45.1- populations in myeloid cells. Frequency reported represents the average frequency of CD45.1+ and CD45.1- determined from the blood, bone marrow and spleen. C) Frequency of CD19+ B cells in blood of irradiated chimeras, showing lymphoid compartment is CD45.1 (all host B cells are ablated). D) RT-qPCR levels of Stat6 in whole and cultured bone marrow from chimeric mice, normalized to GAPDH. Percentage of M-Stat6+/+ is reported. Error is SEM, n = 2 mice for D, and n > 5 mice per group for B and C. WT = wild type.  51 2.3.3 Body Weight and Total Plasma Cholesterol Measurements in M-Stat6-/- Mice Fed an Atherogenic Diet for 8 or 14 Weeks Body weight and plasma cholesterol levels are factors that can influence atherosclerosis. To monitor weight changes and plasma cholesterol content, we measured these parameters during and after the atherogenic diet, respectively. During the induction of atherosclerosis, mice were weighed twice per week to monitor weight changes. At the time of sacrifice, plasma was collected and total cholesterol was measured enzymatically. There were no significant differences total plasma cholesterol levels (Figure 2.4 C) between M-Stat6+/+ and M-Stat6-/- mice fed an atherogenic diet for either 8 or 14 weeks. 52 0 10 20 30 40 501820222410 20 30 40 50-101234Weight (g)Weight Gain (g)0 20 40 60 80 10020253020 40 60 80 100-20246810Weight Gain (g)Weight (g)Days on atherogenic diet8 weeks 14 weeksDays on atherogenic dietDays on atherogenic dietDays on atherogenic dietM-Stat6-/-M-Stat6+/+M-Stat6+/+M-Stat6-/-Cholesterol (mg/dL)Cholesterol (mg/dL)M-Stat6+/+M-Stat6-/-A)B)C)8 weeks 14 weeks8 weeks 14 weeks Figure 2.4 Body Weight and Total Plasma Cholesterol Measurements in M-Stat6-/- Mice Fed an Atherogenic Diet for 8 or 14 Weeks Mice were fed an atherogenic diet for 8 or 14 weeks prior to sacrifice. A) and B) Total body weight (A) and weight gain (B) during the induction of atherosclerosis. C) Enzymatic detection of blood plasma cholesterol content at the time of sacrifice. Error bars represent SD. n = 12-15 per group.    53 2.3.4 Assessment of Bone Marrow Reconstitution in M-Stat6+/+ and M-Stat6-/- Mice To assess the relative contribution of BoyJ donor-derived cells in the blood, bone marrow, and spleens of M-Stat6+/+ and M-Stat6-/- mice, we examined the cellular expression of CD45.1 and/or CD45.2 in these tissues by flow cytometry. Dead cells and cells that were negative for both CD45.1 and CD45.2 were excluded from the analysis. Since only cells derived from wild type BoyJ donors express CD45.1, the levels of CD45.1 and CD45.2 were used as an indirect marker for Stat6 expression in myeloid cells. At 8 weeks following the initiation of atherogenic diet, less than 10% of monocytes in the blood were positive for CD45.1, with Ly6ClowCD62L- monocytes in M-Stat6+/+ mice being slightly higher, at 12?5% positive for CD45.1 (Figure 2.5 A and B). Furthermore, greater than 90% of CD11b+ myeloid cells in the blood were CD45.2+ (Figure 2.5 C and D). By contrast, approximately 80% of CD4+ T cells, 60% of CD8+ T cells, and 95% of B cells in the blood were CD45.1+; the expression pattern of CD45.2 in these lymphocytes was reciprocal to their CD45.1 expression (Figure 2.5). Importantly, since Stat6-/-Rag1-/- bone marrow cannot generate mature T and B cells, all T and B cells found in the recipient are wild type for STAT6, regardless of whether they are CD45.1+ or CD45.2+. In the bone marrow, less than 10% of all monocytes were positive for CD45.1 (Figure 2.6 A and B) and greater than 90% of CD11b+ myeloid cells were positive for CD45.2 (Figure 2.6 C and D). In M-Stat6+/+ mice, 86?3% of inflammatory and 71?13% of patrolling monocytes in the spleen were CD45.1-. Similarly, the percentage of CD45.1- inflammatory and patrolling monocytes in M-Stat6-/- mice were 91?3% and 81?10%, respectively (Figure 2.7 A and C). In splenic DCs, ~55% of MHCII+CD11c+ DCs were CD45.1- while ~70%  54 are CD45.2+. Greater than 90% of MHCII-CD11c+ cells were CD45.1- and CD45.2+. In addition, ~55% of MHCII-CD11c- cells were CD45.1- and CD45.2+ (Figure 2.7). Approximately 75% of CD4+ T cells, 80% of CD8+ T cells, and 95% of B cells in the spleen were CD45.1+ while 65% of CD4+ T cells, 80% of CD8+ T cells, and 95% of B cells were CD45.2- (Figure 2.7). Greater than 75% of NK1.1+CD49b+ NK cells in the spleen were devoid of CD45.1 expression and ~90% were positive for CD45.2. Lastly, >80% of CD11b+Gr-1hi neutrophils were CD45.1- while >95% were CD45.2+ (Figure 2.7). The discrepancy between CD45.1- and CD45.2+ populations in a given cell type was due to the presence of CD45.1+CD45.2+ cells, which were not excluded in our analysis.     55   A)93 2 7 2Ly6ChiCD62L+Ly6ClowCD62L-88 5 12 591 4 9 494 3 6 3CD3+CD4+CD3-CD19+CD3+CD8a+21 9 79 9 38 10 62 10 3 2 97 218 5 82 5 42 10 58 10 6 4 93 4M-Stat6-/-M-Stat6+/+CD45.1CD45.1-CD45.1+CD3+CD4+CD3+CD8a+CD3-CD19+M-Stat6+/+Frequency (%)Ly6ClowCD62L-Ly6ChiCD62L+Frequency (%)M-Stat6-/-CD3+CD4+CD3+CD8a+CD3-CD19+Ly6ClowCD62L-Ly6ChiCD62L+B) 56 CD45.2-CD45.2+C)CD45.2M-Stat6+/+M-Stat6-/-CD3+CD4+CD3-CD19+CD3+CD8a+78 10 22 1081 4 19 461 10 39 10 96 2 4 293 3 7 357 9 43 9CD11b+CD3+CD4+CD3+CD8a+CD3-CD19+M-Stat6+/+Frequency (%)CD11b+Frequency (%)M-Stat6-/-D)05010000100CD3+CD4+CD3+CD8a+CD3-CD19+CD11b+8 3 92 36 3 94 3  Figure 2.5 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Blood of M-Stat6+/+ and M-Stat6-/- Mice Following 8 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Blood CD11b+ cells, monocytes (Ly6C/CD62L) and lymphocytes (CD3+CD4+, CD3+CD8a+, CD3-CD19+) were stained with anti-CD45.1 and/or anti-CD45.2 antibodies for flow cytometry. A) and C) Representative histograms and gating schemes of CD45.1+ (A) and CD45.2+ (C) populations for each cell type. The frequency of events within each gated region ? SD was shown. B) and D) Bar graphs showing CD45.1 (B) and CD45.2 (D) expression of each cell type in the blood. Error bars represent SD. n = 13 mice for each cell type.   57 050100M-Stat6+/+M-Stat6-/-M-Stat6+/+CD45.1Ly6ChiCD62L+97 1 3 1Ly6ClowCD62L-90 3 10 393 3 7 397 2 3 2Frequency (%)Ly6ClowCD62L-Ly6ChiCD62L+M-Stat6-/-Ly6ClowCD62L-Ly6ChiCD62L+Frequency (%)CD45.1-CD45.1+A) B)M-Stat6+/+M-Stat6+/+CD45.2CD11b+2 1 98 15 3 95 3Frequency (%)CD45.2-CD45.2+C) D)CD11b+M-Stat6-/-M-Stat6-/- Figure 2.6 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Bone Marrow of M-Stat6+/+ and M-Stat6-/- Mice Following 8 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Bone marrow monocytes (Ly6C/CD62L) and CD11b+ cells were stained with anti-CD45.1 or anti-CD45.2 antibodies for flow cytometry. A) and C) Representative histograms and gating schemes of CD45.1+ (A) and CD45.2+ (C) cells. The frequency of events within each gated region ? SD was shown. B) and D) Bar graphs showing CD45.1 (B) and CD45.2 (D) expression of each cell type in the bone marrow. Error bars represent SD. n = 11 mice for each group.  58  Ly6ChiCD62L+Ly6ClowCD62L-MHCII+CD11c+MHCII-CD11c-MHCII-CD11c+CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiM-Stat6+/+86 3 14 3 71 13 29 13 55 9 45 9 56 4 44 4 92 4 8 428 7 72 7 20 18 80 18 3 3 97 3 76 4 24 4 82 6 18 6Ly6ChiCD62L+Ly6ClowCD62L-MHCII+CD11c+MHCII-CD11c-MHCII-CD11c+CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiCD45.1M-Stat6-/-91 3 9 3 81 10 19 10 54 2 46 2 54 7 46 7 90 6 9 624 4 76 4 18 15 82 15 4 5 96 5 83 2 17 2 83 5 17 5MHCII+CD11c+MHCII-CD11c-MHCII-CD11c+CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiM-Stat6+/+28 1172 1143 6 59 4 3 3 96 3 64 736 775 2125 2193 2 7 2 11 5 89 5 3 297 2MHCII+CD11c+MHCII-CD11c-MHCII-CD11c+CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiCD45.2M-Stat6-/-31 4 69 4 44 7 56 7 6 6 94 6 66 1034 1079 1721 1792 5 8 5 9 1 91 1 1 199 1A)B) 59   CD45.1- CD45.1+CD3+CD4+CD3+CD8a+CD3-CD19+Ly6ClowCD62L-Ly6ChiCD62L+MHCII-CD11c+NK1.1+CD49b+CD11b+Gr-1hiMHCII-CD11c-MHCII+CD11c+Frequency (%)M-Stat6+/+M-Stat6-/-Frequency (%)CD45.2- CD45.2+CD3+CD4+CD3+CD8a+CD3-CD19+MHCII-CD11c+NK1.1+CD49b+CD11b+Gr-1hiMHCII-CD11c-MHCII+CD11c+Frequency (%)Frequency (%)M-Stat6-/-M-Stat6+/+C)D)CD3+CD4+CD3+CD8a+CD3-CD19+Ly6ClowCD62L-Ly6ChiCD62L+MHCII-CD11c+NK1.1+CD49b+CD11b+Gr-1hiMHCII-CD11c-MHCII+CD11c+CD3+CD4+CD3+CD8a+CD3-CD19+MHCII-CD11c+NK1.1+CD49b+CD11b+Gr-1hiMHCII-CD11c-MHCII+CD11c+ 60 Figure 2.7 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Spleens of M-Stat6+/+ and M-Stat6-/- Mice Following 8 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Spleen myeloid (Ly6C/CD62L, MHCII/CD11c, CD11b+Gr-1hi) and lymphoid (CD3+CD4+, CD3+CD8a+, CD3-CD19+, NK1.1+CD49b+) cells were stained with anti-CD45.1 and/or anti-CD45.2 antibodies for flow cytometry. A) and B) Representative histograms and gating schemes of CD45.1+ (A) and CD45.2+ (B) populations for each cell type. The frequency of events within each gated region ? SD was shown. C) and D) Bar graphs showing CD45.1 (C) and CD45.2 (D) expression of each cell type in the spleen. Error bars represent SD. n = 5-8 mice for each group.   The contribution of BoyJ-derived cells in the blood and bone marrow of M-Stat6+/+ and M-Stat6-/- mice at 14 weeks after atherogenic diet was similar to that at 8 weeks, with only ~10% of myeloid cells originating from the wild type BoyJ donor (Figure 2.8 and Figure 2.9). In contrast, in the spleen of mice fed an atherogenic diet for 14 weeks, as many as 23?7% of Ly6ChiCD62L+ and 39?12% of Ly6ClowCD62L- monocytes stained positive for CD45.1 (Figure 2.10 A and C). Furthermore, the proportions of CD8+ T cells positive for CD45.1 at 14 weeks were 56?5% and 49?7% in M-Stat6+/+ and M-Stat6-/- mice, respectively, compared to the >80% CD45.1 positivity in CD8+ T cells at 8 weeks (Figure 2.10 A and C). The most dramatic difference, however, was seen in the CD45.2 expression of CD8+ T cells. Whereas 80% of CD8+ T cells were CD45.2- at 8 weeks, less than 35% of CD8+ T cells were CD45.2- at 14 weeks (Figure 2.10 B and D). Although these results suggest that the reconstitution of recipient lymphoid compartments by donor CD8+ T cells was imperfect, it is important to point out once again that all T and B cells in the recipient are wild type for STAT6 regardless of their origin. Finally, the expression patterns of CD45.1 and CD45.2 in NK cells and neutrophils at 14 weeks closely mirrored those at 8 weeks (Figure 2.10). 61  A)94 2 6 2Ly6ChiCD62L+Ly6ClowCD62L-89 4 11 490 4 10 494 2 6 2CD3+CD4+CD3-CD19+CD3+CD8a+21 9 79 9 40 12 61 10 3 2 97 222 8 78 8 47 7 53 7 9 4 91 4M-Stat6-/-M-Stat6+/+CD45.1CD45.1-CD45.1+CD3+CD4+CD3+CD8a+CD3-CD19+M-Stat6+/+Frequency (%)Ly6ClowCD62L-Ly6ChiCD62L+Frequency (%)M-Stat6-/-CD3+CD4+CD3+CD8a+CD3-CD19+Ly6ClowCD62L-Ly6ChiCD62L+B) 62 C)CD45.2M-Stat6+/+M-Stat6-/-CD3+CD4+CD3-CD19+CD3+CD8a+79 9 21 977 8 23 860 10 40 10 96 2 5 189 4 11 452 6 48 68 3 92 37 2 93 2CD11b+050100050100CD45.2-CD45.2+M-Stat6+/+Frequency (%)Frequency (%)M-Stat6-/-D)CD3+CD4+CD3+CD8a+CD3-CD19+CD3+CD4+CD3+CD8a+CD3-CD19+CD11b+ CD11b+  Figure 2.8 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Blood of M-Stat6+/+ and M-Stat6-/- Mice Following 14 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 14 weeks prior to sacrifice. Blood CD11b+ cells, monocytes (Ly6C/CD62L) and lymphocytes (CD3+CD4+, CD3+CD8a+, CD3-CD19+) were stained with anti-CD45.1 and/or anti-CD45.2 antibodies for flow cytometry. A) and C) Representative histograms and gating schemes of CD45.1+ (A) and CD45.2+ (C) populations for each cell type. The frequency of events within each gated region ? SD is shown. B) and D) Bar graphs showing CD45.1 (B) and CD45.2 (D) expression of each cell type in the blood. Error bars represent SD. n = 9 mice for each cell type.   63 M-Stat6+/+M-Stat6-/-M-Stat6+/+CD45.1Ly6ChiCD62L+97 1 3 1Ly6ClowCD62L-89 4 11 497 1 3 196 3 4 3Frequency (%)Ly6ClowCD62L-Ly6ChiCD62L+M-Stat6-/-Ly6ClowCD62L-Ly6ChiCD62L+Frequency (%)CD45.1-CD45.1+A) B)M-Stat6-/-M-Stat6+/+CD45.2CD11b+2 198 14 396 3CD45.2-CD45.2+C) D)050100M-Stat6+/+Frequency (%)CD11b+M-Stat6-/- Figure 2.9 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Bone Marrow of M-Stat6+/+ and M-Stat6-/- Mice Following 14 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 14 weeks prior to sacrifice. Bone marrow monocytes (Ly6C/CD62L) and CD11b+ cells were stained with anti-CD45.1 or anti-CD45.2antibodies for flow cytometry. A) and C) Representative histograms and gating schemes of CD45.1+ (A) and CD45.2+ (C) cells. The frequency of events within each gated region ? SD is shown. B) and D) Bar graphs showing CD45.1 (B) and CD45.2 (D) expression of each cell type in the bone marrow. Error bars represent SD. n = 9 mice for each group.  64  Ly6ChiCD62L+Ly6ClowCD62L-CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiM-Stat6+/+78 6 22 6 61 12 39 12 23 8 77 8 44 5 56 57 3 93 3 76 2 24 2 74 5 26 5Ly6ChiCD62L+Ly6ClowCD62L-CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiCD45.1M-Stat6-/-77 7 23 7 65 5 35 5 27 7 73 7 51 7 49 78 2 92 2 85 3 15 3 81 4 19 4A)B)CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiM-Stat6+/+66 10 34 10 32 668 692 2 8 2 6 1 94 1 2 1 98 1CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiCD45.2M-Stat6-/-60 9 41 8 23 5 77 5 90 3 10 3 4 2 96 2 2 1 98 1 65 CD45.1-CD45.1+CD3+CD4+CD3+CD8a+CD3-CD19+Ly6ClowCD62L-Ly6ChiCD62L+NK1.1+CD49b+CD11b+Gr-1hiFrequency (%)M-Stat6+/+M-Stat6-/-Frequency (%)CD45.2-CD45.2+CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiFrequency (%)Frequency (%)M-Stat6-/-M-Stat6+/+C)D)CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiCD3+CD4+CD3+CD8a+CD3-CD19+Ly6ClowCD62L-Ly6ChiCD62L+NK1.1+CD49b+CD11b+Gr-1hi  66 Figure 2.10 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Spleens of M-Stat6+/+ and M-Stat6-/- Mice Following 14 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 14 weeks prior to sacrifice. Spleen myeloid (Ly6C/CD62L, CD11b+Gr-1hi) and lymphoid (CD3+CD4+, CD3+CD8a+, CD3-CD19+, NK1.1+CD49b+) cells were stained with anti-CD45.1 and/or anti-CD45.2 antibodies for flow cytometry. A) and B) Representative histograms and gating schemes of CD45.1+ (A) and CD45.2+ (B) populations for each cell type. The frequency of events within each gated region ? SD is shown. C) and D) Bar graphs showing CD45.1 (C) and CD45.2 (D) expression of each cell type in the spleen. Error bars represent SD. n = 3 mice for CD3+CD8+ and NK1.1+CD49b+ cells. n = 6-9 mice for other cell types.   2.3.5 The Effects of Myeloid-Specific STAT6 Depletion on Monocyte Populations in the Blood, Bone Marrow, and Spleens of Ldlr-/- Mice To determine whether myeloid STAT6 expression affects monocyte populations in mice, we stained blood, bone marrow, and spleen cells from M-Stat6+/+ and M-Stat6-/- mice for flow cytometry analysis of total monocytes (CD11b+CD115+), inflammatory monocytes (Ly6ChiCD62L+), and patrolling monocytes (Ly6ClowCD62L-) (Figure 2.11 and Figure 2.12). Following 8 weeks of atherogenic diet, monocyte populations were identical between M-Stat6+/+ and M-Stat6-/- mice, with the exception of the splenic patrolling monocyte frequency (but not absolute number), which was increased by 11% in M-Stat6-/- mice (Figure 2.11 B 4th row). A similar lack of difference in monocyte populations was also observed at 14 weeks following atherogenic diet, where the only notable change was an increase in the frequency of total monocytes in the spleen by 16% in M-Stat6-/- mice compared to M-Stat6+/+ mice; once again, the absolute cell numbers did not reflect the change in frequency (Figure 2.12 B).    67 2.3.6 Myeloid-Specific STAT6 Depletion Does Not Alter Splenic DC Populations in Ldlr-/- Mice Following 8 Weeks of Atherogenic Diet To detect possible influences of myeloid STAT6 on DC development, spleens from M-Stat6+/+ and M-Stat6-/- mice were homogenized and stained with anti-MHCII and anti-CD11c antibodies for flow cytometry analysis of DCs (MHCII+CD11c+), MHCII-CD11c+ cell, and MHCII-CD11c- cell populations. Dead cells and cells negative for both CD45.1 and CD45.2 were excluded (Figure 2.13 A). There were no statistically significant differences between M-Stat6-/- and M-Stat6+/+ mice in the frequency or absolute cell numbers of the aforementioned cell types (Figure 2.13 B). 68 M-Stat6-/-SSCLy6C48 839 6CD11b22 3PIM-Stat6+/+SSCLy6C49 938 6CD11b22 4PIM-Stat6-/-SSCLy6C80 313 3CD11b13 3PIM-Stat6+/+SSCLy6C80 412 4CD11b13 1PIM-Stat6+/+SSCLy6C40 837 2CD11b6.2 4.9PIM-Stat6-/-SSCFSC CD62LLy6C38 541 4CD115CD11b7.0 5.9FSCPIBloodBone MarrowSpleenA)8 Weeks  69     B)Total Monocytes (CD11b+CD115+)Frequency (%)Inflammatory Monocytes (Ly6ChiCD62L+)Frequency (%)Patrolling Monocytes (Ly6ClowCD62L-)Frequency (%)Frequency (%)Frequency (%)Frequency (%)106Cells106Cells106CellsFrequency (%)Frequency (%)Frequency (%)106Cells106Cells106CellsBloodBone MarrowSpleenM-Stat6+/+M-Stat6-/- 70 Figure 2.11 Monocyte Populations in the Blood, Bone Marrow, and Spleens of M-Stat6+/+ and M-Stat6-/- Mice Following 8 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Blood, bone marrow, and spleen cells were stained for flow cytometry analysis of total monocytes (CD11b+CD115+), inflammatory monocytes (Ly6ChiCD62L+), and patrolling monocytes (Ly6ClowCD62L-). A) Representative dot plots and gating schemes of scatter, live, and monocyte populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency (blood, bone marrow, and spleen) and absolute numbers (bone marrow and spleen) of total, inflammatory, and patrolling monocytes. Error bars represent SD. n = 13 mice for blood. n = 10-11 mice for bone marrow. n = 6-8 mice for spleen. * p < 0.05 by two-tailed t-test.  71  M-Stat6-/-SSCLy6C52 536 2CD11b23 2PIM-Stat6+/+SSCLy6C50 938 7CD11b22 4PIM-Stat6-/-SSCLy6C78 412 2CD11b13 3PIM-Stat6+/+SSCLy6C78 412 3CD11b13 1PIM-Stat6+/+SSCLy6C40 630 4CD11b9.5 1.8PIM-Stat6-/-SSCFSC CD62LLy6C41 632 8CD115CD11b11 1FSCPIBloodBone MarrowSpleenA)14 Weeks 72        B)Total Monocytes (CD11b+CD115+)Frequency (%)Inflammatory Monocytes (Ly6ChiCD62L+)Frequency (%)Patrolling Monocytes (Ly6ClowCD62L-)Frequency (%)Frequency (%)Frequency (%)Frequency (%)106Cells106Cells106CellsFrequency (%)Frequency (%)Frequency (%)106Cells106Cells106CellsBloodBone MarrowSpleenM-Stat6+/+M-Stat6-/- 73 Figure 2.12 Monocyte Populations in the Blood, Bone Marrow, and Spleens of M-Stat6+/+ and M-Stat6-/- Mice Following 14 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 14 weeks prior to sacrifice. Blood, bone marrow, and spleen cells were stained for flow cytometry analysis of total monocytes (CD11b+CD115+), inflammatory monocytes (Ly6ChiCD62L+), and patrolling monocytes (Ly6ClowCD62L-). A) Representative dot plots and gating schemes of scatter, live, and monocyte populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency (blood, bone marrow, and spleen) and absolute numbers (bone marrow and spleen) of total, inflammatory, and patrolling monocytes. Error bars represent SD. n = 8-9 mice for blood and bone marrow. n = 6 mice for spleen. * p < 0.05 by two-tailed t-test.  74   CD45.1CD45.2 CD11cMHCIIMHCII47?1148?941?342?31.8?1.01.4?0.63.4?0.93.5?0.7M-Stat6-/-M-Stat6+/+Frequency (%)Frequency (%)Frequency (%)Frequency (%)CD45.1+and/or CD45.2+DCs (MHCII+CD11c+)MHCII-CD11c-MHCII-CD11c+106Cells106Cells106Cells106CellsM-Stat6+/+M-Stat6-/-A)B) 75 Figure 2.13 Myeloid-Specific STAT6 Depletion Does Not Alter Splenic DC Populations in Ldlr-/- Mice Following 8 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Spleens were homogenized and cells were stained with anti-MHCII and anti-CD11c antibodies for flow cytometry analysis of DC (MHCII+CD11c+), MHCII-CD11c+ cell, and MHCII-CD11c- cell populations. Dead cells, RBCs, and cells negative for both CD45.1 and CD45.2 were excluded. A) Representative dot plot and gating schemes of CD45.1/CD45.2 and DC populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency and absolute numbers of CD45+ cells, DCs, MHCII-CD11c+ cells, and MHCII-CD11c- cells in the spleen. Error bars represent SD. n = 5 mice for each group.   2.3.7 Myeloid-Specific STAT6 Depletion Does Not Affect NK Cell and Neutrophil Populations in the Spleen of Ldlr-/- Mice To study potential influences of myeloid STAT6 expression on NK cell and neutrophil development, spleens from M-Stat6+/+ and M-Stat6-/- mice were homogenized and cells were stained for flow cytometry analysis of NK cell (NK1.1+CD49b+) and neutrophil (CD11b+Gr-1hi) populations. Dead cells, RBCs, and cells negative for both CD45.1 and CD45.2 were excluded (8 weeks: Figure 2.8 A; 14 weeks: Figure 2.9 A). There were no statistically significant differences between M-Stat6-/- and M-Stat6+/+ mice in the frequency or absolute cell numbers of splenic NK cells or neutrophils following 8 or 14 weeks of atherogenic diet (8 weeks: Figure 2.8 B; 14 weeks: Figure 2.9 B). However, there was a trend towards increasing neutrophil frequency and absolute numbers in M-Stat6-/- mice compared to M-Stat6+/+ mice at both 8 and 14 week time points (Figure 2.8 B and Figure 2.9 B 3rd column). 76 CD45.1M-Stat6-/-M-Stat6+/+NK1.1NK1.1Gr-1Gr-140?1038?111.5?0.61.8?0.93.9?1.45.6?4.1CD45.2 CD49bCD11bCD45.1+and/or CD45.2+NK Cells (NK1.1+CD49b+)Neutrophils (CD11b+Gr-1hi)Frequency (%)Frequency (%)Frequency (%)106Cells106Cells106CellsA)B)M-Stat6+/+M-Stat6-/-8 Weeks Figure 2.14 Myeloid-Specific STAT6 Depletion Does Not Affect Splenic NK Cell and Neutrophil Populations in Ldlr-/- Mice Following 8 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Splenocytes were stained for flow cytometry analysis of NK cell (NK1.1+CD49b+) and neutrophil (CD11b+Gr-1hi) populations. Dead cells, RBCs, and cells negative for both CD45.1 and CD45.2 were excluded. A) Representative dot plot and gating schemes of CD45.1/CD45.2, NK cell, and neutrophil populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency and absolute numbers of CD45+ cells, NK cells, and neutrophils in the spleen. Error bars represent SD. n = 8 mice for each group.  77 CD45.1M-Stat6-/-M-Stat6+/+NK1.1NK1.1Gr-1Gr-156?1158?192.7?0.33.4?0.34.9?1.78.5?4.2CD45.2 CD49bCD11bCD45.1+and/or CD45.2+NK Cells (NK1.1+CD49b+)Neutrophils (CD11b+Gr-1hi)Frequency (%)Frequency (%)Frequency (%)106Cells106Cells106CellsA)B) M-Stat6+/+M-Stat6-/-14 Weeks Figure 2.15 Myeloid-Specific STAT6 Depletion Does Not Affect Splenic NK Cell and Neutrophil Populations in Ldlr-/- Mice Following 14 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 14 weeks prior to sacrifice. Splenocytes were stained for flow cytometry analysis of NK cell (NK1.1+CD49b+) and neutrophil (CD11b+Gr-1hi) populations. Dead cells, RBCs, and cells negative for both CD45.1 and CD45.2 were excluded. A) Representative dot plot and gating schemes of CD45.1/CD45.2, NK cell, and neutrophil populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency and absolute numbers of CD45+ cells, NK cells, and neutrophils in the spleen. Error bars represent SD. n = 6 mice for each group.  78 2.3.8 The Effects of Myeloid-Specific STAT6 Depletion on Lymphopoiesis in the Blood, Bone Marrow, and Spleens of Ldlr-/- Mice In addition to studying myelopoiesis, we also analyzed the effects of myeloid-specific STAT6 depletion on the development of lymphocytes in Ldlr-/- mice. Blood cells and splenocytes from M-Stat6+/+ and M-Stat6-/- mice were stained for flow cytometry analysis of CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8a+), and B cells (CD3-CD19+). Dead cells, RBCs, and cells negative for both CD45.1 and CD45.2 were excluded (8 weeks: Figure 2.16 A; 14 weeks: Figure 2.17 A). M-Stat6-/- mice fed an atherogenic diet for 8 weeks showed a slight decrease (by 12%) in splenic B cell frequencies compared to M-Stat6+/+ mice; however, this difference was not seen in the absolute numbers of splenic B cells. No differences in either CD4+ T cells or CD8+ T cells were detected at 8 weeks (Figure 2.16 B). Interestingly, we observed a similar decrease (by 18%) in splenic B cell frequencies in M-Stat6-/- mice compared to M-Stat6+/+ mice at 14 weeks. A noticeable trend towards a decrease in the absolute number of splenic B cells was also noted, though the difference did not reach statistical significance. In addition, at 14 weeks, the frequency of CD4+ T cells in the blood of M-Stat6-/- was 33% less than that of M-Stat6+/+ mice (Figure 2.17 B).  79   A)CD45.1CD3CD3M-Stat6-/-M-Stat6+/+96 397 111 310 38.6 1.88.8 2.417 713 4CD45.2CD3CD4 CD8a CD19CD3M-Stat6-/-M-Stat6+/+42 1040 1118 217 25.3 0.95.6 2.348 443 6CD45.1CD45.1CD45.1BloodSpleen8 Weeks 80  Frequency (%)Frequency (%)Frequency (%)Frequency (%)106Cells106Cells106Cells106CellsM-Stat6+/+M-Stat6-/-CD45.1+and/or CD45.2+CD4+T CellsB Cells (CD3-CD19+)CD8+T CellsFrequency (%)Frequency (%)Frequency (%)Frequency (%)SpleenSpleenBloodBloodB) 81 Figure 2.16 Lymphocyte Populations in the Blood and Spleens of M-Stat6+/+ and M-Stat6-/- Mice Following 8 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Blood cells and splenocytes were stained for flow cytometry analysis of CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8a+), and B cells (CD3-CD19+). Dead cells, RBCs, and cells negative for both CD45.1 and CD45.2 were excluded. A) Representative dot plots and gating schemes of CD45+ cell, CD4+ T cell, CD8+ T cell, and B cell populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency (blood and spleen) and absolute numbers (spleen) of CD45+ cells, CD4+ T cells, CD8+ T cells, and B cells. Error bars represent SD. n = 13 mice for blood. n = 8 mice for spleen. * p < 0.05 by two-tailed t-test.  82   A)CD45.1CD3CD3M-Stat6-/-M-Stat6+/+97 197 112 39 29.1 1.78.9 1.116 612 4CD45.2CD3CD4 CD8a CD19CD3M-Stat6-/-M-Stat6+/+59 1856 1215 213 314 213 252 444 4CD45.1CD45.1CD45.1BloodSpleen14 Weeks 83  Frequency (%)Frequency (%)Frequency (%)Frequency (%)106Cells106Cells106Cells106CellsM-Stat6+/+M-Stat6-/-CD45.1+and/or CD45.2+CD4+T CellsB Cells (CD3-CD19+)CD8+T CellsFrequency (%)Frequency (%)Frequency (%)Frequency (%)SpleenSpleenBloodBloodB) 84 Figure 2.17 Lymphocyte Populations in the Blood and Spleens of M-Stat6+/+ and M-Stat6-/- Mice Following 14 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 14 weeks prior to sacrifice. Blood cells and splenocytes were stained for flow cytometry analysis of CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8a+), and B cells (CD3-CD19+). Dead cells, RBCs, and cells negative for both CD45.1 and CD45.2 were excluded. A) Representative dot plots and gating schemes of CD45+ cell, CD4+ T cell, CD8+ T cell, and B cell populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency (blood and spleen) and absolute numbers (spleen) of CD45+ cells, CD4+ T cells, CD8+ T cells, and B cells. Error bars represent SD. n = 8-9 mice for blood. n = 3-6 mice for spleen. * p < 0.05, ** p < 0.01 by two-tailed t-test.    85 2.3.9 Myeloid-Specific STAT6 Depletion Does Not Affect Atherosclerosis in Ldlr-/- Mice  To study whether myeloid STAT6 plays significant roles in lesion formation in mice, M-Stat6+/+ and M-Stat6-/- mice were fed an atherogenic diet for 8 or 14 weeks and then sacrificed for analysis of atherosclerotic disease burden. For quantification of aortic surface area covered by lesions, entire aortas were sliced open longitudinally, pinned flat onto a dissection pad, and stained with Sudan IV to highlight atherosclerotic regions (Figure 2.18 B). The percentage of total aortic surface area covered by atherosclerotic lesion was measured using the ImagePro Plus software. There were no statistically significant differences in aortic lesion area between M-Stat6+/+ and M-Stat6-/- mice at either 8 or 14 weeks after the initiation of atherogenic diet (Figure 2.18 A).  To measure atherosclerotic plaque area, necrosis, and lipid content in the aortic root, hearts were collected at the time of sacrifice and the aortic root region of each heart was sectioned and stained with Oil Red O and/or H&E. The aortic root lesion areas, lipid content, and necrotic core areas were quantified by manual tracing using the Aperio ImageScope software. There were no statistically significant differences in the lesion area, lipid content, or necrotic core area between M-Stat6+/+ and M-Stat6-/- mice at either of the time points we measured (Figure 2.19 and Figure 2.20). 86 8 weeksA)B)% Plaque AreaM-Stat6+/+M-Stat6-/-M-Stat6+/+M-Stat6-/-14 weeks% Plaque AreaM-Stat6+/+M-Stat6-/-M-Stat6+/+M-Stat6-/- Figure 2.18 Myeloid-Specific STAT6 Depletion in Ldlr-/- Mice Does Not Influence Aortic Atherosclerosis in Mice Fed an Atherogenic Diet for 8 or 14 Weeks Mice were fed an atherogenic diet for 8 or 14 weeks prior to sacrifice. Entire aortas were excised, opened longitudinally, and stained with Sudan IV to reveal atherosclerotic areas (red regions). A) Each data point represents the plaque area of a single animal as a percentage of intimal surface area. Line and whiskers show the mean and SD of each group. B) Representative images of Sudan IV staining of aortas. Scale bars represent 5 mm. n = 14 for 8 weeks. n = 13 for M-Stat6+/+ 14 weeks. n = 15 for M-Stat6-/- 14 weeks. 87 Lesion Area (100 ?m2)8 weeks 14 weeksM-Stat6+/+M-Stat6-/-M-Stat6+/+M-Stat6-/-8 weeks 14 weeksM-Stat6-/-M-Stat6+/+A)C)B)8 weeks 14 weeksM-Stat6+/+M-Stat6-/-M-Stat6+/+M-Stat6-/-Lipid Content (%)  Figure 2.19 Myeloid-Specific STAT6 Depletion in Ldlr-/- Mice Does Not Significantly Influence Aortic Root Lesion Area or Lipid Content in Mice Fed an Atherogenic Diet for 8 or 14 Weeks Mice were fed an atherogenic diet for 8 or 14 weeks prior to sacrifice. Aortic roots were stained with Oil Red O. A) and B) Each data point represents the mean lesion area or lipid content, as a percentage of lesion area, of a single mouse. Line and whiskers show the mean and SD of each group C) Representative images of Oil Red O-stained aortic root sections. Scale bars represent 250 ?m. n = 14 for 8 weeks. n = 13 for M-Stat6+/+ 14 weeks. n = 15 for M-Stat6-/- 14 weeks.  88 A)B) 8 weeks 14 weeksM-Stat6-/-M-Stat6+/+++++M-Stat6-/-8 weeks 14 weeksM-Stat6+/+M-Stat6-/-M-Stat6+/+Necrotic Core (%)  Figure 2.20 Myeloid-Specific STAT6 Depletion in Ldlr-/- Mice Does Not Significantly Influence Aortic Root Necrosis in Mice Fed an Atherogenic Diet for 8 or 14 Weeks Mice were fed an atherogenic diet for 8 or 14 weeks prior to sacrifice. Aortic roots were stained with H&E. A) Each data point represents the mean necrotic core area of one mouse as a percentage of total lesion area. Line and whiskers show the mean and SD of each group. B) Representative images of H&E-stained aortic root sections. +, examples of necrotic areas. Scale bars represent 200 ?m. n = 14 for 8 weeks. n = 13 for M-Stat6+/+ 14 weeks. n = 15 for M-Stat6-/- 14 weeks.  89 2.4 Discussion IL-4 has been shown to up-regulate CD36 expression in the murine RAW264.7 macrophage cell line through PPAR-? activation.308 In this thesis, we showed that treatment with IL-4 increases cholesterol accumulation in murine BMDMs. Since STAT6 facilitates the transcription of PPAR-?-regulated genes in macrophages, 236 we hypothesized that IL-4-induced cholesterol accumulation would be diminished in STAT6-deficient BMDMs. Indeed, IL-4 treatment did not enhance cholesterol accumulation in Stat6-/- BMDMs. Although statistically significant differences were observed, the small magnitude of the difference leads me to question its biological importance.  The systemic knockout of STAT6 in the BALB/c mouse strain impairs the Th2 response and leads to an increase in atherosclerosis.158 Since the expression of STAT6 is ubiquitous, it is unclear which cell types are responsible for this change in phenotype. Therefore, we further explored the functions of STAT6 in murine atherogenesis by selectively decreasing STAT6 expression in the myeloid lineage of Ldlr-/- mice. To test whether we could effectively abolish STAT6 expression in myeloid cells, we transplanted a mixture of bone marrow cells composed of 90% Stat6-/-Rag1-/- plus 10% wild type bone marrow, from the congenic mouse strain, BoyJ, into lethally irradiated Ldlr-/- mice. Since bone marrow from mice deficient in Rag1 are incapable of producing mature T and B cells,310 all mature T and B cells found in the blood and spleen of recipient mice following recovery would have originated from the wild type donor (CD45.1 BoyJ) or from host cells that survived lethal irradiation (CD45.2). Through measuring the surface expression of CD45.1, the congenic marker found on BoyJ- 90 derived leukocytes, we were able to conclude that less than 10% of CD115+CD11b+ myeloid cells and greater than 95% of B cells in recipient Ldlr-/- mice were derived from the wild type donor. We also showed that the mRNA expression of Stat6 in the bone marrow from M-Stat6-/- mice was less than 10% of that seen in the bone marrow of M-Stat6+/+ mice.  Current studies have yet to reach a consensus on the involvement of IL-4 in atherosclerosis. However, there is evidence to suggest that the effects of IL-4 may be dependent upon the length of the atherogenic diet as well as the anatomical locations (for example, the aortic root compared to the aortic arch) at which atherosclerosis is measured.12, 15 Since STAT6 is activated in response to IL-4, it is plausible that STAT6 may exert differential effects on atherosclerosis in a pattern similar to IL-4. We therefore analyzed atherosclerosis in M-Stat6-/- mice at two different time points: 8 weeks and 14 weeks following the initiation of the atherogenic diet. The choice of these two time points was based on a survey of existing literature which revealed that 8 and 14 weeks are suitable early and intermediate times points, respectively, to assess atherosclerosis in Ldlr-/- mice.305, 306, 307  We first assessed the relative contribution of BoyJ-derived donor cells to the immune cell populations in recipient Ldlr-/- mice using CD45.1 and CD45.2 expression. Assuming complete ablation of the host myeloid compartment following lethal irradiation, the only source of Stat6+/+ myeloid cells would be from the wild type CD45.1+ BoyJ donor following bone marrow reconstitution; therefore, we used CD45.1 as a marker for myeloid STAT6 expression. In the ideal scenario, no more than 10% of myeloid cells in the recipient would stain positively for CD45.1 (Stat6+/+) or be negative  91 for CD45.2. Moreover, we expected all lymphocytes to be derived from the wild type CD45.1+ BoyJ donor and be CD45.1+ and CD45.2-. At both 8 and 14 weeks, blood and bone marrow monocytes; blood and spleen B cells; as well as spleen NK cells and neutrophils responded well to irradiation and reconstituted the host immune compartments in the correct proportions. Unexpectedly, we found that splenic monocytes (especially patrolling monocytes) and DCs (at 8 weeks) possessed high levels of CD45.1 expression and, where applicable, correspondingly low levels of CD45.2 expression, suggesting that a significant portion of these cells were derived from the wild type BoyJ bone marrow. In fact, the proportion of CD45.1+ monocytes and DCs exceeded the 10% of CD45.1+ bone marrow transplanted into the recipients. This phenomenon is independent of STAT6 expression, as both M-Stat6+/+ and M-Stat6-/- mice exhibit the same trend. The presence of Rag1 in BoyJ bone marrow is also unlikely to account for this difference, as Rag1-/- bone marrow is equally effective at repopulating myeloid cells as wild type bone marrow.312 Another distinction between Stat6+/+Rag1-/- or Stat6-/-Rag1-/- bone marrow and BoyJ bone marrow is the differential expression of CD45.1 and CD45.2 antigens. Importantly, a recent report showed, for the first time, that CD45.1+ and CD45.2+ cells are not identical in their capability to reconstitute CD45.2+ recipient mice.313 However, the same study noted that CD45.1+ cells are at a competitive disadvantage compared to CD45.2+ cells, which does not account for the enhanced frequency of CD45.1+ monocytes and DCs seen in the spleen of our recipient mice. Taken together, the reasons for increased CD45.1+ splenic cell populations in our mice need to be identified in future experiments.  92 Since Stat6+/+Rag1-/- or Stat6-/-Rag1-/- donors and Ldlr-/- recipients both express CD45.2, an important piece of missing data that will enhance the validity of our results is the direct assessment of STAT6 expression in the immune cells of our mice by RT-qPCR, Western blot, and flow cytometry. While we have shown in an earlier experiment that the mRNA expression of Stat6 in the bone marrow from M-Stat6-/- mice was less than 10% of that seen in the bone marrow of M-Stat6+/+ mice, data for Stat6 expression in specific cell types is lacking. Although there is ample evidence to suggest that radiation doses of 7 to 13 Gy are myeloablative in C57BL/6 mice,314?318 it may be desirable to eliminate the possibility that technical challenges may have caused incomplete ablation of host bone marrow, which are wild type for STAT6. Future studies will attempt to incorporate a STAT6 stain into our flow cytometry protocol, which will allow the clear assessment of cell type-specific STAT6 expression in the blood, lymphoid organs, and atherosclerotic lesions of transplanted recipients. Blood and spleen CD4+ T cells and CD8+ T cells appear to be resistant to radiation. Indeed, at 14 weeks following atherogenic diet, nearly half of the CD8+ T cells in the blood and the spleen expressed CD45.1. The observation that host T cells can persist despite lethal doses of radiation is supported by a previous study that showed CD4+ and CD8+ T cells are less susceptible to radiation-induced apoptosis compared to monocytes and B cells.319 It has also been shown that even at 6 months post-transplantation, a significant proportion of recipient CD3+ cells can be detected in various organs, including the blood and the spleen.320 The majority of these host-derived T cells originate from the host thymus, with a minor population coming from mature, radio-resistant peripheral T cells.320 However, the reasons why certain T cell  93 populations survive lethal radiation remain unclear. Importantly, since both donor and host T cells are wild type for STAT6 expression, the persistence of host T cells is unlikely to affect our results. The immune system plays pivotal roles in all stages of atherosclerosis. Prior to measuring atherosclerotic disease burden in M-Stat6+/+ and M-Stat6-/- mice following 8 or 14 weeks of atherogenic diet, we characterized the changes in major immune cell populations, including inflammatory and patrolling monocytes; dendritic cells; neutrophils; natural killer cells; CD4+ T cells; CD8+ T cells; and B cells,  in these mice. We found that myeloid-specific STAT6 depletion led to a very minor phenotype in Ldlr-/- mice with regards to hematopoiesis. In M-Stat6-/- mice fed the atherogenic diet for 8 weeks, the only statistically significant differences were slight increases in splenic patrolling monocyte frequencies and slight decreases in the splenic B cell frequency. At 14 weeks, there was an increase in the frequency of total spleen monocytes and decreases in the frequencies of blood CD4+ T cells and spleen B cells. In no instance, at either time point, were the differences in frequency reflected in the absolute cell counts. However, since accurate absolute cells counts depend on consistent tissue handling and processing, I consider changes in frequency to be more reliable than changes in absolute cell counts. The decrease in B cell frequency following STAT6 depletion was the only difference present at both 8 and 14 weeks.  Although the mechanism of this phenomenon was not further explored in this thesis, I speculate that this difference may be due to increased B cell apoptosis in M-Stat6-/- mice. Indeed, IL-4 has been shown to stimulate the STAT6-dependent production of IL-4 by murine dendritic cells. In addition, IL-4 promotes splenic B cell survival by up-regulating anti- 94 apoptotic proteins such as Bcl-xL.321, 322 It is possible that myeloid STAT6 depletion reduced IL-4 production by myeloid cells which in turn increased the susceptibility of B cells to apoptosis. Despite the statistically significant changes in monocyte, CD4+ T cell, and B cell frequencies following myeloid STAT6-depletion, the modest differences led me to conclude that myeloid STAT6 deficiency does not induce major phenotypic alterations in hematopoiesis in our experimental model. Therefore, it is also unlikely that these immune cell population changes influenced atherosclerosis in our mice. Our observation that STAT6 is not a major factor in hematopoiesis is consistent with a previous report that showed no differences in neutrophil, monocyte, and lymphocyte counts in Stat6-/- mice compared to wild type mice.239 Intriguingly, the authors noticed that a number of Stat6-/- mice had elevated absolute neutrophil counts in the blood, leading them to hypothesize that Stat6-/- bone marrow may be capable of producing large numbers of neutrophils in response to inflammatory stimuli.239 Hypercholesterolemia induces neutrophilia in the blood, bone marrow, and spleen, which is associated with increased atherosclerosis.323, 324 In our study, we also observed a clear tendency towards increased neutrophil frequency and absolute counts in the spleens of M-Stat6-/- mice at both time points; however, the differences did not reach statistical significance. It is important to point out that our results do not reveal whether circulating neutrophil numbers in the blood, or neutrophil content in atherosclerotic lesions, are altered in M-Stat6-/- mice in response to the atherogenic diet; this may be confirmed via flow cytometry analysis of blood and dissolved lesions. We hypothesized that Ldlr-/- mice deficient in myeloid STAT6 would develop larger and more advanced atherosclerotic lesions compared to controls. To test this  95 hypothesis, we measured atherosclerosis at two separate time points, as mentioned previously, as well as two anatomical locations (the aortic root and the entire aorta) to account for potential age- and site-dependent variations in lesions formation. Plasma total cholesterol levels were not altered in myeloid STAT6-deficient mice. Compared to mice fed an atherogenic diet for 8 weeks, the en face aortic lesion areas in 14 week mice were significantly greater. However, contrary to our hypothesis, STAT6-depletion did not affect the aortic lesion area at either time point. The degree of aortic atherosclerosis observed in our mice is consistent with a recent study that elegantly investigated the extent of lesion formation in Ldlr-/- mice fed a high fat diet for various lengths of time.325 Similar to the trend observed in the aorta, aortic root lesion area; lipid content; and necrotic core size were enlarged with increasing time on the atherogenic diet; however, no phenotypic differences were detected between M-Stat6+/+ and M-Stat6-/- mice at either the 8 or 14 week time points. Necrotic core size and lipid content are common markers used to identify advanced and vulnerable atherosclerotic lesions in humans and mice.326?332 One must keep in mind, however, that the classification of vulnerable lesions in mice is based on the observation that these parameters are associated with plaque rupture in humans, as murine lesions are, by nature, stable.303 Despite rejecting our hypothesis that myeloid STAT6 deficiency will exacerbate atherosclerosis in Ldlr-/- mice, our results are consistent with previous reports that IL-4 does not influence murine fatty streak formation or atherosclerotic lesion area.14, 242 An important question not addressed in our current study is whether the deficiency of STAT6 in myeloid cells directly affects immune cell populations within  96 atherosclerotic lesions. We were especially interested in whether lesional alternatively-activated macrophage populations were perturbed in the absence of STAT6. We attempted to answer this question by staining our formalin-fixed frozen aortic root sections with various antibodies, including anti-F4/80; -Ym-1; -LDLR; CD45; MOMA-2; and Mic-1, using various antigen retrieval methods, incubation times, and detection methods. However, due to the harsh nature of formalin fixation and the low tolerance of frozen tissue to harsh antigen retrieval regimes, we were unable to achieve any quantifiable staining in our sections. A repeat of the current study using a milder fixation protocol (i.e. frozen sections without formalin fixation) will be required to address this issue. In this chapter, we demonstrated that STAT6 is essential for IL-4-induced cholesterol accumulation in murine BMDMs but this did not translate into phenotypic changes in atherosclerosis in vivo. Furthermore, we demonstrated that myeloid-specific STAT6 deficiency does not induce major phenotypic alterations in Ldlr-/- mice with regards to hematopoiesis and atherosclerosis, which limits the value of myeloid STAT6 as a potential therapeutic target in atherosclerotic diseases.  97 CHAPTER 3: Investigation of STAT4 in a Mouse Model of Atherosclerosis 3.1 Background The IL-12/STAT4 signaling pathway mediates the differentiation of CD4+ T cells into Th1 cells and stimulates IFN-? production by NK cells, T cells, B cells, and APCs.265?268 The majority of current studies point toward a pro-atherogenic role for IL-12; indeed, intraperitoneal injections of IL-12 enhances lesion size and complexity whereas IL-12 blockade or knockout attenuates atherosclerosis in mice.12, 13, 289 Interestingly, similar to IL-4, the effects of IL-12 on atherosclerosis appear to be time-dependent. Notably, aortic root atherosclerosis is reduced in Il-12-/-ApoE-/- mice at 30 but not at 45 weeks of age compared to ApoE-/- mice, suggesting that IL-12 is an important cytokine during early lesion formation.12 However, it is important to keep in mind that IL-12 is capable of up-regulating the expression of a number of genes even in the absence of STAT4.333 Accordingly, one must exercise caution when extrapolating the functional significance of STAT4 in atherosclerosis using data from IL-12. In addition to IL-12, STAT4 is also phosphorylated, albeit to a lesser extent, in response to other cytokines in the IL-12 family, including IL-23; IL-27; and IL-35.258, 259, 260 IL-23 is an inflammatory cytokine involved in the development of Th17 cells while IL-27 and IL-35 are immunoregulatory cytokines.334 Although the importance of IL-23 and IL-35 in murine atherosclerosis have not been studied, IL-27 has recently been shown to attenuate atherosclerosis in Ldlr-/- mice by inhibiting myeloid cell recruitment and activation, decreasing T cell accumulation, and preventing inflammatory cytokine production during atherosclerosis.290, 291 Therefore, the ultimate consequence of STAT4  98 activation on atherosclerosis may be the result of a constant tug-of-war between pro- and anti-atherogenic forces acting through the STAT4 signaling pathway. Despite robust evidence that cytokines in the STAT4 signaling pathway play important roles in atherosclerosis, STAT4 itself has not been studied directly. The lack of significant phenotypic changes following myeloid STAT6 depletion prompted us to expand beyond myeloid cells for our STAT4 studies. In the following chapter, I outline the experiments conducted to investigate the ways in which STAT4 deficiency in 1) all hematopoietic cells or 2) bone marrow-derived myeloid cells affects atherosclerosis development in Ldlr-/- mice. The specific aims for this chapter are 1. To assess the effects of total hematopoietic system STAT4 depletion on atherosclerotic lesion size and complexity in Ldlr-/- mice fed an atherogenic diet. 2. To assess the effects of myeloid STAT4 depletion on atherosclerotic lesion size and complexity in Ldlr-/- mice fed an atherogenic diet. 3. To determine whether total hematopoietic system or myeloid-specific STAT4 affects the immune cell populations in the blood, bone marrow, and spleen of Ldlr-/- mice fed an atherogenic diet. Since the primary function of STAT4 is mediating the IL-12-induced production of IFN-? by inflammatory cells, and both IL-12 and IFN-? have been shown to be atherogenic in mice,335 we hypothesized that Ldlr-/- mice transplanted with Stat4-/- bone marrow (designated Stat4-/-) or Stat4-/-Rag1-/- bone marrow (myeloid STAT4 deficient, designated M-Stat4-/-) would be protected from atherosclerosis compared to control animals. We speculated that decreases in atherosclerosis following STAT4 depletion  99 would be a consequence of a blunted Th1 response with decreased inflammatory cell recruitment and infiltration and reduced inflammatory cytokine production within lesions. Furthermore, since IL-12 stimulates IFN-? production in both myeloid and lymphoid cells,265?268 we hypothesized that total hematopoietic system STAT4 deficient mice would be more protected from atherosclerosis compared to mice in which STAT4 expression is reduced only in the myeloid compartment.  As is the case with STAT6, STAT4 appears to be dispensable for the development of mature hematopoietic cells under basal conditions.240, 265, 273 Unlike STAT6 knockout mice, however, STAT4 knockout mice have reduced numbers, as well as cycling activity, of hematopoietic progenitor cells in the bone marrow and spleen.240 Since immune cells recruited to atherosclerotic lesions must eventually be replenished by progenitor cells, decreased availability of progenitor cells following STAT4 depletion may decrease the number of cells available for recruitment. Therefore, we examined whether inflammation due to diet-induced hypercholesterolemia would reveal differences in hematopoiesis in Stat4-/- or M-Stat4-/- mice compared to wild type mice. Interestingly, the effects of STAT4 depletion on progenitor cell homeostasis appear to be T cell-dependent, as T cell-specific STAT4 expression restores normal progenitor cell numbers and cycling status. 240 Therefore, we also expected that any alterations in hematopoiesis would be more pronounced in total hematopoietic system STAT4 depletion compared to myeloid STAT4 depletion.     100 3.2 Materials and Methods 3.2.1 Animals Stat4-/- mice on a C57BL/6 background were rederived at the Centre for Disease Modeling at the University of British Columbia from Stat4-/- embryos generously donated by Dr. Andrew Churg. Stat4-/- mice were crossed with Rag1-/- mice to generate the Stat4-/-Rag1-/- genotype. The animals were housed under standard pathogen-free conditions. All experimental protocols were performed in compliance with the University of British Columbia Animal Care Committee and the Canadian Council of Animal Care.  3.2.2 Bone Marrow Transplantation To induce bone marrow aplasia, 8 week-old female Ldlr-/- mice on a C57BL/6 background were exposed to two doses of 6.5 Gy total body irradiation 4 hours apart. To generate total hematopoietic system STAT4-deficient animals, irradiated mice were transplanted with 1 x 107 total bone marrow cells from Stat4-/- C57BL/6 mice. Mice transplanted with wild type C57BL/6 bone marrow were used as controls. For myeloid-specific STAT4 depletion, we transplanted Ldlr-/- mice with 90% of Stat4-/-Rag1-/- bone marrow plus 10% of wild type bone marrow from the congenic BoyJ strain. Mice transplanted with 90% of Stat4+/+Rag1-/- and 10% wild type BoyJ bone marrow were used as controls. Following bone marrow transplantation, animals were given normal chow diet and fresh water ad libitum for 6 weeks to allow for the full reconstitution of the immune compartments with donor bone marrow (Figure 3.1).     101 3.2.3 Induction of Atherosclerosis Following the recovery period, the chow diet was substituted with an atherogenic diet (TD.94059, Harlan Teklad) containing 15.8% fat (w/w) and 1.25% cholesterol (w/w) for 8 weeks (Figure 3.1). Mice were monitored twice per day and weighed twice per week for the duration of the diet.  Stat4-/-Rag1-/-orStat4+/+Rag1-/-(CD45.2)Wild Type BoyJ (CD45.1)Lethally-Irradiated Ldlr-/-10%90%SacrificeAndAnalysisAtherogenicDiet8 weeksRecovery6 weeksStat4-/-orWild Type C57BL/6Lethally-Irradiated Ldlr-/-SacrificeAndAnalysisAtherogenicDiet8 weeksRecovery6 weeksA)B)   102 Figure 3.1 Experimental Design for in vivo STAT4 Studies. Ldlr-/- mice were lethally irradiated and reconstituted with A) Stat4-/- or wild type C57BL/6 bone marrow for total hematopoietic system STAT4 depletion, or B) 90% Stat4-/-Rag1-/- or Stat4+/+Rag1-/- bone marrow plus 10% wild type BoyJ bone marrow for myeloid-specific STAT4 depletion. Following a 6-week recovery period, the mice were fed a high fat diet for 8 weeks prior to sacrifice.  3.2.4 Atherosclerosis Measurements and Flow Cytometry Please refer to pages 40-46 of this thesis for the methods used to quantify atherosclerotic lesion area and complexity; stain and analyze flow cytometry; and perform statistical analyses.  3.3 Results 3.3.1 Body Weight and Total Plasma Cholesterol Measurements in Total Hematopoietic System STAT4 Deficient Mice During the induction of atherosclerosis, mice were weighed twice per week to monitor weight changes. At the time of sacrifice, plasma was collected and total plasma cholesterol was measured enzymatically. There were no statistically significant differences in total plasma cholesterol levels (Figure 3.2 C) between mice transplanted with wild type or Stat4-/- bone marrow.  3.3.2 Changes in Monocyte Populations in Hematopoietic System STAT4-Deficient Mice Following 8 Weeks of Atherogenic Diet To determine whether STAT4 expression affects monocyte development in mice, we measured total (CD11b+CD115+), inflammatory (Ly6ChiCD62L+), and patrolling (Ly6ClowCD62L-) monocyte populations in the blood, bone marrow, and spleens of mice transplanted with wild type or Stat4-/- bone marrow (Figure 3.3). In mice transplanted  103 with Stat4-/- bone marrow, blood inflammatory monocyte frequencies were decreased while blood patrolling, splenic total, and splenic patrolling monocyte frequencies were increased (Figure 3.3 B 1st and 4th row). The absolute cell number of bone marrow inflammatory monocytes was also increased in Stat4-/- mice, although the frequency remained unchanged (Figure 3.3 B 3rd row).  104 B)A)C)10 20 30 40 50-2-101230 10 20 30 40 501618202224Weight (g)Days on atherogenic dietWeight Grain (g)Days on atherogenic dietCholesterol (mg/dL)Wild Type Stat4-/-Wild Type Stat4-/- Figure 3.2 Body Weight and Total Plasma Cholesterol Measurements in Total Hematopoietic System STAT4 Deficient Mice Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. A) and B) Total body weight (A) and weight gain (B) during the induction of atherosclerosis. C) Enzymatic detection of blood plasma cholesterol content at the time of sacrifice. Error bars represent SD. n = 14 mice for each group. 105 Stat4-/-SSCLy6C46 942 9CD11b7.8 3.4PIWild TypeSSCLy6C56 632 5CD11b6.7 2.5PIStat4-/-SSCLy6C80 512 4CD11b13 2PIWild TypeSSCLy6C79 412 3CD11b13 3PIWild TypeSSCLy6C40 443 5CD11b6.3 1.0PIStat4-/-SSCFSC CD62LLy6C35 648 6CD115CD11b7.3 1.1FSCPIBloodBone MarrowSpleenA) 106         B)Total Monocytes (CD11b+CD115+)Frequency (%)Inflammatory Monocytes (Ly6ChiCD62L+)Frequency (%)Patrolling Monocytes (Ly6ClowCD62L-)Frequency (%)Frequency (%)Frequency (%)Frequency (%)106Cells106Cells106CellsFrequency (%)Frequency (%)Frequency (%)106Cells106Cells106CellsBloodBone MarrowSpleenWild Type Stat4-/- 107 Figure 3.3 Changes in Monocyte Populations in Total Hematopoietic System STAT4-Deficient Mice Following 8 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Blood, bone marrow, and spleen cells were stained for flow cytometry analysis of total monocytes (CD11b+CD115+), inflammatory monocytes (Ly6ChiCD62L+), and patrolling monocytes (Ly6ClowCD62L-). A) Representative dot plots and gating schemes of scatter, live, and monocyte populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency (blood, bone marrow, and spleen) and absolute numbers (bone marrow and spleen) of total, inflammatory, and patrolling monocytes. Error bars represent SD. n = 11-12 mice for each group. * p < 0.05, ** p < 0.01 by two-tailed t-test.    3.3.3 Total Hematopoietic System STAT4 Deficiency Reduces DC Frequency in the Spleens of Ldlr-/- Mice To assess changes in splenic DC populations in mice transplanted with Stat4-/- bone marrow, spleens were homogenized and stained with anti-MHCII and anti-CD11c antibodies for flow cytometry analysis of DC (MHCII+CD11c+) populations. Cells negative for CD45.2 were excluded (Figure 3.4 A). Compared to mice transplanted with wild type bone marrow, mice transplanted with Stat4-/- bone marrow show a reduction in the frequency, but not absolute cell numbers, of DCs in the spleen (Figure 3.4 B 1st row). In addition, the frequency of a population of MHCII-CD11c- cells was significantly increased in the spleen of Stat4-/- transplanted mice (Figure 3.4 B 3rd row). However, it is yet unknown what type of cells this group represents and further characterization must be performed.  108   CD45.2 CD11cMHCIIMHCII52?548?441?550?40.1?0.10.2?0.14.9?0.74.3?0.7Stat4-/-Wild TypeFrequency (%)Frequency (%)Frequency (%)CD45.2+DCs (MHCII+CD11c+)MHCII-CD11c-106Cells106Cells106CellsWild Type Stat4-/-A)B) 109 Figure 3.4 Total Hematopoietic System STAT4 Deficiency Reduces DC Frequency in the Spleens of Ldlr-/- Mice Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Spleens were homogenized and stained with anti-MHCII and anti-CD11c antibodies for flow cytometry analysis of DC (MHCII+CD11c+) populations. Dead cells, RBCs, and cells negative for CD45.2 were excluded. A) Representative plots and gating schemes of CD45.2+ and DC populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency and absolute numbers of CD45.2+ cells, DCs and MHCII-CD11c- cells in the spleen. Error bars represent SD. n = 11 mice for each group. * p < 0.05, *** p < 0.001 by two-tailed t-test.   3.3.4 Total Hematopoietic System STAT4 Deficiency Enhances NK Cell and Neutrophil Populations in the Spleens of Ldlr-/- Mice Next, we determined whether STAT4 expression influences NK cell and neutrophil development in mice. Spleens were homogenized and cells were stained for flow cytometry analysis of NK cell (NK1.1+CD49b+) and neutrophil (CD11b+Gr-1hi) populations. Dead cells, RBCs, and cells negative for CD45.2 were excluded (Figure 3.5 A). In mice transplanted with Stat4-/- bone marrow, there was an increase in the frequency as well as absolute cell number of NK cells compared to wild type mice. In addition, the frequency of neutrophils in mice transplanted with Stat4-/- bone marrow was also significantly elevated. There was a trend towards increasing absolute neutrophil numbers in mice transplanted with Stat4-/- bone marrow compared to wild type mice; however, the difference did not reach statistical significance (Figure 3.5 B).    110 Stat4-/-Wild TypeNK1.1NK1.1Gr-1Gr-152?647?61.0?0.31.2?0.28.3?1.612.5?4.1CD45.2 CD49bCD11bCD45.1+and/or CD45.2+NK Cells (NK1.1+CD49b+)Neutrophils (CD11b+Gr-1hi)Frequency (%)Frequency (%)Frequency (%)106Cells106Cells106CellsA)B)Wild Type Stat4-/- Figure 3.5 Total Hematopoietic System STAT4 Deficiency Enhances NK Cell and Neutrophil Populations in the Spleen Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Spleens were stained for flow cytometry analysis of NK cell (NK1.1+CD49b+) and neutrophil (CD11b+Gr-1hi) populations. Dead cells, RBCs, and cells negative for CD45.2 were excluded. A) Representative plots and gating schemes of CD45.2+, NK cell, and neutrophil populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency and absolute numbers of CD45.2+ cells, NK cells, and neutrophils in the spleen. Error bars represent SD. n = 11 mice for each group. * p < 0.05, ** p < 0.01 by two-tailed t-test.  111 3.3.5 Lymphocyte Development is Impaired in Total Hematopoietic System STAT4-Deficient Mice   To reveal potential effects of STAT4 expression on lymphopoiesis, blood cells and splenocytes were stained for flow cytometry analysis of CD4+ T cells, CD8+ T cells, and B cells. Dead cells, RBCs, and cells negative for CD45.2 were excluded (Figure 3.6 A). In mice transplanted with Stat4-/- bone marrow, there was a significant reduction of CD8+ T cell frequencies in the blood and absolute numbers in the spleens compared to mice transplanted with wild type bone marrow (Figure 3.6 B). Moreover, the absolute cell count of B cells was lower in the spleens of total hematopoietic system STAT4-deficient mice (Figure 3.6 B). The most dramatic difference, however, was seen in the CD4+ T cell population. The lack of STAT4 greatly impairs the development of CD4+ T cells, as there were highly significant and consistent reductions in the frequency of CD4+ T cells in the blood and spleens of mice transplanted with Stat4-/- bone marrow. This difference was further reflected in the absolute cell numbers of splenic CD4+ T cells (Figure 3.6 B).  112  A)CD3CD3Stat4-/-Wild Type96 297 111 27 36.3 1.74.1 1.559 660 10CD45.2CD3CD4 CD8a CD19CD3Stat4-/-Wild Type51 642 87.6 1.25.6 1.25.2 1.34.5 1.753 449 5BloodSpleen 113 Frequency (%)Frequency (%)Frequency (%)Frequency (%)106Cells106Cells106Cells106CellsWild Type Stat4-/-CD45.2+CD4+T CellsB Cells (CD3-CD19+)CD8+T CellsFrequency (%)Frequency (%)Frequency (%)Frequency (%)SpleenSpleenBloodBloodB)  114 Figure 3.6 Lymphocyte Development is Impaired in Total Hematopoietic System STAT4-Deficient Mice Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Blood cells and splenocytes were stained for flow cytometry analysis of CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8a+), and B cells (CD3-CD19+). Dead cells, RBCs, and cells negative for CD45.2 were excluded. A) Representative plots and gating schemes of CD45.2+ cell, CD4+ T cell, CD8+ T cell, and B cell populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency (blood and spleen) and absolute numbers (spleen) of CD45.2+ cells, CD4+ T cells, CD8+ T cells, and B cells. Error bars represent SD. n = 8 mice for blood. n = 11 mice for spleen. * p < 0.05, *** p < 0.001 by two-tailed t-test.    115 3.3.6 Total Hematopoietic System STAT4 Deficiency Exacerbates Atherosclerosis in Ldlr-/- Mice To elucidate the functional significance of STAT4 in atherosclerotic lesion formation in mice, Ldlr-/- mice transplanted with Stat4-/- bone marrow were given an atherogenic diet for 8 weeks to induce atherosclerosis. To measure lesion formation in the aorta, entire aortas were excised at the time of sacrifice, cut open longitudinally, pinned flat onto a dissection pad, and stained with Sudan IV to highlight atherosclerotic areas (Figure 3.7 B). The percentage of total aorta area covered by atherosclerotic lesions was then quantified using the ImagePro Plus software. We found that mice transplanted with Stat4-/- bone marrow showed a dramatic 60% increase in the aortic lesion area compared to mice transplanted with wild type bone marrow (Figure 3.7 A). To measure aortic root atherosclerotic lesion area, lipid content, and necrosis in bone marrow STAT4-deficient Ldlr-/- mice, aortic roots were sectioned, stained with Oil Red O or H&E, and then scanned and quantified using the Aperio ImageScope. There was a very significant 56% increase in aortic root lesion area (Figure 3.8 A and D) and a 49% increase in aortic root lesion necrosis (Figure 3.8 C and E) compared to mice transplanted with wild type bone marrow. There were no differences in the lipid content between wild type and Stat4-/- bone marrow-transplanted mice. 116 Stat4-/-Wild TypeA) B)% Plaque AreaWild Type Stat4-/- Figure 3.7 Total Hematopoietic System STAT4 Deficiency in Ldlr-/- Mice Increases Aortic Atherosclerosis Lesion Area Following 8 Weeks of Atherogenic Diet. Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Entire aortas were excised, opened longitudinally, and stained with Sudan IV to reveal atherosclerotic areas (red regions). A) Each data point represents the plaque area of a single animal as a percentage of intimal surface area. Line and whiskers show the mean and SD of each group. B) Representative images of Sudan IV staining of aortas. Scale bars represent 5mm. n = 13 for each group. **, p < 0.01 by two-tailed t-test.   117       Wild TypeStat4-/-Oil Red OH+E++A)C)D)E)B)Lesion Area (100 ?m2)Lipid Content (%)Necrotic Core (%)Wild Type Stat4-/-Wild Type Stat4-/-Wild Type Stat4-/-Wild TypeStat4-/- 118 Figure 3.8 Total Hematopoietic System STAT4 Deficiency in Ldlr-/- Mice Increases Aortic Root Lesion Area and Necrosis Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Aortic roots were stained with Oil Red O or H&E. A), B), and C) Each data point represents the mean lesion area, necrotic core area, or lipid content of a single mouse. Line and whiskers show the mean and SD of each group. D) and E) Representative images of Oil Red O or H&E-stained aortic root sections. +, examples of necrotic areas. Scale bars represent 250 ?m in D) and 200 ?m in E). n = 13 per group. **, p < 0.01; ****, p < 0.0001 by two-tailed t-test.    3.3.7 Body Weight and Total Plasma Cholesterol Measurements M-Stat4-/- Mice During the induction of atherosclerosis, mice were weighed twice per week to monitor weight changes. At the time of sacrifice, plasma was collected and total cholesterol was measured enzymatically. There were no significant differences in total plasma cholesterol levels (Figure 3.9 C) between M-Stat4-/- and M-Stat4+/+ mice.    119 M-Stat4+/+Weight (g)Days on atherogenic diet0 20 40 60161820222420 40 60-3-2-10123Weight Grain (g)Days on atherogenic dietCholesterol (mg/dL)M-Stat4+/+M-Stat4-/-B)A)C)M-Stat4-/- Figure 3.9 Body Weight and Total Plasma Cholesterol Measurements M-Stat4-/- Mice Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. A) and B) Total body weight (A) and weight gain (B) during the induction of atherosclerosis. C) Enzymatic detection of blood plasma cholesterol content at the time of sacrifice. Error bars represent SD. n = 14 mice for M-Stat4-/- and n = 13 for M-Stat4+/+.  120 3.3.8 Assessment of Bone Marrow Reconstitution in M-Stat4+/+ and M-Stat4-/- Mice To assess the relative contribution of BoyJ donor-derived cells in the blood, bone marrow, and spleens of M-Stat4+/+ and M-Stat4-/- mice, we examined the cellular expression of CD45.1 and/or CD45.2 in these tissues by flow cytometry. As mentioned in the previous chapter, since only cells derived from wild type BoyJ donors express CD45.1, the levels of CD45.1 and CD45.2 were used as an indirect marker for Stat4 expression. We found that less than 5% of Ly6ChiCD62L+ inflammatory and 10% of Ly6ClowCD62L- patrolling monocytes in the blood expressed CD45.1 (Figure 3.10 A and B). Furthermore, 95% of CD11b+ myeloid cells in the blood were CD45.2+ (Figure 3.10 C and D). In blood T and B cells, approximately 75% of CD4+ T cells, 60% of CD8+ T cells, and 95% of B cells were CD45.1+ (Figure 3.10 A and B). Similarly, the proportion of CD4+ T cells, CD8+ T cells, and B cells in the blood that lacked CD45.2 expression was approximately 75%, 60%, and 95%, respectively (Figure 3.10 C and D). Myeloid reconstitution in the bone marrow was reminiscent of that in the blood, with only ~5% of monocytes being CD45.1+ (Figure 3.11 A and B) while greater than 95% of CD11b+ myeloid cells were positive for CD45.2 (Figure 3.11 C and D). With the exception of splenic patrolling monocytes in M-Stat4-/- mice, which were 86?6% CD45.1-, >95% of monocytes in the spleen lacked CD45.1 expression (Figure 3.12 A and C). In splenic DCs, ~80% of MHCII+CD11c+ DCs were CD45.1- while ~90% expressed CD45.2. In MHCII-CD11c+ cells, however, only ~65% were CD45.1- and CD45.2+. In addition, >90% of MHCII-CD11c- cells were CD45.1- and CD45.2+ (Figure 3.12). Greater than 80% of NK1.1+CD49b+ NK cells in the spleen were devoid of  121 CD45.1 expression and >90% of them were positive for CD45.2. Finally, ~90% of CD11b+Gr-1hi neutrophils were CD45.1- and >95% of them were CD45.2+ (Figure 3.12).  In splenic T and B cells, ~70% of CD4+ T cells, ~60% of CD8+ T cells and >90% of B cells were CD45.1+ (Figure 3.12 A and C). At the same time, ~65% of CD4+ T cells, 60% of CD8+ T cells, and 90% of B cells were CD45.2- (Figure 3.12 B and D). Since T and B cells can only be derived from the wild type BoyJ donor or from radioresistant host cells, all T and B cells found in recipient mice following reconstitution are wild type for STAT4 regardless of whether they are CD45.1+ or CD45.2+.  122  A)96 2 4 2Ly6ChiCD62L+Ly6ClowCD62L-93 5 7 593 3 7 396 2 4 2CD3+CD4+CD3-CD19+CD3+CD8a+24 8 76 8 43 5 57 5 5 2 95 227 12 73 1239 12 61 12 5 4 95 4M-Stat4-/-M-Stat4+/+CD45.1CD45.1-CD45.1+CD3+CD4+CD3+CD8a+CD3-CD19+M-Stat4+/+Frequency (%)Ly6ClowCD62L-Ly6ChiCD62L+Frequency (%)M-Stat4-/-CD3+CD4+CD3+CD8a+CD3-CD19+Ly6ClowCD62L-Ly6ChiCD62L+B)  123   Figure 3.10 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Blood of M-Stat4+/+ and M-Stat4-/- Mice  Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Blood CD11b+ cells, monocytes (Ly6C/CD62L) and lymphocytes (CD3+CD4+, CD3+CD8a+, CD3-CD19+) were stained with anti-CD45.1 and/or anti-CD45.2 antibodies for flow cytometry. A) and C) Representative histograms and gating schemes of CD45.1+ (A) and CD45.2+ (C) populations for each cell type. The frequency of events within each gated region ? SD is shown. B) and D) Bar graphs showing CD45.1 (B) and CD45.2 (D) expression of each cell type in the blood. Error bars represent SD. n = 8-10 mice for each cell type. C)CD45.2-CD45.2+M-Stat4+/+Frequency (%)Frequency (%)M-Stat4-/-D)020406080100020406080100CD3+CD4+CD3+CD8a+CD3-CD19+CD11b+CD3+CD4+CD3+CD8a+CD3-CD19+CD11b+CD45.2M-Stat4+/+M-Stat4-/-CD3+CD4+ CD3-CD19+CD3+CD8a+75 9 25 972 12 28 1256 5 44 5 94 3 6 393 4 7 461 12 39 12CD11b+6 4 94 45 2 95 2 124  Figure 3.11 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Bone Marrow of M-Stat4+/+ and M-Stat4-/- Mice  Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Bone marrow monocytes (Ly6C/CD62L) and CD11b+ cells were stained with anti-CD45.1 antibodies for flow cytometry. A) and C) Representative histograms and gating schemes of CD45.1+ (A) and CD45.2+ (C) cells. The frequency of events within each gated region ? SD is shown. B) and D) Bar graphs showing CD45.1 (B) and CD45.2 (D) expression of each cell type in the bone marrow. Error bars represent SD. n = 12-14 for each group. 0255075100125M-Stat4+/+M-Stat4-/-M-Stat4+/+CD45.1Ly6ChiCD62L+98 1 2 1Ly6ClowCD62L-95 1 5 193 2 7 297 2 3 2Frequency (%)Ly6ClowCD62L-Ly6ChiCD62L+M-Stat4-/-Ly6ClowCD62L-Ly6ChiCD62L+Frequency (%)CD45.1-CD45.1+A) B)M-Stat4-/-M-Stat4+/+CD45.2CD11b+2 1 98 13 3 97 3CD45.2-CD45.2+C) D)M-Stat4+/+Frequency (%)CD11b+M-Stat4-/- 125 Ly6ChiCD62L+Ly6ClowCD62L-MHCII+CD11c+MHCII-CD11c-MHCII-CD11c+CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiM-Stat4+/+98 1 2 1 95 1 5 2 80 8 20 8 94 4 6 4 63 7 37 729 9 71 9 37 8 63 8 7 3 93 3 82 7 18 7 88 4 12 3Ly6ChiCD62L+Ly6ClowCD62L-MHCII+CD11c+MHCII-CD11c-MHCII-CD11c+CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiCD45.1M-Stat4-/-95 2 5 2 86 6 14 6 79 8 21 8 93 9 7 9 66 11 34 1129 8 71 8 41 11 59 11 8 4 92 4 86 6 14 6 90 5 10 5MHCII+CD11c+MHCII-CD11c-MHCII-CD11c+CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiM-Stat4+/+8 3 92 3 2 2 98 2 35 7 65 7 66 9 34 959 9 41 9 90 4 10 4 8 4 92 4 3 2 97 2MHCII+CD11c+MHCII-CD11c-MHCII-CD11c+CD3+CD4+CD3+CD8a+CD3-CD19+NK1.1+CD49b+CD11b+Gr-1hiCD45.2M-Stat4-/-11 5 89 5 4 996 933 11 67 11 67 8 33 855 11 45 12 89 5 11 5 7 3 93 3 3 3 97 3A)B) 126 CD3+CD4+CD3+CD8a+CD3-CD19+Ly6ClowCD62L-Ly6ChiCD62L+MHCII-CD11c+NK1.1+CD49b+CD11b+Gr-1hiMHCII-CD11c-MHCII+CD11c+Frequency (%)M-Stat4+/+M-Stat4-/-Frequency (%)CD3+CD4+CD3+CD8a+CD3-CD19+MHCII-CD11c+NK1.1+CD49b+CD11b+Gr-1hiMHCII-CD11c-MHCII+CD11c+Frequency (%)Frequency (%)M-Stat4-/-M-Stat4+/+C)D)CD3+CD4+CD3+CD8a+CD3-CD19+Ly6ClowCD62L-Ly6ChiCD62L+MHCII-CD11c+NK1.1+CD49b+CD11b+Gr-1hiMHCII-CD11c-MHCII+CD11c+CD45.1-CD45.1+CD45.2-CD45.2+CD3+CD4+CD3+CD8a+CD3-CD19+MHCII-CD11c+NK1.1+CD49b+CD11b+Gr-1hiMHCII-CD11c-MHCII+CD11c+  127 Figure 3.12 Assessment of the Relative Contribution of BoyJ Donor-Derived Cells in the Spleens of M-Stat4+/+ and M-Stat4-/- Mice Following 8 Weeks of Atherogenic Diet  Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Spleen myeloid (Ly6C/CD62L, MHCII/CD11c, CD11b+Gr-1hi) and lymphoid (CD3+CD4+, CD3+CD8a+, CD3-CD19+, NK1.1+CD49b+) cells were stained with anti-CD45.1 and/or anti-CD45.2 antibodies for flow cytometry. A) and B) Representative histograms and gating schemes of CD45.1+ (A) and CD45.2+ (B) populations for each cell type. The frequency of events within each gated region ? SD is shown. C) and D) Bar graphs showing CD45.1 (C) and CD45.2 (D) expression of each cell type in the spleen. Error bars represent SD. n = 13-14 mice for each group.   3.3.9 Myeloid STAT4 Deficiency Increases Absolute Monocyte Counts in the Spleen of Ldlr-/- Mice To determine whether myeloid STAT4 expression affects monocyte development in mice, we measured total (CD11b+CD115+), inflammatory (Ly6ChiCD62L+), and patrolling (Ly6ClowCD62L-) monocyte populations in the blood, bone marrow, and spleen of M-Stat4+/+ and M-Stat4-/- mice (Figure 3.13 A). We found that M-Stat4-/- mice had significantly elevated total, inflammatory, and patrolling monocyte counts in the spleen (Figure 3.13 B 5th row) compared to M-Stat4+/+ mice. These differences, however, were not reflected in the frequency of monocytes in the spleen (Figure 3.13 B 4th row).  128    M-Stat4-/-SSCLy6C55 431 4CD11b21 3PIM-Stat4+/+SSCLy6C56 729 4CD11b19 6PIM-Stat4-/-SSCLy6C79 213 2CD11b12 1PIM-Stat4+/+SSCLy6C79 313 2CD11b13 1PIM-Stat4+/+SSCLy6C40 545 4CD11b6.0 1.1PIM-Stat4-/-SSCFSC CD62LLy6C37 547 4CD115CD11b6.8 1.7FSCPIBloodBone MarrowSpleenA) 129   B)Total Monocytes (CD11b+CD115+)Frequency (%)Inflammatory Monocytes (Ly6ChiCD62L+)Frequency (%)Patrolling Monocytes (Ly6ClowCD62L-)Frequency (%)Frequency (%)Frequency (%)Frequency (%)106Cells106Cells106CellsFrequency (%)Frequency (%)Frequency (%)106Cells106Cells106CellsBloodBone MarrowSpleenM-Stat4+/+M-Stat4-/- 130 Figure 3.13 Myeloid STAT4 Deficiency Increases Absolute Monocyte Counts in the Spleens of Ldlr-/- Mice Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Blood, bone marrow, and spleen cells were stained for flow cytometry analysis of total monocytes (CD11b+CD115+), pro-inflammatory monocytes (Ly6ChiCD62L+), and patrolling monocytes (Ly6ClowCD62L-). A) Representative dot plots and gating schemes of scatter, live, and monocyte populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency (blood, bone marrow, and spleen) and absolute numbers (bone marrow and spleen) of total, inflammatory, and patrolling monocytes. Error bars represent SD. n = 9-10 mice for blood. n = 12-14 mice for bone marrow. n = 13-14 for spleen. * p < 0.05 by two-tailed t-test.     3.3.10 Myeloid STAT4 Deficiency Increases Absolute DC Counts in the Spleens of Ldlr-/- Mice  To assess changes in splenic DC populations in M-Stat4-/- mice, spleens were homogenized and stained with anti-MHCII and anti-CD11c antibodies for flow cytometry analysis of DC (MHCII+CD11c+) populations. Dead cells, RBCs, and cells negative for both CD45.1 and CD45.2 were excluded (Figure 3.14 A). Compared to M-Stat4+/+ mice, M-Stat4-/- mice have increased absolute DC counts in the spleen. (Figure 3.14 B). As is the case with monocytes, differences in the frequency of DCs did not reach statistical significance.   131  CD45.1CD45.2 CD11cMHCIIMHCII68?570?655?454?71.0?1.11.5?1.72.9?0.53.1?0.9M-Stat4-/-M-Stat4+/+Frequency (%)Frequency (%)Frequency (%)Frequency (%)CD45.1+and/or CD45.2+DCs (MHCII+CD11c+)MHCII-CD11c-MHCII-CD11c+106Cells106Cells106Cells106CellsM-Stat4+/+M-Stat4-/-A)B) 132 Figure 3.14 Myeloid STAT4 Deficiency Increases Absolute DC Counts in the Spleens of Ldlr-/- Mice Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Spleens were homogenized and cells were stained with anti-MHCII and anti-CD11c antibodies for flow cytometry analysis of DC (MHCII+CD11c+), MHCII-CD11c+ cell, and MHCII-CD11c- cell populations. Dead cells, RBCs, and cells negative for both CD45.1 and CD45.2 were excluded. A) Representative dot plot and gating schemes of CD45.1/CD45.2 and DC populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency and absolute numbers of CD45+ cells, DCs, MHCII-CD11c+ cells, and MHCII-CD11c- cells in the spleen. Error bars represent SD. n = 13-14 mice for each group. * p < 0.05, ** p < 0.01 by two-tailed t-test.     3.3.11 Splenic NK Cell Frequency and Absolute Numbers are Elevated in M-Stat4-/- Mice Next, we studied the development of NK cells and neutrophils in M-Stat4+/+ and M-Stat4-/- mice. Spleens were homogenized and cells were stained for flow cytometry analysis of NK cell (NK1.1+CD49b+) and neutrophil (CD11b+Gr-1hi) populations. Dead cells, RBCs, and cells negative for both CD45.1 and CD45.2 were excluded (Figure 3.15 A). In M-Stat4-/- mice, there was a significant increase in both the frequency and absolute numbers of NK cells compared to M-Stat4+/+ mice (Figure 3.15 B). The frequency and absolute numbers of neutrophils were not significantly different.  133 CD45.1M-Stat4-/-M-Stat4+/+NK1.1NK1.1Gr-1Gr-167?667?102.2?1.03.8?1.88.5?1.28.6?3.2CD45.2 CD49bCD11bCD45.1+and/or CD45.2+NK Cells (NK1.1+CD49b+)Neutrophils (CD11b+Gr-1hi)Frequency (%)Frequency (%)Frequency (%)106Cells106Cells106CellsA)B)M-Stat4+/+M-Stat4-/-  Figure 3.15 Splenic NK Cell Frequency and Absolute Numbers are Elevated in M-Stat4-/- Mice Following 8 Weeks of Atherogenic Diet Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Splenocytes were stained for flow cytometry analysis of NK cell (NK1.1+CD49b+) and neutrophil (CD11b+Gr-1hi) populations. Dead cells, RBCs, and cells negative for both CD45.1 and CD45.2 were excluded. A) Representative dot plot and gating schemes of CD45.1/CD45.2, NK cell, and neutrophil populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency and absolute numbers of CD45+ cells, NK cells, and neutrophils in the spleen. Error bars represent SD. n = 13-14 mice for each group. * p < 0.05 by two-tailed t-test.  134 3.3.12 Myeloid STAT4 Deficiency Does Not Affect Lymphocyte Populations in Ldlr-/- Mice To evaluate potential effects of bone marrow STAT4 expression on lymphopoiesis, blood cells and splenocytes from transplanted mice were stained for flow cytometry analysis of CD4+ T cells, CD8+ T cells, and B cells. Dead cells, RBCs, and cells negative for both CD45.1 and CD45.2 were excluded (Figure 3.16 A). There were no statistically significant differences in CD4+ T cell, CD8+ T cell, and B cell populations in M-Stat4-/- mice compared to M-Stat4+/+ mice (Figure 3.16 B).    135 A)CD45.1CD3CD3M-Stat4-/-M-Stat4+/+97 195 413 311 39.0 2.09.4 0.916 108.8 3.6CD45.2CD3CD4 CD8a CD19CD3M-Stat4-/-M-Stat4+/+68 668 916 215 38.8 1.38.4 1.639 441 5CD45.1CD45.1CD45.1BloodSpleen 136  Frequency (%)Frequency (%)Frequency (%)Frequency (%)106Cells106Cells106Cells106CellsM-Stat4+/+M-Stat4-/-CD45.1+and/or CD45.2+CD4+T CellsB Cells (CD3-CD19+)CD8+T CellsFrequency (%)Frequency (%)Frequency (%)Frequency (%)SpleenSpleenBloodBloodB) 137 Figure 3.16 Myeloid STAT4 Deficiency Does Not Affect Lymphocyte Populations in Ldlr-/- Mice Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Blood cells and splenocytes were stained for flow cytometry analysis of CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8a+), and B cells (CD3-CD19+). Dead cells, RBCs, and cells negative for both CD45.1 and CD45.2 were excluded. A) Representative dot plots and gating schemes of CD45+ cell, CD4+ T cell, CD8+ T cell, and B cell populations. The frequency of events within each gated region ? SD is shown. B) Bar graphs showing the average frequency (blood and spleen) and absolute numbers (spleen) of CD45+ cells, CD4+ T cells, CD8+ T cells, and B cells. Error bars represent SD. n = 13 mice for blood. n = 8 mice for spleen. * p < 0.05 by two-tailed t-test.    3.3.13 Myeloid STAT4 Deficiency Does Not Significantly Affect Aortic Atherosclerosis in Ldlr-/- Mice To study whether the selective depletion of STAT4 in myeloid cells affects atherosclerotic lesion formation in mice, M-Stat4-/- mice were given an atherogenic diet for 8 weeks for induction of atherosclerosis. At the time of sacrifice, entire aortas were excised, opened longitudinally, pinned onto a dissection pad, and stained with Sudan IV to highlight atherosclerotic areas. Lesion areas, as a percentage of total aortic surface area, were then quantified using the ImagePro Plus software. There was a 35% increase in aortic lesion area in M-Stat4-/- mice compared to M-Stat4+/+ mice; however, the difference was not statistically significant (Figure 3.17).   138 A) B)M-Stat4-/-M-Stat4+/+% Plaque AreaM-Stat4+/+M-Stat4-/- Figure 3.17 Myeloid STAT4 Deficiency Does Not Affect Aortic Atherosclerosis in Ldlr-/- Mice Fed an Atherogenic Diet for 8 Weeks Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Entire aortas were excised, opened longitudinally, and stained with Sudan IV to reveal atherosclerotic areas (red regions). A) Each data point represents the plaque area of a single animal as a percentage of intimal surface area. Line and whiskers show the mean and SD of each group. B) Representative images of Sudan IV staining of aortas. Scale bars represent 5mm. n = 13 mice for M-Stat4+/+ and n = 14 mice for M-Stat4-/-.    139 3.3.14 Myeloid STAT4 Deficiency Increases Aortic Root Lesion Area but Does Not Affect Lesion Lipid Content or Necrosis in Ldlr-/- Mice To measure aortic root atherosclerotic lesion area, lipid content, and necrosis in M-Stat4-/- mice following 8 weeks of atherogenic diet, the aortic root region of hearts was sectioned and stained with Oil Red O and/or H&E. Images were digitally scanned and then quantified using the Aperio ImageScope. In M-Stat4-/- mice, there was a statistically significant 27% increase in aortic root lesion area compared to M-Stat4+/+ mice (Figure 3.18 A and D). Although there is a notable trend towards increased lesion necrosis in M-Stat4-/- mice, the differences in lipid content and plaque necrosis in M-Stat4+/+ and M-Stat4-/- mice were not statistically significant (Figure 3.18 B-E). 140   M-Stat4+/+M-Stat4-/-Oil Red OH + EA)C)D)E)++B)Lesion Area (100 ?m2)Lipid Content (%)Necrotic Core (%)M-Stat4+/+M-Stat4-/-M-Stat4+/+M-Stat4-/-M-Stat4+/+M-Stat4-/-M-Stat4+/+M-Stat4-/-p = 0.12 141 Figure 3.18 Myeloid STAT4 Deficiency Increases Aortic Root Lesion Area but Does Not Affect Lesion Lipid Content or Necrosis in Ldlr-/- Mice Mice were fed an atherogenic diet for 8 weeks prior to sacrifice. Aortic roots were stained with Oil Red O or H&E. A), B), and C) Each data point represents the mean lesion area, necrotic core area, or lipid content of a single mouse. Line and whiskers show the mean and SD of each group. D) and E) Representative images of Oil Red O or H&E-stained aortic root sections. +, examples of necrotic areas. Scale bars represent 250 ?m in D) and 200 ?m in E). n = 11-13 mice for M-Stat4+/+ and n = 12-14 mice for M-Stat4-/-. * p < 0.05 by two-tailed t-test.   142 3.4 Discussion The IL-12/STAT4 signaling pathway orchestrates the Th1 response and is essential for the optimal production of IFN-? by effector cells.265?268 Although IL-12 has been shown to exacerbate atherosclerosis, at least during early stages of lesion formation, the requirement for STAT4 in this process is unknown.12, 13, 289 Moreover, IL-27, an IL-12 family cytokine that can activate STAT4 (along with other STATs), has recently been shown to have protective effects against atherosclerosis.290, 291 Indeed, the functional significance of STAT4 itself in atherosclerosis has not been explored previously. Using an experimental approach analogous to the one we employed to study STAT6, we investigated the consequences of total hematopoietic system STAT4 as well as myeloid-specific STAT4 depletion on atherosclerosis and hematopoiesis on Ldlr-/- mice following 8 weeks of atherogenic diet. To generate total hematopoietic system STAT4 knockout mice, we transplanted Stat4-/- bone marrow into lethally irradiated Ldlr-/- mice. To specifically deplete STAT4 in the myeloid compartment, we used a mixture of 90% Stat4-/-Rag1-/- bone marrow combined with 10% of wild type BoyJ bone marrow, as described in Chapter 2. We hypothesized that mice transplanted with Stat4-/- bone marrow or Stat4-/-Rag1-/- bone marrow would be protected from atherosclerosis compared to control animals due to a blunted Th1 response with decreased inflammatory cell recruitment and infiltration and reduced inflammatory cytokine production within lesions. Furthermore, since IL-12 stimulates IFN-? production in both myeloid and lymphoid cells,265?268 we hypothesized that total hematopoietic system STAT4 deficient mice would be more protected from  143 atherosclerosis compared to mice in which STAT4 expression is reduced only in the myeloid compartment. Contrary to our hypothesis that STAT4 depletion would be atheroprotective, we observed significant increases in atherosclerotic lesion area in the aorta and the aortic root in Ldlr-/- mice transplanted with Stat4-/- bone marrow compared to control mice. In addition, Stat4-/- bone marrow-transplanted mice developed larger necrotic cores compared to mice transplanted with wild type bone marrow. The changes in lesion area and necrosis were independent of plasma total cholesterol levels. Lesional lipid content was not significantly different between Stat4-/- and wild type mice. Although lipid content is a marker used to differentiate stable and vulnerable atherosclerotic lesions in humans and mice,326?329 there have been reports where the lipid content was not in agreement with other markers of plaque stability.336 Intriguingly, the pattern of atherosclerosis in M-Stat4-/- mice is in agreement with our data in total hematopoietic system STAT4-deficient mice. Indeed, we observed a trend towards increased atherosclerosis in the aorta of M-Stat4-/- mice compared to M-Stat4+/+ mice, although this difference did not reach statistical significance. On the other hand, the aortic root atherosclerotic lesion area in M-Stat4-/- mice was significantly greater than that of M-Stat4+/+ mice. The necrotic core size was also noticeably enlarged in M-Stat4-/- mice compared to M-Stat4+/+ mice; however, once again, the difference was not statistically significant. Finally, lesional lipid content was not significantly different between M-Stat4+/+ and M-Stat4-/- mice. Despite the lack of statistical significance in aortic lesion area and aortic root necrosis, the clear and consistent trends lead me to believe that the loss of myeloid STAT4 does indeed cause  144 a biologically significant increase in atherosclerotic lesion area and vulnerability; a repeat of the experiment with a larger sample size may reveal these differences. We originally hypothesized that total hematopoietic system STAT4 deficient mice would be more protected from atherosclerosis compared to M-Stat4-/- mice because IL-12 stimulates IFN-? production in both myeloid and lymphoid cells.265?268 Although the observed effects of total hematopoietic system and myeloid-specific STAT4 deficiency on murine atherosclerosis was in contrast to our original hypothesis, they were consistent with our expectation that myeloid STAT4-depletion would exert similar but less pronounced effects on atherosclerosis compared to total hematopoietic system STAT4 depletion. The production of IFN-? in atherosclerotic lesions can be enhanced by IL-12 injections and inhibited by IL-12-blocking antibodies.13, 289 Although STAT4 is necessary for IL-12-induced IFN-? production in cultured murine cells,265, 273 the requirement of STAT4 for IFN-? secretion within atherosclerotic plaques has never been demonstrated. Not only is there evidence to suggest that IL-12/STAT4-independent pathways can induce IFN-? production in T cells,337 a previous study also found that IFN-? alone is capable of inducing its own production in CD4+ splenocytes from Stat4-/- mice but not in cells from wild type mice.333 In fact, the level of IFN-? production in IFN-?-treated Stat4-/- T cells is comparable to that of wild type T cells stimulated with IL-12, implying that IFN-?-induced IFN-? production is normally inhibited by a STAT4-dependent mechanism.333 This prompted us to hypothesize that the lack of STAT4 in our mice may have allowed IFN-? to potently enhance its own production, which at least partially accounts for the increase in atherosclerosis in Stat4-/- and M-Stat4-/- mice. Furthermore, since T cells are  145 major sources of IFN-?, STAT4 deficiency in both lymphoid and myeloid lineages may explain the reason behind the more pronounced phenotype in total hematopoietic system STAT4-deficient mice compared to M-Stat4-/- mice. An important experiment to test these hypotheses would be to measure the local production of IFN-? within atherosclerotic lesions using immunohistochemistry. We expect to observe higher concentrations of IFN-? within the plaques of STAT4-deficient mice compared to wild type mice. The patterns of hematopoietic perturbations in our mice support our speculation that IFN-? activity is preserved even in the absence of STAT4. Under basal conditions, STAT4 knockout C57BL/6 mice possess reduced numbers of hematopoietic progenitor cells but no abnormalities in mature cells relative to controls.240, 265 On the other hand, in vivo IL-12 administration in mice has profound effects on hematopoiesis in an IFN-?-dependent manner.338, 339, 340 Wild type mice treated with IL-12 have reduced circulating lymphocyte numbers in the blood; increased monocytic precursors in the bone marrow; and increased NK cell populations in the spleen; these changes are absent in IFN-? receptor-deficient mice treated with IL-12.340 Interestingly, in Stat4-/- bone marrow-transplanted Ldlr-/- mice, we observed significant reductions in CD4+ and CD8+ T cells in the blood and spleen; increased numbers of inflammatory monocytes in the bone marrow (in addition to non-statistically significant increases in total and patrolling monocytes); and elevated frequency and absolute numbers of NK cells in the spleen compared to wild type mice. Since STAT4 knockout mice have no alterations in hematopoiesis under normal conditions,265 it is likely that IFN-? produced in response to hypercholesterolemia, instead of endogenous IFN-?, is the cause of the immune cell  146 changes in Ldlr-/- mice transplanted with Stat4-/- bone marrow. A likely initial source of hypercholesterolemia-induced IFN-? in bone marrow STAT4-deficient mice is the population of host T cells that was not completely ablated by lethal irradiation. Compared to total hematopoietic system STAT4-deficient mice, the phenotype in M-Stat4-/- mice was less dramatic. There were no changes in immune cell populations in the blood and the bone marrow. However, in the spleen, absolute numbers of total monocytes, inflammatory monocytes, patrolling monocytes, and DCs were elevated in M-Stat4-/- mice compared to M-Stat4+/+ mice. Importantly, similar to the total hematopoietic system STAT4-deficient mice, splenic NK cell frequency and absolute numbers in M-Stat4-/- were also increased relative to M-Stat4+/+ mice. NK cells can potentially influence atherosclerosis through their secretion of inflammatory cytokines, including IFN-? and TNF-?.114 Indeed, a previous study demonstrated that NK cell numbers in the plaque correlates with atherosclerosis and the depletion of functional NK cells significantly reduces aortic and aortic root lesion area in Ldlr-/- mice.115 That being said, our study does not reveal whether increases in splenic NK cell populations are indicative of elevated NK cell numbers in the lesion. A potential future research aim would be to evaluate NK cell infiltration and production of inflammatory cytokines within the atherosclerotic plaques of STAT4-deficient mice. We also assessed the relative contribution of BoyJ-derived donor cells to the immune cell populations in M-Stat4+/+ and M-Stat4-/- mice using CD45.1 and CD45.2 expression. The majority of myeloid cells as well as B lymphocytes were reconstituted at the correct proportions in recipient mice. The CD4+ T cell and CD8+ T cell populations in the blood and spleen once again proved to be radioresistant. Speculations of why  147 CD45.1+ cells constitute greater than 10% of cells in some myeloid populations as well as the high resistance of T lymphocytes to radiation have been discussed in detail in Chapter 2 (page 89-91 of this thesis) and will not be addressed again here. Regardless, both radioresistant host lymphocytes and lymphocytes from the BoyJ donor are wild type for STAT4. Therefore, the persistence of radioresistant lymphocytes in the host is unlikely to significantly affect atherosclerosis in our mice in a STAT4-dependent manner. An important future study would be to investigate the effects of STAT4 during intermediate or later stages of atherosclerotic plaque development in Ldlr-/- mice. In the current study, we exclusively evaluated lesion formation at 8 weeks after the initiation of the atherogenic diet. It has been shown previously in Il-12-/-ApoE-/- mice that IL-12 deficiency can inhibit atherosclerosis at earlier but not at later time points.12 Although our results in STAT4-deficient mice suggest that STAT4 and IL-12 may exert differential effects on atherosclerosis, whether the influences of STAT4 on atherosclerosis are time-dependent remains an interesting and important question. In addition to IL-12, STAT4 is also phosphorylated and activated in response to other cytokines in the IL-12 family, including IL-23; IL-27; and IL-35.258, 259, 260 However, these cytokines also employ other STAT family members, such as STAT1, STAT3, and STAT5, for their signaling. Indeed, the activation of STAT4 by the aforementioned cytokines is considerably weaker than that compared to IL-12.258, 259, 260  Nonetheless, while the effects of IL-23 and IL-35 on atherosclerosis are unknown, IL-27 has been shown in two separate studies to inhibit atherosclerosis in mice.290, 291 Although the importance of STAT4 in the atheroprotective functions of IL-27 was not addressed in  148 those studies, it is plausible that STAT4 depletion in our animals eliminated, or perhaps diminished, the protective effects of IL-27 which partially accounts for increased atherosclerosis in our mice. In conclusion, we demonstrated that total hematopoietic system STAT4 as well as myeloid-specific STAT4 deficiency exacerbates atherosclerosis in Ldlr-/- mice fed an atherogenic diet for 8 weeks. We hypothesize that this phenomenon is due, at least in part, to enhanced IFN-?-induced IFN-? production in response to hypercholesterolemia, and to the diminished atheroprotective effects of IL-27 in the absence of STAT4. Importantly, our data does not necessarily conclude that STAT4 is protective against atherosclerosis. Instead, it is likely that any significant deviations from the regular physiological functions of STAT4 may profoundly enhance atherosclerosis.    149 CHAPTER 4: Conclusions and Future Directions 4.1 Overall Summary The primary goal of the current thesis was to evaluate the impact of perturbations in STAT4 and STAT6, transcription factors that are central to the Th1 and Th2 response, respectively, on atherosclerosis with the intention of identifying novel therapeutic targets for cardiovascular disease.304 To address our research aims, we generated Ldlr-/- mice that are deficient in STAT6 or STAT4 via bone marrow transplantation and measured atherosclerosis in these mice. We hypothesized that atherosclerosis will be exacerbated in STAT6-deficient mice and attenuated in STAT4-deficient mice. Contrary to our hypothesis, we found that myeloid-specific STAT6 depletion did not significantly impact atherosclerotic lesion area, necrosis, or lipid content at early and intermediate stages of lesions formation. The lack of differences in atherosclerosis was accompanied by very modest phenotypic changes in hematopoiesis. The only significant differences in M-Stat6-/- mice compared to M-Stat6+/+ mice were slight increases in splenic patrolling monocyte and decreases in splenic B cell frequencies at 8 weeks, and higher total splenic monocyte and lower splenic B cell and blood CD4+ T cell frequencies at 14 weeks. Although these cell types have been implicated in atherosclerosis, the biological significance of changes in these cell types in our mice appears to be limited. Our observation that myeloid STAT6 depletion is not a significant factor in atherosclerosis is consistent with studies published by King and colleagues in 2007, which showed that IL-4 does not influence lesion size or composition in ApoE-/- or Ldlr-/-  150 mice.14 However, even in studies where IL-4 affected atherosclerosis, the cellular composition of the lesions, with regards to macrophage, CD4+ T cell, and CD8+ T cell accumulation, were not affected, and detailed mechanistic explanations for the effects of IL-4 on atherosclerosis in vivo remain elusive.12, 15 Importantly, in addition to IL-4, STAT6 is also phosphorylated and activated in response to IL-13.214 The reconstitution of Ldlr-/- mice with Il-13-/- bone marrow enhances atherosclerotic lesion size and necrosis and decreases lesional M2 macrophage content. On the other hand, exogenous IL-13 administration for 5 weeks decreases lesional macrophage content but does not affect lesion area.243 It will be interesting to determine, as a future aim, whether lesional composition in M-Stat6-/- mice, with regards to macrophage and collagen content, are perturbed despite a lack of significant difference in lesion area. BALB/c Stat6 knockout mice develop larger lesions compared to BALB/c wild type mice, which seemingly contradicts our observations that myeloid STAT6 is not involved in atherosclerosis.158 It is important to note, however, that STAT6 depletion in BALB/c mice, which inhibits their natural tendency to mount a Th2 response and enhances their Th1 response, was only able to increase atherosclerosis to a level comparable to wild type C57Bl/6 mice.158 Therefore, it is possible that STAT6 can exacerbate atherosclerosis, but only in mouse models that are normally resistant to atherosclerosis due to their natural predisposition to produce a Th2 response. The depletion of myeloid STAT6 in a strain already prone to a Th1 response, such as C57Bl/6, might not significantly affect atherosclerosis. Our focus on myeloid-specific STAT6, as opposed to systemic STAT6, depletion is another potential reason for the discrepancy between our study and previous literature. Following initial IL-4 stimulation,  151 CD4+ T lymphocytes become efficient producers of IL-4, which up-regulates the expression of GATA3 and represses Th1 differentiation by inhibiting STAT4 signaling and IFN-? production.154, 341 Since the STAT6 signaling pathway remains intact in lymphocytes in M-Stat6-/- mice, the impact of myeloid STAT6 depletion on atherosclerosis may be less substantial compared to germline STAT6 knockout. Total hematopoietic system STAT4 depletion profoundly exacerbated atherosclerosis in Ldlr-/- mice fed an atherogenic diet for 8 weeks, which was unexpected as we hypothesized that the pro-atherogenic Th1 response would be blunted in the absence of STAT4. We speculated that this phenomenon may be due to enhanced IFN-?-induced IFN-? production, which is normally inhibited by STAT4.333 This explanation can also account for the reason why IL-12 knockout is atheroprotective while STAT4 depletion exacerbates atherosclerosis.12 It has recently been shown that IL-27, an IL-12 family cytokine that can activate STAT4, protects mice against atherosclerosis by inhibiting myeloid cell recruitment and activation, decreasing T cell accumulation, and preventing inflammatory cytokine production.290, 291 However, IL-27 preferentially signals through STAT1, STAT3, and STAT5 and only weakly phosphorylates STAT4, limiting the likelihood that defective IL-27 signaling is the main culprit for the increase in atherosclerosis in STAT4-deficient mice.259 Although the roles of IL-35 in atherosclerosis have not been investigated, IL-35 is a potent immunoregulatory cytokine that suppresses Th1 and Th17 cell development. Since IL-35 mainly signals through a STAT1/STAT4 heterodimer, I hypothesize that STAT4 depletion would disrupt IL-35 signaling, which could potentially increase atherosclerosis by promoting T cell proliferation and inflammation.334 Our understanding and  152 interpretation of the roles of STAT4 in atherosclerosis must be supplemented by future investigations into the functions of other STAT4-activating cytokines, such as IL-35, in atherosclerotic disease. Immune cell populations in total hematopoietic system STAT4-deficient mice are reminiscent of the IFN-?-dependent hematopoietic changes in IL-12-treated wild type mice, providing indirect evidence for the hypothesis that IFN-? levels may be elevated in response to hypercholesterolemia in Stat4-/- bone marrow-transplanted mice.340 Compared to total hematopoietic system STAT4, myeloid-specific STAT4-deficient mice exhibited similar, though less dramatic, phenotypic changes in atherosclerosis. Interestingly, we found that NK cell populations were increased in total hematopoietic system as well as myeloid-specific STAT4-deficient mice. Since NK cell numbers are positively correlated with atherosclerosis, the increased NK cell populations may also partially account for the augmented atherosclerosis in our mice.115  4.2 Limitations and Future Directions An important limitation of bone marrow transplantation studies is the imperfect reconstitution of recipient immune compartments by donor-derived cells. Since the myeloid cells of Ldlr-/- recipients are wild type for STAT expression, their presence can potentially confound our results by being a source of unwanted STAT proteins in the bone marrow recipients. To minimize the interference from radioresistant host cells, we used a radiation dose of 13 Gy, which has been shown to be myeloablative.314?318 In addition to myeloid cells, T lymphocytes were also resistant to radiation, as evidenced by the high frequency of CD45.2+ T cells in the Ldlr-/- recipients. Although studies have found that radiosensitive T cells produce more TNF-?, IL-12, and IL6 in response to  153 bacterial flagellin compared to radioresistant T cells, these cells were present in approximately equal numbers in mice transplanted with wild type or STAT-deficient bone marrow and therefore unlikely to skew our results.320, 342 Furthermore, as both donor and recipient T cells are wild type for STAT expression, the incomplete reconstitution of the recipient by donor T cells bears minimal consequence. Nonetheless, should radioresistant T cells become a concern, we may be able to eliminate them using Ldlr-/- mice crossed with Rag1-/- mice. Since Rag1-/- animals are devoid of mature lymphocytes, transplantation of Stat4/6-/-Rag1-/- plus wild type bone marrow into Ldlr-/-Rag1-/- mice would allow the reconstitution of the lymphoid compartment exclusively by wild type lymphocytes. In addition to radioresistant host myeloid cells, another source of unwanted STAT expression is the wild type bone marrow transplanted into the Ldlr-/- recipients during our myeloid-specific STAT depletion studies. Although we have confirmed, by flow cytometry analysis of CD45.1/2 surface expression, that the reconstitution of the myeloid compartment by donor cells is generally successful, the minimum level of STAT expression required to maintain normal STAT-mediated responses in vivo is currently unknown. There remains the possibility that the 10% of wild type bone marrow transplanted into our recipient mice is sufficient for a physiologically-relevant STAT response. Therefore, in order to prove that STAT protein-mediated immunity is effectively inhibited in our model system, we need to measure STAT4/6 expression and determine whether markers of Th1/Th2 immune responses, such as IFN-?, TNF-?, IL-4, IL-5, IL-13, IL-10, and M1/M2 macrophage ratio, are altered within atherosclerotic lesions. Due to challenges regarding tissue fixation, addressed in Chapter 2, I was  154 unable to include this data in the thesis; consequently, this issue must be addressed in future studies. An alternative to achieving tissue-specific depletion of a target protein without using bone marrow transplantation is to utilize the Cre/loxP recombination system. The Cre recombinase enzyme catalyzes site-specific recombination of genes flanked between 34-bp sequences known as loxP sites.346 Using transgenic technology, it is possible to generate mice in which a portion of the Stat4 or Stat6 gene is situated between loxP sites. Crossing these mice with mice in which the expression of Cre recombinase is controlled by a tissue-specific promoter may allow the targeted deletion of STAT4 or STAT6 in specific cell types. The expression of the murine lysozyme M gene is restricted to cells of the myeloid lineage.347, 348 Indeed, mice expressing Cre recombinase under the control of the lysozyme M promoter (LysM-Cre) are commercially available from the Jackson Laboratories and have been used extensively to study myeloid-specific deletion of target genes, even in the context of atherosclerosis.349, 350 Despite its usefulness, the LysM-Cre model isn?t without its limitations. For example, the efficiency of gene deletion is dependent upon the target gene; whether Stat4 and Stat6 expression can be effectively inhibited requires additional validation. Furthermore, LysM is not uniformly expressed in all myeloid cells and the expression level is variable between individual animals.351 These factors may introduce additional challenges that warrant careful consideration when interpreting data from LysM-Cre mouse models. A major assumption made during our study was that the ratio of CD45.1+ and CD45.2+ cells in the blood, bone marrow, and spleen was representative of the  155 proportions of Stat4/6+/+ and Stat4/6-/- cells in atherosclerotic lesions. This assumption could only be true if lesional immune cells originate from circulating cells, if Stat4/6+/+ and Stat4/6-/- cells are equally recruited to sites of atherosclerosis, and if all lesional CD45.2+ myeloid cells are derived from the Rag1-/- donor and not from radioresistant recipient myeloid cells. However, it has been shown that IL-4 signaling enhances bone marrow-derived DC trafficking in vivo, and the expression of certain chemokine receptors (CCR3, CCR5, and CCR7), albeit in T cells, are dependent upon STAT4 and STAT6.343, 344 In addition, a recent publication by Robbins and colleagues found that local proliferation, rather than continuous monocyte influx, is the dominant source of lesional macrophages.345 Therefore, the lack of data confirming STAT4 or STAT6 depletion within the atherosclerotic lesions of our mice becomes a potential limitation. This limitation can be addressed in future studies by staining STAT4 or STAT6 using immunohistochemistry or flow cytometry analysis of dissolved aortas. Due to reasons mentioned above, even though myeloid-STAT6 depletion did not significantly affect atherosclerotic lesion size and vulnerability in our experimental model, several issues must be addressed in future experiments before we can confidently conclude that myeloid STAT6 plays no roles in atherosclerosis. First, we must confirm that the majority of lesional myeloid cells are STAT6-deficient. This is important in order to validate that the ratio of 10% wild type plus 90% Stat6-/-Rag1-/- myeloid cells is preserved in the lesion. Secondly, we need to determine whether myeloid STAT6-depletion alters the lesional inflammatory environment with regards to the cytokine milieu and the immune cell populations. It is possible that myeloid-STAT6 depletion alone is not sufficient to significantly affect the Th2 response within  156 atherosclerotic lesions. Lesional detection of cytokines (IFN-?, IL-12, IL-4, IL-13, IL-10 etc.) and M1/M2 macrophage markers (Ym-1, arginase I/II etc.) by flow cytometry, immunohistochemistry, or RT-PCR will aid in answering these questions. A similar approach can be used to provide mechanistic insight into the roles of STAT4 in atherosclerosis. In addition to studying the consequences of total hematopoietic system or myeloid-specific depletion of STAT proteins on atherosclerosis, a logical future aim is to elucidate the functional significance of lymphocyte-specific expression of STAT4 or STAT6 in atherosclerotic disease. This aim can be explored using commercially-available transgenic mice expressing Cre recombinase in lymphocytes under the control of the human CD2 promoter and locus control region (LCR).352 Crossing these mice with an athero-susceptible mouse model containing loxP-flanked Stat4 or Stat6 will allow us to investigate the effects of lymphoid-specific STAT4/6 deletion on murine atherosclerosis. Since STAT6 orchestrates Th2 cell proliferation and production of anti-inflammatory cytokines, including IL-4 and IL-13, the deletion of STAT6 in lymphocytes may attenuate the Th2 response, enhance the Th1 response, and exacerbate atherosclerosis.158, 226, 227 On the other hand, we have shown that total hematopoietic system and myeloid-specific STAT4-depletion is potentially atherogenic. Since T cells are major producers of IFN-?, lymphoid-specific STAT4 deletion may enhance IFN-?-induced IFN-? production by T cells, which may exacerbate atherosclerosis. Whether we will witness this predicted outcome following lymphoid-specific STAT4 deletion awaits further experimentation.  157 Further characterization of immune cell changes is another important future study. For example, we observed a decrease in the CD4+ T cell population following total hematopoietic STAT4 depletion, but it is unclear whether T cell subsets, such as Th1, Th2, Th17, or Treg cells, which may have opposing functions in atherosclerosis, are selectively affected. Similarly, we did not distinguish between the atheroprotective B1 B cells and the atherogenic B2 B cells, nor did we characterize DC subsets (CD8?+ or CD8?-; Flt3-dependent and independent, for example) beyond MHCII and CD11c expression.99, 167 Since different immune cell subsets may exhibit functional diversity in atherosclerosis, detailed investigations into immune cell subset changes is necessary to fully understand the roles of STAT4 and STAT6 in atherosclerosis. A potential limitation intrinsic to all animal studies is the translatability of the results into the human system. Although the JAK/STAT signaling pathway is evolutionarily conserved, there have been reports that type I IFNs are capable of activating STAT4 and promoting Th cell development in humans but not in mice.194, 343, 344 With regards to atherosclerosis, Ldlr-/- mice are essentially models for human homozygous familial hypercholesterolemia. Interestingly, in patients with homozygous familial hypercholesterolemia, the preference for lesion formation in the aortic root, as well as the relatively low susceptibility to plaque rupture despite extensive atherosclerosis, more closely resembles atherosclerosis in Ldlr-/- mice rather than humans.301 This observation prompts the yet-unanswered question of whether current mouse models correctly represent atherosclerosis in the general human population. Despite these limitations, the low cost of maintenance, rapid breeding, and ease of  158 genetic manipulation makes the mouse a valuable model organism in the identification of potential therapeutic targets for atherosclerosis.  4.3 Concluding Statement This thesis explored the plausibility of modulating the immune response through STAT4 and STAT6 as a prospective treatment strategy for atherosclerosis. We hypothesized that STAT6 deficiency would enhance atherosclerosis while STAT4 deficiency would be protective. Using bone marrow transplantation into the Ldlr-/- mouse model, we demonstrated that myeloid STAT6 does not significantly affect atherosclerotic lesion area or vulnerability. On the other hand, a loss of STAT4 in all hematopoietic cells or in all myeloid cells paradoxically exacerbates atherosclerotic disease. 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