Therapies that promote tolerance will improve outcomes in solid organ transplantation by eliminating the need for long-term immunosuppression. This thesis investigates two possible tolerance induction mechanisms: rapamycin induced expression of regulatory T cells and re-education of the immune system using syngeneic hematopoietic stem cell transplantation. Fibrinogen-like protein 2, a effector molecule of regulatory T cells, was also determined as a key mediator in the tolerance induction pathway as depletion of fibrinogen-like protein 2 lead to allograft rejection. The feasibility of using syngeneic hematopoietic stem cells for inducing allograft tolerance was studied by setting up a murine heart and bone marrow transplant model. Syngeneic T-depleted bone marrow transplantation resulted in a slight prolongation of the graft survival time compared to the animals reconstituted with total bone marrow cells. We provide compelling evidence to suggest that fibrinogen-like protein 2 and syngeneic hematopoietic stem cells can possibly be used to induce transplantation tolerance.

Therapies that promote tolerance will improve outcomes in solid organ transplantation by eliminating the need for long-term immunosuppression. This thesis investigates two possible tolerance induction mechanisms: rapamycin induced expression of regulatory T cells and re-education of the immune system using syngeneic hematopoietic stem cell transplantation. Fibrinogen-like protein 2, a effector molecule of regulatory T cells, was also determined as a key mediator in the tolerance induction pathway as depletion of fibrinogen-like protein 2 lead to allograft rejection. The feasibility of using syngeneic hematopoietic stem cells for inducing allograft tolerance was studied by setting up a murine heart and bone marrow transplant model. Syngeneic T-depleted bone marrow transplantation resulted in a slight prolongation of the graft survival time compared to the animals reconstituted with total bone marrow cells. We provide compelling evidence to suggest that fibrinogen-like protein 2 and syngeneic hematopoietic stem cells can possibly be used to induce transplantation tolerance.

Systemic lupus erythematosus (SLE) is a chronic, multi-organ autoimmune disease characterized by the production of antibodies against self nuclear antigens. Genetics play a dominant role in disease pathogenesis and functional examination of spontaneously-arising lupus-prone animal models has provided key insights into understanding the genetic complexity of the disease. The overarching goal of the work presented here is to identify the underlying immunologic abnormalities, together with lupus susceptibility loci that produce them, that promote the development of autoimmunity in the lupus-prone New Zealand Black (NZB) background. Chapter 2 identifies the critical role of CD40-CD40L interactions in the pathogenesis of disease in NZB mice. We showed that this interaction is required for the production of class-switched IgG autoantibodies and development of hemolytic anemia and kidney disease. Polyclonal B cell activation, expansion of plasmacytoid dendritic cells (pDC), and elevated gene expression of baff were maintained in CD40L-deficient NZB mice, despite the lack of IgG immune complexes. Chapter 3 utilizes bicongenic mice carrying both NZB chromosomes 1 and 13 to investigate the genetic complexity in disease pathogenesis. In addition to identifying new phenotypes, examination of bicongenic mice showed that chronic stimulation of pDC due to the persistence of nuclear antigens leads to a refractory state with a failure to produce more IFN-α upon subsequent stimulation. Chapter 4 identifies a novel lupus susceptibility locus on NZB chromosome 13 associated with impaired clearance of apoptotic debris, a key initiating step in the development of autoimmunity. Using subcongenic mice, this locus was localized and examined its impact on immune function. Work from this thesis will contribute to understanding the complex immunogenetic mechanisms that lead to development of SLE.

Nod1 and Nod2 are intracellular pattern recognition receptors that detect specific moieties of peptidoglycan, a critical component of the bacterial cell wall, to initiate host innate immune responses. Importantly, mutations in the human NOD2 gene have been associated with increased risk to develop mucosal auto-inflammatory disorders such as Crohn’s Disease. However, how Nod1 and Nod2 mediate mucosal homeostasis still remains unclear. In Chapter 2, I determined that mice deficient for both Nod1 and Nod2 (Nod1-/-Nod2-/-) exhibited delayed induction of intestinal inflammation at early timepoints after infection with Citrobacter rodentium compared to wild-type mice, which correlated with compromised control of the pathogen at later timepoints. Notably, I determined that induction of the cytokines IL-17 and IL-22 in the cecal lamina propria (LP) was blunted in Nod1-/-Nod2-/- mice after infection with either C. rodentium or Salmonella enterica serovar Typhimurium. Importantly, I found that Th17 cells were the principal producers of IL-17 and IL-22 after infection. Due to the rapid kinetics of activation and the regulation by Nod1 and Nod2, I termed this early mucosal response the innate Th17 (iTh17) response. The iTh17 cells exhibited an effector memory phenotype and required priming from the enteric microbiota for full induction. Therefore, in Chapter 3, I next determined that major histocompatibility complex (MHC) class II expression in hematopoietic cells was required for the induction of LP Th17 responses after infection. Interestingly, I found that the percentage IL-17+CD8+ T cells was strongly upregulated when MHCII signaling was ablated, suggesting a dynamic compensatory mechanism of IL-17-producing T cell responses in the mucosa. In Chapter 4, I identified MDP(D-Glu2)-OCH3 as a synthetic Nod2 agonist that exhibited increased stimulatory ability of Nod2-dependent NF-B activation compared to MDP in an unbiased screen. Moreover, I determined that MDP(D-Glu2)-OCH3 induced more potent inflammatory responses both in vitro and in vivo and was a better adjuvant than MDP. Together, the data presented in this thesis expand our current understanding of the roles of Nod1 and Nod2 in the intestinal LP, the regulation of IL-17 producing T cells in the gut and the therapeutic potential of novel Nod2 agonists.

Nod1 and Nod2 are intracellular pattern recognition receptors that detect specific moieties of peptidoglycan, a critical component of the bacterial cell wall, to initiate host innate immune responses. Importantly, mutations in the human NOD2 gene have been associated with increased risk to develop mucosal auto-inflammatory disorders such as Crohn’s Disease. However, how Nod1 and Nod2 mediate mucosal homeostasis still remains unclear. In Chapter 2, I determined that mice deficient for both Nod1 and Nod2 (Nod1-/-Nod2-/-) exhibited delayed induction of intestinal inflammation at early timepoints after infection with Citrobacter rodentium compared to wild-type mice, which correlated with compromised control of the pathogen at later timepoints. Notably, I determined that induction of the cytokines IL-17 and IL-22 in the cecal lamina propria (LP) was blunted in Nod1-/-Nod2-/- mice after infection with either C. rodentium or Salmonella enterica serovar Typhimurium. Importantly, I found that Th17 cells were the principal producers of IL-17 and IL-22 after infection. Due to the rapid kinetics of activation and the regulation by Nod1 and Nod2, I termed this early mucosal response the innate Th17 (iTh17) response. The iTh17 cells exhibited an effector memory phenotype and required priming from the enteric microbiota for full induction. Therefore, in Chapter 3, I next determined that major histocompatibility complex (MHC) class II expression in hematopoietic cells was required for the induction of LP Th17 responses after infection. Interestingly, I found that the percentage IL-17+CD8+ T cells was strongly upregulated when MHCII signaling was ablated, suggesting a dynamic compensatory mechanism of IL-17-producing T cell responses in the mucosa. In Chapter 4, I identified MDP(D-Glu2)-OCH3 as a synthetic Nod2 agonist that exhibited increased stimulatory ability of Nod2-dependent NF-B activation compared to MDP in an unbiased screen. Moreover, I determined that MDP(D-Glu2)-OCH3 induced more potent inflammatory responses both in vitro and in vivo and was a better adjuvant than MDP. Together, the data presented in this thesis expand our current understanding of the roles of Nod1 and Nod2 in the intestinal LP, the regulation of IL-17 producing T cells in the gut and the therapeutic potential of novel Nod2 agonists.

Kawasaki disease (KD) is a multisystem vasculitis leading to coronary artery aneurysm formation. In a superantigen-mediated murine model of KD, the development of coronary arteritis is mediated by T-cells through the production of TNFα. TNFα localizes to the coronary arteries, where it induces the expression of MMP-9, resulting in the breakdown of elastin and the formation of aneurysms. Statins have been recently shown to have anti-inflammatory and immunomodulatory properties as a result of the inhibition of small GTPases. In our murine model of KD, atorvastatin treatment inhibits superantigen mediated T- cell proliferation and cytokine production, including IL-2 and TNFα. Additionally, statin treatment inhibits TNFα-mediated MMP-9 production by vascular smooth muscle cells, through inhibition of the MEK/ERK pathway. Thus, statins modulate each of the critical steps in the pathogenesis of KD in a disease model, suggesting that statin use could alter the outcome and prognosis of children suffering with this disease.

Lactobacillus casei cell wall extract (LCWE)- induced coronary arteritis in mice models Kawasaki disease (KD). LCWE injections consist of T-cell dependent factors that expand superantigen (SAg)-activated T-cell receptor (TCR) Vβ6+ cells, and T-cell independent factors (i.e. TLR2 activity) that localize and sustain the immune response. TLR2 can upregulate costimulatory molecules to rescue SAg-activated T-cells from apoptosis. Accordingly, SAg-activated costimulation-rescued TCRVβ6+ cells are predicted to express activation markers, produce cytokines and be able to induce coronary arteritis. MAM was identified as a SAg able to activate TCRVβ6+ cells in a manner similar to LCWE; however a combination of MAM and TLR2 agonist Pam3Cys could not induce coronary arteritis. As another marker of disease, leukocyte recruitment molecule expression in the hearts of MAM+Pam3Cys- injected mice was found to be lower than in LCWE- injected mice. Therefore, LCWE contains unique features beyond TCRVβ6 stimulation and TLR2 activity that are important for disease induction.

Systemic lupus erythematosus (SLE) is a chronic, multi-organ autoimmune disease characterized by the production of antibodies against self nuclear antigens. Genetics play a dominant role in disease pathogenesis and functional examination of spontaneously-arising lupus-prone animal models has provided key insights into understanding the genetic complexity of the disease. The overarching goal of the work presented here is to identify the underlying immunologic abnormalities, together with lupus susceptibility loci that produce them, that promote the development of autoimmunity in the lupus-prone New Zealand Black (NZB) background. Chapter 2 identifies the critical role of CD40-CD40L interactions in the pathogenesis of disease in NZB mice. We showed that this interaction is required for the production of class-switched IgG autoantibodies and development of hemolytic anemia and kidney disease. Polyclonal B cell activation, expansion of plasmacytoid dendritic cells (pDC), and elevated gene expression of baff were maintained in CD40L-deficient NZB mice, despite the lack of IgG immune complexes. Chapter 3 utilizes bicongenic mice carrying both NZB chromosomes 1 and 13 to investigate the genetic complexity in disease pathogenesis. In addition to identifying new phenotypes, examination of bicongenic mice showed that chronic stimulation of pDC due to the persistence of nuclear antigens leads to a refractory state with a failure to produce more IFN-α upon subsequent stimulation. Chapter 4 identifies a novel lupus susceptibility locus on NZB chromosome 13 associated with impaired clearance of apoptotic debris, a key initiating step in the development of autoimmunity. Using subcongenic mice, this locus was localized and examined its impact on immune function. Work from this thesis will contribute to understanding the complex immunogenetic mechanisms that lead to development of SLE.

Leukocyte exit from peripheral tissues is fundamental to host defense, yet little is known about the role of adhesive molecules in this process. In my thesis I ask the question “can an integrin regulate leukocyte exit from inflamed peripheral tissues” and specifically investigate the leukocyte integrin α1β1. This is an important question because leukocyte exit, or persistence, at an inflammatory lesion can have a profound effect on the immune response. In addition, I present special in situ staining techniques which had to be developed in order to assay endogenous leukocyte migration in a murine model. The introductory sections review functional differences between myeloid and lymphoid leukocyte subsets, the leukocyte adhesion cascade, integrins, chemokine receptors and the essential concepts of signaling and the relationship between chemokines and integrin activation. I also discuss the pro-migratory paradigm of leukocyte integrins, in other words that integrin adhesion is equated with leukocyte migration. The current literature regarding what is known about integrin function in peripheral tissues and leukocyte migration is also discussed. Chapter 2 characterizes my inflammatory model and implicates α1β1integrin and macrophages as important molecular and cellular entities respectively, involved in sustaining the inflammatory response. Chapter 3 develops endogenous in-situ labeling in the blood compartment, establishing the fundamentals of my in-situ approach. Chapter 4 extends this and establishes in-situ pulse labeling (ISPL) to label endogenous leukocytes in peripheral tissues. Chapter 4 then goes on to combine the technological advances and conceptual framework established in the previous chapters to elucidate a role for α1β1integrin in the exit of macrophages from inflamed peripheral tissues. Finally, in Chapter 5 I discuss the implications of my results in the context of the host defense, how it might impact the immune response and future directions for this research.

Viral hepatitis infection represents a significant epidemiological and economic burden on society. Following infection, some patients mount a blunted immune response to the virus, which ultimately can result in chronic infection. FGL2, a member of the fibrinogen-related protein superfamily, has been implicated in vitro in suppressing both innate and adaptive immune responses. In a murine model of acute viral hepatitis caused by Lymphocytic Choriomeningitis Virus strain WE, we demonstrate that FGL2 expressed by reticuloendothelial cells limits viral spread. When expressed by Treg cells FGL2 binds to FCγRIIB and prevents DC maturation and suppresses virus-specific T and B cell responses. We provide compelling evidence to suggest that hepatitis viruses utilize the FGL2-FCγRIIB pathway to evade immune detection. Inhibition of this pathway restores effective cellular and humoral antiviral immune responses towards hepatitis viruses.