Sub-acute/chronic inflammatory diseases are often associated with altered inflammatory response, leading to increased host vulnerability to secondary inflammatory challenges. In the first study, by employing streptozotocin (STZ)-induced diabetes in mice, we further investigate mechanisms leading to enhanced polymorphonuclear leukocytes (PMN) response under hyperglycemia. We show that existence of a proinflammatory state associated with broad increases of macrophages in various organs plays a dominant role in promoting PMN response in diabetic mice. Studies of PMN infiltration during zymosan-induced peritonitis reveal that hyperglycemia enhances PMN recruitment through increasing F4/80+ macrophages in the peritoneal cavity. Insulin reversal of hyperglycemia reduces peritoneal macrophage numbers and ameliorates PMN infiltration. Significantly increased macrophages are also observed in the liver, kidneys, and intestines under hyperglycemia, and are attributable to exacerbated nephropathy and colitis when respective inflammatory conditions are induced. We also find that significant monocytosis of inflammatory F4/80+Gr-1+ monocytes from the spleen and macrophage proliferation in situ synergistically contribute to the increased macrophage population under hyperglycemia. In conclusion, our results demonstrate that STZ-induced hyperglycemic/diabetic mice develop a systemic proinflammatory state mediated by broad infiltration of macrophages. In the second study, we focus on the identification of the carrier that binds to and delivers Shiga toxin 2(Stx2) to the target organ causing hemolytic uremic syndrome (HUS). By employing a murine HUS model through co-injection of LPS-Stx2, we show that, adoptive transfer of CD11b+ leukocytes, but not CD11b- leukocytes, RBC, platelets or plasma, isolated from mice with HUS induces HUS in healthy recipients. Interestingly, we find that LPS priming of mice significantly promotes CD11b+ leukocytes binding to Stx2. Compared to CD11b+ leukocytes from mice without LPS priming, CD11b+ leukocytes isolated from mice after LPS priming demonstrate higher frequencies of toxin binding and augmented potency to induce HUS. In sum, our results demonstrate peripheral CD11b+ myeloid leukocytes act as effective Stx2 carriers that deliver toxin to kidneys causing HUS and that LPS-induced inflammation enhances the carrier capacity and aggravates HUS.

The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Macrophage foam cells are the major culprits in atherosclerotic lesions, having a prominent role in both lesion initiation and progression. With atherosclerosis being the main factor underlying cardiovascular complications, there is a long-standing interest on finding ways to reverse lipid buildup in plaques. Studies have shown that promoting reverse cholesterol transport (RCT) from macrophage foam cells is anti-atherogenic because it alleviates the cholesterol burden of the plaques. The goal of this thesis was to gain insight into the mechanisms that govern cholesterol efflux from macrophage foam cells. The first part of this study looked at the ability of different oxysterols to promote cholesterol efflux in unloaded as compared to lipid-loaded macrophages, and our major finding here is that epoxycholesterol decreases efflux in lipid-loaded macrophages. It appears that epoxycholesterol does so by impairing the release cholesterol from its cellular storage site, the lipid droplet (LD), where it accumulates in the form of cholesteryl esters (CE). These results highlighted the importance of cholesterol release from LDs for efflux; indeed, this process is increasingly being recognized as the rate-limiting step for RCT in vivo. Subsequent experiments aimed at elucidating the mechanisms that govern LD CE hydrolysis in macrophage foam cells lead to the discovery of a novel pathway involved in cholesterol efflux. Macrophage CE hydrolysis is classically defined as being entirely dependent on neutral CE hydrolases. In the second part of this study, we demonstrate that in addition to the canonical CE hydrolases, which mediate neutral lipid hydrolysis, lysosomal acid lipase (LAL) also participates in the hydrolysis of cytoplasmic CE. Autophagy is specifically triggered in macrophages by atherogenic lipoproteins and delivers LD CE to LAL in lysosomes, thus generating free cholesterol for efflux. This autophagy-mediated cholesterol efflux is a process that is primarily dependant on the ABCA1 transporter and, importantly, is important for whole-body RCT. Overall, the studies presented in this thesis support that macrophage LD CE hydrolysis is rate-limiting for cholesterol efflux and shed light on the mechanisms of cholesterol mobilization for efflux in macrophage foam cells.

The binding of CD80/CD86 on the APC to CD28 on the T cell surface provides a second signal for T cell activation. While it was once believed that this interaction represented a one-way signal, resulting in T cell activation, recently, it has been investigated as a bidirectional signaling process. CD80/86 activation produces IL-6 in DCs, but its role in macrophage activation is unknown. Dysregulation of CD80/86 expression has been observed in autoimmune disorders and cancer, and may also influence the development of immune responses including production of cytokines in response to stimulation with TLR-4 ligand, LPS. Therefore, the focus of my project was twofold: 1) to investigate the role of CD80/86 as signaling receptors capable of transmitting extracellular signals, and 2) to determine the TLR-4 activated pathways that regulate CD80/86 expression in human monocyte-derived macrophages (MDMs). Since I demonstrated that activation of CD80/86 alone did not induce expression of the four cytokines investigated, I hypothesized that CD80/86 synergizes with other signaling pathways. I show for the first time that CD80/86 activation synergizes with TLR-4 signaling to produce IL-27 and IL-10 in human MDMs. Since cIAPs play a key role in TLR-4-mediated signaling, I investigated their role in TLR-4- and CD80/86-activated production of IL-10 and IL-27. Degradation of IAPs by SMAC mimetics inhibited LPS-induced IL-10 and IL-27 production in MDMs. However, it did not alter the TLR-4 and CD80/86 synergistic effect on IL-10 and IL-27 production suggesting that IAPs may not play a role in CD80/86 activation of macrophages. Since I have demonstrated this role for IAPs, I extended my studies by examining the involvement of IAPs and other upstream signaling molecules such as SHP-1, RIP1, TRAF2, in modulating the LPS-induced CD80/86 expression. I showed that cIAP2, SHP-1, RIP1, TRAF2 co-localize to form a complex that regulates the LPS-induced CD80 and CD86 expression through AKT-activated p38 MAPK in human macrophages. These findings may lead to the development of novel therapeutic interventions in the treatment of autoimmune diseases.

Cell death plays a vital role throughout the immune response, from the onset of inflammation to the elimination of primed T cells. Understanding the regulation of cell death within immune cells is of vital importance to understanding the immune system and developing therapies against various immune-disorders. In this thesis I have investigated the regulation of cell death and its functional role in of the innate and adaptive arms of the immune system. The mechanisms that govern expansion and contraction of antigen stimulated CD8+ T cells are not well understood. In the first section of this thesis, I show that caspase-3 becomes activated in proliferating CD8+ proliferation, yet this does not result in cell death. I used both in vivo and in vitro models to demonstrate that caspase-3 activation is specifically driven by antigen presentation and not inflammation, and that it likely plays a role in promoting T cell proliferation. Next, I present novel data regarding the regulation of a newly identified form of programmed cell death via necrosis, known as necroptosis. I show that the cellular inhibitor of apoptosis (cIAP) proteins act to limit activation of key necroptosis proteins in macrophage cells. Furthermore, I show that necroptosis can be exploited by intracellular bacterial pathogens to escape removal by the immune system. I also demonstrate that necroptosis is highly intertwined with the pathway of inflammation, and the autocrine production of type-I interferon constitutes a vital positive feedback loop in the induction of inflammatory cell death. In the final section of my thesis work, I delve into the specific regulation of Rip1 kinase and demonstrate that in addition to previously demonstrated regulation by caspase-8, cathepsins are also able to cleave Rip1 kinase and limit necroptosis. This thesis presents a wide variety of novel data regarding the regulation of cell death within immune cells. In total, the results reveal a picture of two divergent forms of programmed cell death, apoptosis and necroptosis. Through improving the understanding of the cross-regulation of these two key cell death pathways this work aims to improve the understanding of the immune function.

Introduction: Increased activation of inflammatory pathways is associated with elevated metabolic stress, which leads to a constellation of metabolic pathologies like fatty liver, insulin resistance and atherosclerosis. Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a novel transcription co-factor that binds to and inhibits two main pro-inflammatory transcription factors, IRF2 and NFAT1. IRF2BP2 genetic variants are also linked to increased human serum cholesterol level in GWAS studies. Therefore, we hypothesized that IRF2BP2 may inhibit macrophage polarization to pro-inflammatory phenotype and considering the remarkable overlap between inflammatory and metabolic sensors, alter their metabolic function. We sought to determine if specific ablation IRF2BP2 in the mouse myeloid lineage (IRF2BP2MKO) leads to development of metabolic symptoms and alters the risk of atherosclerosis. Results: Our results indicate that IRF2BP2 ablation impairs macrophage polarization to the anti-inflammatory phenotype. IRF2BP2MKO bone marrow derived macrophages (BMDM) show increased oxidized LDL-cholesterol uptake and decreased cholesterol efflux. Also, mice with specific ablation of IRF2BP2 in macrophages are more susceptible to obesity, insulin resistance and hepatic steatosis compared to control mice, when fed high fat diet (HFD). However, LDLR-/- mice transplanted with IRF2BP2MKO bone marrow demonstrate similar extent of atherosclerotic lesions compared to LDLR-/- mice transplanted with control bone marrow, reflecting increased IRF2BP2MKO macrophage apoptosis. Conclusion: In conclusion, this is the first study to identify the metabolic and inflammatory functions of IRF2BP2 protein in macrophages, with important implications in metabolic syndrome and atherosclerosis.

L'athérosclérose est un processus physiopathologique chronique impliqué dans la majorité des maladies cardio-vasculaires. Le développement des lésions d'athérosclérose est caractérisé par l'accumulation de lipides extra et intracellulaires dans la paroi artérielle à l'origine d'une forte réponse inflammatoire impliquant notamment les macrophages. Les macrophages sont considérés comme des acteurs clés dans le développement des plaques d'athérosclérose. En effet, de par leur capacité à métaboliser le cholestérol (captation, stockage, efflux), à réguler l'inflammation et à phagocyter les cellules apoptotiques, ils exercent des fonctions pro et/ou anti-athèrogènes qui peuvent être modulées à des fins thérapeutiques. Dans cette perspective, nous avons évalué le pouvoir thérapeutique des " macrophages protégés de l'apoptose " sur la progression des lésions d'athérosclérose constituées. Nous avons démontré que l'augmentation de la survie des macrophages permet de ralentir la progression des lésions, de stabiliser les lésions et de diminuer la cholestérolémie. Ces effets athéro-protecteurs sont attribués à l'augmentation des cellules de Kupffer et des monocytes Ly-6Clow en partie par leur capacité à produire de l'apolipoprotéine E. Nous montrons également que les cellules de Kupffer participent à la clairance des lipoprotéines pro-athérogènes. L'augmentation du pool d'apoE ainsi que l'augmentation des cellules de Kupffer permettent de diminuer la cholestérolémie et ainsi de diminuer la progression des lésions. / Atherosclerosis represents a chronic pathophysiological process implicated in the majority of cardiovascular diseases. The development of atherosclerotic lesions is characterized by an accumulation of extra and intracellular lipids in the arterial wall at the origin of a strong inflammatory response involving macrophages.Macrophages are considered key actors in the development of atherosclerotic plaques. Indeed, because of their ability to metabolize cholesterol (capture, storage, efflux), to regulate inflammation and to phagocyte apoptotic cells, they exert pro and/or anti-atherogenic functions that may be modulated therapeutically. In this context, we evaluated the therapeutic potential of macrophages protected against apoptosis, on the progression of established atherosclerotic lesions.We have demonstrated that increased macrophage survival can slow down the progression of established lesions, stabilize lesion and reduce cholesterol levels. These athero-protective effects are attributed to the increase in Kupffer cells and Ly-6Clow monocytes partly due to their ability to produce apolipoprotein E. We also show that Kupffer cells are involved in the clearance of pro-atherogenic lipoproteins. The increase in ApoE pool and in Kupffer cells reduces cholesterol levels and thus lesion progression.

Les caspases sont des protéases à cystéine, très étudiées dans la mort cellulaire par apoptose et impliquées dans de nombreux autres processus de survie tels que la prolifération, l’inflammation et la différenciation. Au cours de la différenciation des monocytes en macrophages induite par le M-CSF, les caspases sont activées au sein d’un complexe multiprotéique composé de FADD, de RIP1, de FLIP et de la caspase-8. Ce complexe se forme indépendamment des récepteurs de mort et en réponse à des vagues d’activation de PI3K/Akt successives d’amplitudes et de durées croissantes prenant naissance au niveau du récepteur au M-CSF. La caspase-8, activée dans ce complexe, active par clivage les caspases effectrices, qui clivent à leur tour des protéines dont la Nucléophosmine. La Nucléophosmine, aussi appelée NPM, B23, NO38 ou Numatrine est une protéine chaperonne nucléolaire effectuant la navette entre noyau et cytoplasme. Cette protéine est impliquée dans de nombreux processus dont la ribogenèse, la duplication du centrosome ou la régulation de la transcription. Au cours de ma thèse, j’ai décrit le clivage de NPM au cours de la différenciation macrophagique. NPM est clivée une première fois par les caspases sur le site NGKD213 puis pas la cathepsine B. La surexpression du fragment de clivage généré par les caspases inhibe la phagocytose de bactéries ainsi que la migration cellulaire, sans affecter la phagocytose des corps apoptotiques suggérant que le clivage de NPM par les caspase participe au maintien au repos des macrophages. La protéolyse de NPM est arrêtée lorsque les macrophages sont activés par des ligands des TLR comme les LPS. La forme native s’accumule et inhibe la synthèse de cytokines in vitro et in vivo. NPM est recrutée sur le promoteur de MCP-1 en présence de LPS suggérant une fonction transcriptionnelle de NPM. / Caspases are cystein proteases whose function in apoptosis has been largely explored. Caspases play a role in various other cellular processes, such as proliferation, inflammation or differentiation. During M-CSF-driven macrophagic differentiation, caspases are activated in a multimolecular complex, including FADD, RIP1, FLIP and caspase-8. This complex is death receptor independent and is linked to M-CSFR by successive PI3K/Akt waves of increasing amplitude and duration. Caspase-8 is activated in this complex, activates downstream effector caspases, which in turn cleaves cellular targets such as Nucleophosmin. Nucleophsomin (also called NPM, B23, NO38 or Numatrin) is a nucleolar chaperon protein that shuttles between nucleus and cytoplasm. This protein is involved in various cellular processes such as ribogenesis, centrosome duplication or transcription regulation. During my PhD, I described NPM cleavages during macrophagic differentiation. NPM is first cleaved by caspases at NGKD213 site and then by cathepsin B. Caspases-generated fragment overexpression inhibits bacteria phagoctosis and migration without affecting apoptotic bodies engulfment, suggesting that caspase-mediated NPM cleavage maintains macrophages at steady-state. NPM proteolysis is abrogated upon macrophage activation by TLR ligands such as LPS. Full-length NPM accumulates and inhibits cytokine synthesis in vitro and in vivo. NPM is recruited on MCP-1 promoter, suggesting a transcriptionnal function for NPM.

Trichuris trichiura (T. Trichiura) is a large-intestinal dwelling nematode that affects over 1 billion people world-wide and thus has large global significance. Much of our understanding of T. trichiura infection comes from the study of the mouse model Trichuris muris (T. muris). However, how the immune system is initiated in response to helminth threat and how inflammation and pathology are resolved in T. muris infection still remain to be addressed. Here, I have attempted to provide insight into these questions. Previous work has shown resistance to T. muris infection is associated with the rapid recruitment of dendritic cells (DCs) to the colonic epithelium via epithelial production of CCL5 and CCL20. However, the epithelial-parasite interaction that drives chemokine production is not known. Pattern recognition receptor (PRRS) are critical mediators of pathogen recognition but there is no known (PRR) specific for T. muris. Here, we address the role of the cytosolic pattern recognition receptor Nod2, the location of which within the crypts correlates with the T. muris niche. In WT mice, in response to infection, there was a rapid influx of CD103+CD11c+ DCs into the colonic epithelium, whereas, this recruitment was impaired in Nod2 /- animals. In vitro and in vivo experiments confirmed the impairment in DC recruitment in Nod2-/- mice was attributable to the epithelial compartment. Subsequent work revealed decreased production of epithelial chemokines in the absence of functional Nod2. Thus, we have shown a novel role for Nod2 in the initiation the immune response to T. muris. We next addressed how pathology is regulated during T. muris infection. Firstly we investigated the role of arginase and Arg1-expressing macrophages in regulating pathology. My data showed that, unlike other gastrointestinal helminths, arginase and Arg1-expressing macrophages are not essential for resistance to T. muris or effective resolution of helminth-induced inflammation. I also addressed the role of DCs in the resolution of infection. DCs can regulate immune responses via the anti-inflammatory cytokine IL-10 and induction of regulatory T cells (Treg). I used an IL 10flox/floxCD11cCre transgenic model in which mice have DCs that cannot make IL-10. I found no role for CD11c+ cell mediated IL-10 production in the regulation of pathogen induced pathology in chronic T. muris infection. In summary I have been able to identify factors in the initiation of immunity to T. muris namely epithelial expression of Nod2. However, as arginase, Arg1-expressing macrophages and DC derived IL-10 appeared to play a redundant role in T. muris infection, the question as to how infection induced inflammation is resolved remains elusive.

No description available.