Inflammatory immune response is essential for maintaining the defense against invading pathogens, although its aberrant activation leads to impaired self tolerance and development of autoimmune pathologies. Interleukin-17A (also known as IL-17), is a major proinflammatory cytokine, which contributes to the development and maintenance of inflammation and provides protection against several bacterial and yeast infections. However, extreme activation of IL-17 signaling leads to autoimmune pathologies. Thus, a strict regulation of IL-17 signal transduction is crucial to prevent progression of autoimmunity. Non-degradative ubiquitination is one of the main mechanisms regulating IL-17 signaling. Main E3 ubiquitin ligase within this signal transduction is TRAF6, which is also participating in several signaling pathways within the immune system. Non-degradative polyubiquitin chains created within inflammatory signaling complexes recruit signaling proteins such as IKK complex and TAK1 kinase, crucial for triggering of NF-κB and MAPKs downstream pathways. However, activation of these pathways upon IL-17 is very weak in comparison with other inflammatory stimuli, indicating a presence of a strong negative feedback loop. In this thesis, we establish the role of several regulatory molecules in IL-17 signaling....

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21634号 / 医博第4440号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 三森 経世, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Interleukin-33 (IL-33) is a multifunctional cytokine that mediates local inflammation upon tissue damage. IL-33 is known to act on multiple cell types including group 2 innate lymphoid cells (ILC2s), Th2 cells, and mast cells to drive production of Th2 cytokines including IL-5 and IL-13. IL-33 signaling activity through transmembrane ST2L can be inhibited by soluble ST2 (sST2), which acts as a decoy receptor. Previous findings suggested that modulation of IL-13 levels in mice lacking decoy IL-13Rα2, or mice lacking IL-13, impacted responsiveness to IL-33. In this study, we used Il13-/- mice to investigate whether IL-13 regulates IL-33 activity by modulating the transmembrane and soluble forms of ST2. In Il13-/- mice, the effects of IL-33 administration were exacerbated relative to wild type (WT). Il13-/- mice administered IL-33 i.p. had heightened splenomegaly, more immune cells in the peritoneum including an expanded ST2L+ ILC2 population, increased eosinophilia in the spleen and peritoneum, and reduced sST2 in the circulation and peritoneum. In the spleen, lung, and liver of mice given IL-33, gene expression of both isoforms of ST2 was increased in Il13-/- mice relative to WT. Because IL-13 and IL-4 signal through a shared receptor complex IL-13Rα1/IL-4Rα, we also studied the combined deficiency of IL-4 and IL-13 using Il4rα-/- mice which are defective in both IL-4 and IL-13 signaling. Responses of Il4rα-/- mice were indistinguishable from those of Il13-/- mice in our model system of IL-33-induced inflammation, suggesting that IL-4 does not play a distinct role separate from IL-13 in regulation of IL-33 activity. Through in vitro experiments, we confirmed fibroblasts to be an IL-13-responsive cell type that can regulate IL-33 activity through production of sST2. This study elucidates the important regulatory activity that IL-13 exerts on IL-33 through induction of IL-33 decoy receptor sST2 and through modulation of ST2L+ ILC2s.

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Peripheral arterial disease (PAD) is a worldwide disease caused by atherosclerosis. It is a circulatory condition where narrowed blood vessels reduce blood flow to the peripheral such as legs. Although current gold standard treatment for advanced PAD patients is still based on surgical revascularization, there is no effective therapy for many patients that are not suitable for surgery. In addition, better recovery from surgical revascularization largely relies on angiogenesis in the adjacent ischemic tissue. Thus, novel pro-angiogenic therapies to improve post-ischemic neovascularization are urgently desired. However, current poor understanding of the roles of anti-inflammatory cytokines in angiogenesis prevents the development of these new therapies. We and others have reported that IL-35 is a newly identified inducible immunosuppressive heterodimeric cytokine in the IL-12 family. IL-35 is composed of p35 (IL-12A) and EBI3, and its receptors are comprised of homodimers or heterodimer of IL-12Rb2 and gp130 (IL-6ST). We have shown that IL-35 inhibits endothelial cell (EC) activation induced by lipopolysaccharide (LPS) or atherogenic lysophosphatidylcholine (LPC). At least partially through these new EC-dependent mechanisms, IL-35 inhibits inflammation in autoimmune diseases, infectious diseases, atherosclerosis, and tumors. Recent studies have indicated the role of IL-35 in angiogenesis in rheumatoid arthritis and different tumors. However, whether and how IL-35 regulates post-ischemic angiogenesis in peripheral artery disease are unrevealed. In our study, we used hindlimb ischemia (HLI) and Matrigel plug assay as in vivo angiogenesis models and wound healing assay as in vitro angiogenesis model to study the role and underlying mechanisms of IL-35-mediated angiogenesis. We made the following findings: 1) muscle in human and mouse has high angiogenic potential in physiological conditions; 2) angiogenic cytokines and chemokines including anti-inflammatory cytokines are predominantly regulated by inflammatory transcription factors; 3) IL-35 signaling is induced in ischemic muscle; 4) IL-12Rb2, but not IL-6ST, is the key receptor component of IL-35 signaling in ischemic muscle and hypoxic human microvascular endothelial cells (HMVECs); 5) hyperlipidemia (atherogenic factor) impairs angiogenesis in vivo and in vitro, which partially acts through the induction of IL-35; 6) IL-12Rb2 deficiency improves HLI-induced angiogenesis in both WT or apolipoprotein E (ApoE) -/- mice (an atherosclerosis model); 7) IL-35 injection inhibits HLI-induced angiogenesis in WT mice but not that in the IL-12Rb2 deficient mice; 8) IL-35 injection enlarges the avascular area in gastrocnemius muscle after HLI; 9) IL-35 obstructs fibroblast growth factor-2 (FGF2)-induced angiogenesis in Matrigel plug assay in vivo; 10) CD45-CD31+ ECs from the IL-35-injected ischemic muscle at day 14 of HLI have an abnormal extracellular matrix organization, activated integrin pathways (cell-matrix adhesions), disrupted vascular endothelial (VE)-cadherin-plakoglobin complex (cell-cell adhesions), and increased infiltration and migration of bone marrow-derived leukocytes; 11) IL-35 inhibits HMVEC migration in wound healing assay in vitro presumably through upregulation of anti-angiogenic proteins including pigment epithelium-derived factor (PEDF), serpin family B member 5 (SERPINB5, Maspin), and thrombospondin (THBS)-1. These results suggest that anti-inflammatory cytokine IL-35, signaling through the key receptor subunit IL-12Rb2, inhibits HLI-induced angiogenesis and delays tissue repair by dysregulating cell-cell and cell-matrix adhesions, which leads to the impaired vascular adhesion junction and maturation of blood vessels. / Biomedical Sciences

Epithelial cells are the first line of defense against invading pathogens and external threats in the environment. Keratinocytes, often not perceived of as immune cells, release cytokines in response to infection or injury to signal danger to neighboring cells and recruit effector leukocytes to prevent further damage to the host. IL-1 and IL-36 cytokines are a group of closely related proteins that share similarities in structure and function and have been shown to play key roles in inflammatory responses of epithelial tissues. While IL-1, consisting of IL-1α and IL-1β, have been widely studied and recognized as pinnacle cytokines in a variety of inflammatory responses, relatively little is understood about IL-36 cytokines since their discovery more than 15 years ago, and how they differ from their better-known IL-1 relatives. IL-36 cytokines, consisting of IL-36α, IL-36β, and IL-36γ, signal through the same receptor, IL-36R, which is expressed most abundantly on epithelial cells. IL-36 proteins garnered attention when it was discovered that a missense mutation in the gene encoding the naturally occurring receptor antagonist, IL-36Ra, was associated with the deadly form of psoriasis, generalized pustular psoriasis (GPP). This disease is characterized by episodic flares of keratinocyte hyperproliferation leading to red scaly lesions all over the body, excessive neutrophil recruitment to the epidermis resulting in pustule formation, and severe fever. Our data presented here demonstrate that IL-36α, but not IL-36β or IL-36γ is critical for the psoriatic phenotype, including epidermal thickening and neutrophil recruitment, generated during a murine model of psoriasis induced by the drug Imiquimod. Furthermore, IL-36α was found to induce IL-1α expression and vice versa through a signaling feedback loop which perpetuated disease. These data provide insight into mechanisms whereby IL-36 signaling can lead to excessive inflammatory effects in patients with pre-existing regulation deficiencies, which can lead to acute flares of disease. Beyond their association with disease, IL-1 has been shown to contribute to anti-bacterial and anti-viral responses of the immune system by upregulating inflammatory signals and chemoattractants. Herpes Simplex Virus-1 (HSV-1) is a human pathogen that has developed several strategies to manipulate elements of the immune system to avoid detection by the host. One such mechanism is the prevention of activation and release of IL-1β from infected cells thereby blocking its pro-inflammatory responses. Our data show that keratinocytes infected with HSV-1 actively release IL-1α to alert danger to neighboring cells to circumvent this blockage of IL-1β signaling. This release of IL-1α initiates recruitment of leukocytes to early HSV-1 microinfection sites resulting in increased protection against disease, as evident by the increased mortality rate of mice deficient in the IL-1 receptor, IL-1R1. This study, for the first time in vivo, demonstrates the ability of IL-1α to act as an alarmin to initiate an immune response to combat infection. The role of IL-36 cytokines during viral infections has been less defined than that of IL-1. Several studies have shown the upregulation of IL-36 expression during viral infections in epithelial tissues, such as HSV-1 and Influenza, yet a direct link has not been established between these proteins and anti-viral responses. Our research presented within this thesis show that IL-36β, but not IL-36α nor IL-36γ, provides protection against the lethal outcome of cutaneous HSV-1 infection, as demonstrated by IL-36β knockout mice dying earlier and more often than wild type mice. Surprisingly, while previous reports have found IL-36 cytokines to be capable of activating the adaptive immune system, our results found no significant differences in development of HSV-1 specific antibodies or CD8+ T cell development between wild type and IL-36β knockout mice. Furthermore, we found no significant differences in viral copy numbers at infection sites between the two groups. Although our data show that IL-36β clearly plays a critical role in controlling the outcome of HSV-1 infection, further studies are necessary to define the mechanisms behind this protection. The final section of this thesis focuses on the endogenous nature of IL-36 cytokines, specifically IL-36γ, and their potential processing. IL-36 cytokines were originally believed to be synthesized as full-length fully active proteins; however, large concentrations of the recombinant proteins were required to elicit cellular responses in vitro. Since then, studies have shown that IL-36 cytokines gained up to 1000-fold increases in reactivity following processing at very specific N-terminal locations of each individual cytokine, however this processing has never been shown to occur in vivo. These studies were recently expanded when neutrophil proteases were found to be responsible for processing of these proteins in vitro. Data presented here show, for the first time, that IL-36γ may be endogenously processed by neutrophils in wounded murine skin in vivo, yet, the amino acid processing site appears to be different from that predicted. Although further studies are required to fully characterize the nature of this processing, these data provide valuable insight into the natural mechanisms involved in the potential activation of these cytokines. Taken together, the research presented within this thesis sheds light on the mechanisms whereby IL-1 and IL-36 cytokines enhance immunological defenses against potential threats, and yet, can contribute to disease if unregulated. Furthermore, these studies demonstrate the evolutionary advantage of producing multiple cytokines that appear to have redundant roles within the body, yet can provide multiple levels of protection to the host. This knowledge contributes to our overall understanding of these proteins and their contribution to immunological systems within the body. / Microbiology and Immunology

Yes / The IL-1 family member cytokine IL-36γ is recognised as key mediator in the immunopathology of psoriasis, hallmarks of which involve the activation of both resident and infiltrating inflammatory myeloid cells and aberrant angiogenesis. This research demonstrates a role for IL-36γ in both myeloid activation and angiogenesis. We show that IL-36γ induces the production of psoriasis-associated cytokines from macrophages (IL-23 and TNFα) and that this response is enhanced in macrophages from psoriasis patients. This effect is specific for IL-36γ and could not be mimicked by other IL-1 family cytokines such as IL-1α. IL-36γ was also demonstrated to induce endothelial tube formation and branching, in a VEGF-A-dependent manner. Furthermore, IL-36γ-stimulated macrophages potently activated endothelial cells and led to increased adherence of monocytes, effects that were markedly more pronounced for psoriatic macrophages. Interestingly, regardless of stimulus, psoriasis monocytes showed increased adherence to both the stimulated and unstimulated endothelium when compared with monocytes from healthy individuals. Collectively, these findings show that IL-36γ has the potential to enhance endothelium directed leucocyte infiltration into the skin and strengthen the IL-23/IL-17 pathway adding to the growing evidence of pathogenetic roles for IL-36γ in psoriatic responses. Our findings also point to a cellular response, which could potentially explain cardiovascular comorbidities in psoriasis in the form of endothelial activation and increased monocyte adherence. / Faculty of Life Sciences, University of Bradford. MRC, Grant/Award Number: MR/M01942X/1; British Skin Foundation, Grant/Award Number: BSF 5035.