Innate Determinants of Microbial Immunity

微生物免疫的先天决定因素

• 批准号: 7732692
• 负责人: Alan Sher
• 金额: $ 64.78万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732692
• 关键词: Ablation; Address; Animals; Antigens; Autoimmune Diseases; Autophagocytosis; Bacterial Infections; Biological Assay; Blood; Blood Cells; Blood Platelets; Bone Marrow; Bone Marrow Cells; CD4 Positive T Lymphocytes; Cell Proliferation; Cell Survival; Cells; Cessation of life; Chemicals; Condition; Data; Defect; Dendritic Cells; Development; Disease; Disease Resistance; Disease model; Effector Cell; Event; Exhibits; Family; Fluorouracil; Freund' s Adjuvant; Generations; Genes; Genus Mycobacterium; Glycoproteins; Goals; Guanosine Triphosphate Phosphohydrolases; Heating; Hematopoietic; Hematopoietic stem cells; Host Defense; Host resistance; Immune; Immune response; Immunity; Immunization; Immunologics; In Vitro; Infection; Inflammatory Response; Interferon Type II; Interferons; Interleukin-1; Interleukin-17; Life; Lymphocyte; Lymphopenia; Mature Lymphocyte; Mediating; Mus; Mutation; Mycobacterium Infections; Mycobacterium avium; Mycobacterium tuberculosis; Natural Immunity; Outcome; Ovalbumin; Ovum; Pancytopenia; Pathway interactions; Peripheral; Pharmaceutical Preparations; Play; Population; Process; Proliferating; Proteins; Receptor Signaling; Regulation; Ribonucleases; Role; Role playing therapy; Schistosoma mansoni; Signal Pathway; Signal Transduction; Stem cells; Stimulus; Stress; T-Lymphocyte; T-Lymphocyte Subsets; TLR2 gene; Toll-Like Receptor 1; Toll-like receptors; Transgenic Organisms; Trypanosoma cruzi; Vaccines; Work; base; cytokine; egg; in vitro Assay; in vivo; in vivo Model; killings; macrophage; microbial; mortality; novel; pathogen; prevent; protein aminoacid sequence; receptor; response; stem

The host defense related cytokine IFN-gamma induces the expression of a broad array of genes in both hematopoietic and nonhematopoietic cells. A dominant set of these genes belong to the p47kDa GTPase family. Mice deficient in one gene in this family, Irgm1 (LRG47), are highly susceptible to a wide variety of different intracellular bacterial and protozoan pathogens and we have been investigating the immunologic mechanisms underlying this profound and far reaching defect in host resistance and its relationship to IFN function. Although Irgm1 has been shown to regulate the killing of pathogens inside IFN-gamma-activated macrophages in vitro, this is not its only function in host resistance. Thus, we have shown previously that Mycobacterium avium and Trypanosoma cruzi infected Irgm1-/- mice undergo a profound loss in lymphocytes, platelets and other bone marrow derived cells, a phenomenon referred to as 'bone marrow failure". In work completed this year we analyzed the mechanism responsible for this defect in blood cell generation/survival in Irgm-1 deficient animals focussing on both the bone marrow itself and the periphery where immune responses to pathogens occur. With a collaborator Margaret Goodell at Baylor, we first examined the role of hematopoietic stem cells (HSCs) which are self-renewing bone marrow cells that give rise to all blood lineages and retain the capacity to proliferate in response to different forms of exogenous stress. We found that Irgm1 inhibits baseline HSC proliferation and is required for a normal HSC response to chemical and infectious stimuli. HSCs from IRGm-1 deficient mice were found to be severely impaired in functional repopulation assays, and when subjected in vivo to hematopoietic ablation with the drug 5-fluorouracil or infection with Mycobacterium avium, the mice failed to undergo the expected expansion in stem and progenitor cell populations. This impaired expansion of HSC and progenitor cells likely contributes to the defective host resistance Irgm1-/- to infection by limiting the generation of new effector cells needed to replace those attacking the pathogen. In the second part of this project, we addressed the possible role of Irgm1 in the IFN-gamma dependent regulation of cell survival in the periphery. Focusing on CD4+ T lymphocytes we showed that IFN-gamma induced Irgm1 is critical for the survival of activated mature lymphocytes since in the absence of Irgm1, IFN-gamma triggers death of CD4+ T cells in vitro by a novel mechanism involving autophagy. Importantly, the lymphopenia and mortality previously observed in M. avium-infected Irgm1-/- animals was completely abrogated in Irgm1-/- IFN-gamma -/-double deficient mice, demonstrating that the presence or absence of Irgm1 determines the host protective vs detrimental outcome of IFN-gamma induction during pathogen encounter. These findings reveal an unexpected regulatory function for Irgm1 in T lymphocyte survival and suggest that the GTPase, in addition to its role in regulating stem cell/progenitor cell renewal, is essential for the peripheral expansion of immune effector cells during Th1 responses to intracellular pathogens Additional projects undertaken during the year addressed the innate pathogen signals responsible for triggering Th2 and Th17 effector cell development. We and other groups had previously shown that Schistosoma mansoni eggs contain factors that trigger potent Th2 responses in vivo and condition murine dendritic cells (DC) to promote Th2 lymphocyte differentiation in vitro. Using the latter assay which involves bystander polarization by DC of OVA specific TCR transgenic T cells, we purified the major Th2-inducing component from egg extracts and identified it by its peptide sequence as the secreted T2 ribonuclease, omega-1 an egg glycoprotein previously characterized by another group. Importantly, omega-1-exposed DC displayed pronounced cytoskeletal changes and exhibited decreased antigen dependent conjugate formation with CD4 T cells an in vitro assay. Based on this evidence we propose that S. mansoni omega-1 acts on DC and limits their interaction with CD4 T lymphocytes. By so doing the molecule lowers the strength of the activation signal that the DC deliver to the cells thereby creating a setting that favors Th2 differentiation. In a related study, we investigated the mechanism by which mycobacteria promote Th17 differentiation. Th17 cells represent a newly discovered CD4+ T cell subset that appears to play an important role in host inflammatory responses and immunity to certain pathogens. Th17 responses have been shown to function in vaccine induced protection against Mycobacterium tuberculosis. In addition heat killed M. tuberculosis is a critical component in Complete Freunds' Adjuvant (CFA) where it promotes the development of Th17 as well as Th1 responses to protein antigens co-administered with it. Indeed, CFA immunization forms the basis of many autoimmune disease models that require Th17 induction. In our work this year we investigated the specific role played by MyD88 dependent signaling pathways in the induction of Ag specific Th17 versus Th1 responses following immunization with ovalbumin in CFA. MyD88 is in adaptor molecule required for signaling by both toll-like receptors (TLR) and the Interleukin-1 (IL-1) receptor and mice deficient in this protein are typically used to assess the role of TLR in immune reponses. As predicted, we observed a major requirement for MyD88 signaling in the induction of Ag specific CD4+ T cell IFN-gamma and IL-17 responses following CFA/Ova immunization. Mice doubly deficient in TLR2 and TLR9, two TLR previously shown to be stimulated by live M. tuberculosis, displayed only partial defects in these CFA induced Th1 and Th17 responses. In contrast, mice deficient in IL-1R, showed a near complete loss in Th17 and a major reduction in Th1 responses. Taken together these findings suggest that TLR signaling may play a limited role in the effects of CFA on Th17 polarization and only partially explain its effects on Th1 induction. Instead they argue that CFA triggers Th17 polarization through an IL-1R dependent signaling cascade. Preliminary data implicate an upstream requirement for inflammasome mediated IL-1 processing in this function. In addition to providing information concerning the innate recognition pathways stimulated by mycobacteria that contribute to adaptive CD4+T responses, these findings establish a convenient and easily manipulated in vivo model for dissecting the unique signals involved in Th17 polarization.

与宿主防御相关的细胞因子IFN-GAMMA诱导造血和非杂造细胞中广泛基因的表达。这些基因的主要集合属于P47KDA GTPase家族。在该家族中缺乏一个基因的小鼠IRGM1（LRG47）非常容易受到各种不同的细胞内细菌和原生动物病原体的影响，我们一直在研究这种宿主耐药性及其与IFN功能的关系，这些免疫机制及其在宿主耐药性及其与IFN功能的关系中所具有的远处的免疫机制。尽管已证明IRGM1在体外调节IFN-gamma激活巨噬细胞内病原体的杀死，但这并不是其在宿主抗性中的唯一功能。因此，我们先前已经表明，硫杆菌和克鲁兹锥虫感染了IRGM1 - / - 小鼠在淋巴细胞，血小板和其他骨髓衍生的细胞中经历了严重损失，这种现象被称为“骨髓失败”，这是“骨髓失败”。在这一年中，我们分析了这种动物的污染，我们分析了动物的污染，使其在流血上的污点上的污染中的污染范围固定在流动中，并在流血中污染了，并在污染中脱颖而出。骨髓本身和对病原体的免疫反应与贝勒的合作者发生的外围，我们首先检查了造血干细胞（HSC）的作用，这是自我更新的骨骨髓细胞，这些细胞会引起所有血统的能力，并促进了对不同的压力的响应。 HSC对化学和感染性刺激的正常反应在功能重生测定中受到严重损害的HSC，并且在体内受到造血的造成造血5-氟尿嘧啶或感染的小鼠的预期群体的繁殖量，并在体内受到严重损害。 HSC和祖细胞的扩张受损可能会导致宿主抗性IRGM1 - / - 通过限制替代攻击病原体所需的新效应细胞的产生来感染。在该项目的第二部分中，我们解决了IRGM1在外围细胞存活的IFN-gamma依赖性调节中的可能作用。专注于CD4+ T淋巴细胞，我们表明IFN-GAMMA诱导的IRGM1对于活化成熟的成熟淋巴细胞的存活至关重要，因为在缺乏IRGM1的情况下，IFN-Gamma触发了涉及自动摄影的新机制在体外触发CD4+ T细胞的死亡。重要的是，在IRGM1 - / - IFN-GAMMA - / - 双重缺陷小鼠中完全废除了先前在雄性雄性雄性雄性捕获狂热的IRGM1-/ - 动物中观察到的淋巴细胞减少症和死亡率，这表明IRGM1的存在或不存在IRGM1的存在确定宿主保护性VS在IFN-Gamma诱导过程中的宿主保护性VS在病理过程中诱导。这些发现揭示了IRGM1在T淋巴细胞存活中的意外调节功能，并表明GTPase除了其在调节干细胞/祖细胞更新中的作用外，对于TH1对细胞内病原体的TH1反应过程中免疫效应细胞的外围膨胀至关重要。 这一年进行的其他项目介绍了负责触发TH2和TH17效应细胞开发的先天病原体信号。我们和其他组先前已经表明，曼森卵血吸虫包含在体内引发有效的Th2反应的因素，并条件鼠树突状细胞（DC）促进体外Th2淋巴细胞分化。使用后一种测定，该测定法涉及OVA特异性TCR转基因T细胞的DC旁观者极化，我们从鸡蛋提取物中纯化了主要的Th2诱导成分，并通过其肽序列将其鉴定为分泌的T2核糖核酸酶，Omega-1，Omega-1蛋白，先前由另一组表征。 重要的是，欧米茄-1暴露的DC显示出明显的细胞骨架变化，并显示出与CD4 T细胞和体外测定法的抗原依赖性共轭形成。基于此证据，我们建议曼氏链球菌对DC作用，并限制其与CD4 T淋巴细胞的相互作用。通过这样做，分子降低了激活信号的强度，即直流传递到细胞，从而产生了有利于Th2分化的设置。 在一项相关研究中，我们研究了分枝杆菌促进Th17分化的机制。 Th17细胞代表了新发现的CD4+ T细胞子集，该子集似乎在宿主炎症反应和对某些病原体的免疫力中起重要作用。 TH17的反应已显示在疫苗诱导的结核分枝杆菌中起作用。另外，杀死结核分枝杆菌的热量是完整弗朗德斯（CFA）中的关键组成部分，在该组件中，它促进了Th17的发展以及对与之共同管理的蛋白质抗原的Th1反应。实际上，CFA免疫构成了许多需要Th17诱导的自身免疫性疾病模型的基础。在今年的工作中，我们调查了MyD88依赖性信号通路在CFA中用椭圆蛋白免疫后的Ag特异性TH17与Th1反应的特定作用。 MyD88是通过Toll样受体（TLR）和白介素-1（IL-1）受体（IL-1）受体和缺乏该蛋白质中的小鼠的信号传导所需的衔接分子中，通常用于评估TLR在免疫再生中的作用。正如预测的那样，我们观察到在CFA/OVA免疫后，在诱导Ag特异性CD4+ T细胞IFN-GAMMA和IL-17响应中，MyD88信号的主要要求。小鼠在TLR2和TLR9中的双重缺陷，先前证明的两个TLR被活结核分枝杆菌刺激，仅在这些CFA诱导的TH1和TH17响应中显示出部分缺陷。相比之下，缺乏IL-1R的小鼠在TH17中显示出几乎完全完全损失，而Th1反应的大大降低。综上所述，这些发现表明TLR信号传导在CFA对TH17极化的影响中起着有限的作用，并且只能部分解释其对TH1诱导的影响。相反，他们认为CFA通过IL-1R依赖的信号级联反应触发TH17极化。初步数据暗示了该功能中炎症体介导的IL-1处理的上游要求。除了提供有关有助于自适应CD4+T响应的分枝杆菌刺激的先天识别途径的信息外，这些发现还建立了一种方便易于操纵的体内模型，以剖析涉及Th17极化涉及的独特信号。