Interaction between microbiota and dendritic cells in mucosal tolerance

描述（由申请人提供）：树突状细胞（dc）是高度特化的抗原呈递细胞（apc），作为先天免疫和适应性免疫之间的细胞桥梁。最近的研究已经开始阐明dc在维持免疫耐受中的关键作用，特别是在粘膜表面，免疫细胞和共生微生物群（以及微生物群衍生的模式相关分子产物（PAMPs））之间发生实质性的相互作用。PAMPs通过dc上的toll样受体（TLRs）等模式识别受体（PRRs）触发激活和调节信号。特殊的粘膜DC亚群可能以某种方式处理信号和复杂的免疫调节因子，将它们与主要对微生物作为病原体作出反应的外周DC区分开来。由于这些背景差异，了解粘膜dc如何以促耐受性的方式与共生微生物群相互作用，对减轻自身免疫性和过敏性疾病具有重要的临床意义。我们之前已经证明TLR信号因子TRAF6是外周dc的关键激活因子，但TRAF6缺失小鼠的多器官缺陷至今仍阻碍了dc表达的TRAF6对免疫耐受的原位影响的检测。我们现在已经产生了dc特异性TRAF6缺陷小鼠(TRAF6？DC)，我们意外地发现其表现为：1)自发性纤维化嗜酸性粒细胞肠炎，2)粘膜th2相关免疫异常，以及3)iTreg发育缺陷。引人注目的是，这些表型需要共生微生物群。因此，我们建议TRAF6？将DC小鼠作为研究DC在粘膜耐受中的新平台，并将追求以下具体目标：研究共生肠道微生物群与dc相互作用以维持免疫稳态的机制。为了确认DC TRAF6、共生菌和免疫耐受之间的联系（并允许有针对性地操纵共生菌群），我们首先提出了TRAF6的重新衍生和表型重新分析。无菌条件下的DC小鼠。我们将进行细菌再引入实验，以跟踪细菌负荷的影响以及TRAF6？DC，免疫耐受，和卫生假说。其次，因为我们观察到TRAF6？我们将对DC小肠进行微生物谱分析，以研究DC固有信号是否影响肠道细菌种群动态。2. 描述驱动TRAF6耐受性丧失的细胞和分子机制？直流老鼠。TRAF6的分子功能与上游TLR适配器MyD88密切相关。TRAF6？DC肠道表型暗示DC TLR信号的关键作用，但我们发现MyD88？在DC区特异性缺乏MyD88的DC小鼠不表型TRAF6？直流。因此，我们将使用遗传模型来确定：1)微生物来源的刺激是否通过myd88独立通路直接通过DC tlr调节肠道免疫稳态，以及/或2)相同的刺激是否在非DC中利用myd88依赖通路来访问DC内traf6依赖的功能。

DESCRIPTION (provided by applicant): Dendritic cells (DCs) are highly specialized antigen-presenting cells (APCs) that act as cellular bridges between innate and adaptive immunity. Recent efforts have begun to clarify the critical role of DCs in maintaining immune tolerance, especially at mucosal surfaces, where substantial interaction occurs between immune cells and commensal microbiota (and microbiota-derived pattern-associated molecular products (PAMPs)). PAMPs trigger both activating and regulatory signals through pattern-recognition receptors (PRRs), like Toll-like receptors (TLRs), on DCs. Specialized mucosal DC subsets may process signals and elaborate immunomodulatory factors in a way that distinguishes them from peripheral DCs that primarily respond to microbes as pathogens. Because of these contextual differences, understanding how mucosal DCs interact with commensal microbiota in a pro-tolerogenic manner has major clinical implications for mitigation of autoimmune and allergic diseases. We have previously shown the TLR signaling factor TRAF6 is a critical activator of peripheral DCs, but multi-organ defects in TRAF6-null mice have until now prevented examination of in situ effects of DC-expressed TRAF6 on immune tolerance. We have now generated DC-specific TRAF6- deficient mice (TRAF6?DC), which we unexpectedly found exhibit: 1) spontaneous fibrotic eosinophilic enteritis, 2) aberrant mucosal Th2-associated immunity, and 3) defective iTreg development. Strikingly, these phenotypes require commensal microbiota. We therefore propose TRAF6?DC mice as a novel platform for study of DCs in mucosal tolerance, and will pursue the following specific aims: 1. Investigate the mechanism(s) by which commensal gut microbiota interact with DCs to maintain immune homeostasis. To confirm links between DC TRAF6, commensals, and immune tolerance in a more robust system (and to allow for targeted manipulation of commensal microbiota), we first propose re-derivation and phenotypic re-analysis of TRAF6?DC mice under germ-free conditions. We will conduct bacterial re-introduction experiments to track both effects on bacterial load and possible links between TRAF6?DC, immune tolerance, and the hygiene hypothesis. Second, because we have observed higher bacterial loads in TRAF6?DC small intestines we will perform microbiotic profiling to investigate whether DC-intrinsic signals affect gut bacterial population dynamics. 2. Characterize the cellular and molecular mechanisms driving loss of tolerance in TRAF6?DC mice. The molecular functions of TRAF6 and the upstream TLR adaptor MyD88 are closely linked. The microbiota-dependence of the TRAF6?DC gut phenotype implies a key role for DC TLR signaling, but we find that MyD88?DC mice, lacking MyD88 specifically in the DC compartment, do not phenocopy TRAF6?DC. Therefore, we will use genetic models to determine whether: 1) microbiota-derived stimuli regulate gut immune homeostasis directly through DC TLRs using a MyD88-independent pathway, and/or 2) the same stimuli utilize MyD88-dependent pathways in non-DCs in order to access TRAF6-dependent functions within DCs.