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），充当先天免疫和适应性免疫之间的细胞桥梁。最近的努力已经开始阐明DC在维持免疫耐受中的关键作用，特别是在粘膜表面，其中免疫细胞和肠道微生物群（和微生物群衍生的模式相关分子产物（PAMP））之间发生实质性相互作用。PAMP通过DC上的模式识别受体（PRR）（如Toll样受体（TLR））触发激活和调节信号。专门的粘膜DC亚群可以处理信号并以一种将其与主要对作为病原体的微生物应答的外周DC区分开的方式精心制作免疫调节因子。由于这些背景差异，理解粘膜DC如何以促耐受性方式与肠道微生物群相互作用对于减轻自身免疫性和过敏性疾病具有重要的临床意义。我们先前已经表明TLR信号传导因子TRAF 6是外周DC的关键激活剂，但是TRAF 6缺失小鼠中的多器官缺陷直到现在还阻止了DC表达的TRAF 6对免疫耐受的原位作用的检查。我们现在已经产生了DC特异性TRAF 6缺陷小鼠（TRAF 6？DC），我们意外地发现其表现出：1）自发性纤维化嗜酸性粒细胞性肠炎，2）异常粘膜Th 2相关免疫，和3）缺陷性iTreg发育。引人注目的是，这些表型需要肠道微生物群。因此，我们建议TRAF 6？DC小鼠作为研究DC在黏膜耐受中作用的新平台，将致力于以下几个方面的研究：1.研究肠道微生物群与DC相互作用以维持免疫稳态的机制。为了确认DC TRAF 6，aptosals和免疫耐受之间的联系，在一个更强大的系统（并允许有针对性地操纵aptosal微生物群），我们首先提出重新推导和表型重新分析TRAF 6？无菌条件下的DC小鼠。我们将进行细菌再引入实验，以跟踪细菌负荷和TRAF 6？DC，免疫耐受和卫生假说。第二，因为我们观察到TRAF 6中的细菌载量较高？DC小肠我们将进行微生物分析，以研究DC内在信号是否影响肠道细菌种群动力学。2.表征TRAF 6耐受性丧失的细胞和分子机制？DC小鼠TRAF 6的分子功能与上游TLR衔接子MyD 88密切相关。TRAF 6的微生物群依赖性？DC肠道表型意味着DC TLR信号转导的关键作用，但我们发现，MyD 88？DC小鼠，缺乏MyD 88特别是在DC室，不表型TRAF 6？DC.因此，我们将使用遗传模型来确定：1）微生物群来源的刺激是否使用MyD 88非依赖性途径直接通过DC TLR调节肠道免疫稳态，和/或2）相同的刺激是否利用非DC中的MyD 88依赖性途径以获得DC内的TRAF 6依赖性功能。

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.