Endosomal Control of Microbe-Host Homeostasis

描述（由申请人提供）：肠上皮（IEC）及其肠道菌群是一种理想的实验和医学模型，在组织再生环境中融合了先天免疫识别。引起不平衡的微生物-宿主相互作用的遗传易感性可能有助于炎症性肠病（IBD）的发病机制。已经确定了克罗恩病和溃疡性结肠炎的各种易感位点；然而，它们对疾病机制的贡献仍然不明确。染色体15q22上的克罗恩病易感位点与RAB11A直接相关。该基因编码一个小的鸟苷三磷酸酶（GTPase），该酶调节再循环内体的功能。申请人实验室对小鼠Rab11a进行了基因定位，并分析了iec特异性Rab11a敲除小鼠。初步数据表明Rab11a控制上皮细胞对肠道微生物群的内在炎症细胞因子反应。果蝇和小鼠肠道中缺乏Rab11a的肠细胞过量产生促炎细胞因子，导致早发性肠炎，对炎性瘤变的易感性更高，以及过早死亡。长期目标是了解IECs中微生物受体的内体分选如何影响先天粘膜免疫和微生物-宿主稳态。本研究的目的是建立Rab11a内体介导的toll样受体（TLRs）分选，特别是TLR9，支持肠道黏膜对微生物群耐受的分子机制。核心假设是Rab11a将TLR9隔离到无活性的内核室，并在稳态条件下抑制对肠道微生物群的不必要免疫反应。了解到的有关iec介导的炎症细胞因子产生和免疫反应的调节的信息可能有助于减少IBD的不良炎症反应。这一假设将通过两个特定目的进行验证：(1)建立Rab11a内体室作为细胞因子对微生物群反应的上皮细胞内在调节剂；(2)确定rab11a控制的TLR9转运和激活响应微生物激动剂的机制。实验将使用新的Rab11a条件小鼠模型，该模型促进诱导Rab11a缺失，iec特异性果蝇Rab11 RNA干扰（RNAi）系，Rab11a缺失的人结肠上皮细胞系以及微生物激动剂的体内灌注。将这些遗传模型与体内生理分析相结合，研究微生物-宿主稳态的内吞控制是创新的，也是了解IBD的重要一步。

DESCRIPTION (provided by applicant): The intestinal epithelium (IEC) with its luminal microflora serves as an ideal experimental and medical model that merges innate immune recognition in the context of a tissue regeneration environment. Genetic predispositions that cause imbalanced microbe-host interaction may contribute to the pathogenesis of inflammatory bowel disease (IBD). Various susceptibility loci for Crohn's disease and ulcerative colitis have been identified; however, their contributions to the disease mechanism remain poorly defined. A Crohn's disease susceptibility locus at chromosome 15q22 is immediately linked to RAB11A. This gene encodes a small guanosine triphosphatase (GTPase) that regulates the recycling endosome function. The applicant's laboratory has genetically targeted the mouse Rab11a, and analyzed IEC-specific Rab11a knockout mice. Preliminary data suggested that Rab11a controls epithelial-cell-intrinsic inflammatory cytokine response to enteric microbiota. Enterocytes deficient in Rab11a in Drosophila and mouse intestines overproduced proinflammatory cytokines, and caused early-onset enteritis, higher susceptibility to inflammatory neoplasia, and premature mortality. The long- term goal is to understand how the endosomal sorting of microbial receptors in IECs influences innate mucosal immunity and microbe-host homeostasis. The objective of this proposal is to establish the molecular mechanisms, by which Rab11a endosome-mediated sorting of Toll-like receptors (TLRs), in particular TLR9, support intestinal mucosal tolerance to microbiota. The central hypothesis is that Rab11a sequesters TLR9 to an inactive endosomeal compartment and dampens unwanted immune response to enteric microbiota at steady state conditions. Information learned about IEC-mediated modulation of inflammatory cytokine production and immune response may help reduce the adverse inflammatory response in IBD. This hypothesis will be tested with 2 specific aims: (1) to establish Rab11a endosomal compartment as an epithelial-cell-intrinsic modulator of cytokine response to microbiota; and (2) to determine the mechanism of Rab11a-controlled TLR9 transport and activation in response to microbial agonists. The experiments will use the novel Rab11a conditional mouse model that facilitates inducible Rab11a deletion, IEC-specific Drosophila Rab11 RNA interference (RNAi) lines, RAB11A-depleted human colon epithelial cell lines, and in vivo perfusion of microbial agonists. The combination of these genetic models with in vivo physiologic analyses in the study of endocytic control of microbe-host homeostasis is innovative and a major step toward understanding IBD.