Endosomal Control of Microbe-Host Homeostasis

描述（由申请人提供）：肠上皮（IEC）及其管腔微生物群作为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。导致微生物与宿主相互作用失衡的遗传倾向可能导致炎症性肠病（IBD）的发病机制。克罗恩病和溃疡性结肠炎的各种易感位点已被确定；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 直接连锁。该基因编码一种小鸟苷三磷酸酶（GTPase），可调节内体循环功能。申请人的实验室对小鼠Rab11a进行了基因靶向，并分析了IEC特异性Rab11a基因敲除小鼠。初步数据表明 Rab11a 控制上皮细胞内在炎症细胞因子对肠道微生物群的反应。果蝇和小鼠肠道中缺乏 Rab11a 的肠细胞会过量产生促炎细胞因子，并导致早发性肠炎、对炎性肿瘤的易感性更高以及过早死亡。长期目标是了解 IEC 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。该提案的目的是建立分子机制，通过 Rab11a 内体介导的 Toll 样受体 (TLR)（特别是 TLR9）分选，支持肠粘膜对微生物群的耐受性。中心假设是 Rab11a 将 TLR9 隔离到不活跃的内体隔室中，并抑制稳态条件下对肠道微生物群的不需要的免疫反应。有关 IEC 介导的炎症细胞因子产生和免疫反应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具体目标进行检验：（1）建立 Rab11a 内体区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 转运和响应微生物激动剂激活的机制。实验将使用新型 Rab11a 条件小鼠模型，该模型促进诱导型 Rab11a 缺失、IEC 特异性果蝇 Rab11 RNA 干扰 (RNAi) 系、RAB11A 耗尽的人结肠上皮细胞系以及微生物激动剂的体内灌注。在微生物-宿主稳态的内吞控制研究中将这些遗传模型与体内生理分析相结合是创新的，也是理解 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.