Endosomal Control of Microbe-Host Homeostasis

描述（由申请人提供）：肠上皮细胞（IEC）及其管腔微生物群落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。引起微生物-宿主相互作用失衡的遗传易感性可能有助于炎症性肠病（IBD）的发病机制。克罗恩病和溃疡性结肠炎的各种易感基因座已被确定，但是，其对疾病机制的贡献仍然不明确。染色体15 q22上的克罗恩病易感基因座与RAB 11 A直接相关。该基因编码一个小的鸟苷三磷酸酶（GT3），调节内体的再循环功能。申请人的实验室已经对小鼠Rab 11 a进行了遗传靶向，并分析了IEC特异性Rab 11 a敲除小鼠。初步数据表明，Rab 11 a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Rab 11 a缺陷的肠细胞过度产生促炎细胞因子，并引起早发性肠炎、对炎性肿瘤的更高易感性和过早死亡。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性粘膜免疫和微生物-宿主稳态。本提案的目的是建立Rab 11 a内体介导的Toll样受体（TLR）（特别是TLR 9）分选支持肠道粘膜对微生物群耐受的分子机制。核心假设是Rab 11 a将TLR 9隔离到非活性的内体区室，并在稳态条件下抑制对肠道微生物群的不必要的免疫应答。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中的不良炎症反应。该假设将以2个特定目的进行测试：（1）建立Rab 11 a内体区室作为细胞因子对微生物群响应的上皮细胞内在调节剂;和（2）确定Rab 11 a控制的TLR 9转运和激活响应微生物激动剂的机制。这些实验将使用新的Rab 11 a条件小鼠模型，该模型有助于诱导Rab 11 a缺失，IEC特异性果蝇Rab 11 RNA干扰（RNAi）系，RAB 11 A耗尽的人结肠上皮细胞系，以及微生物激动剂的体内灌注。这些遗传模型与体内生理分析相结合，研究了微生物-宿主体内平衡的内吞控制，这是创新性的，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.