Regulation of Intestinal Tight Junction Barrier and Inflammation by Autophagy

项目摘要 我们实验室的长期目标是阐明肠道动态平衡的分子基础及其 肠道炎症的调节失调，并开发新的方法来预防和治疗 炎症性肠病(IBD)。位于肠道顶端的细胞间紧密连接(TJ) 上皮起着细胞旁屏障的作用，阻止有害的管腔抗原的渗透。肠失禁 TJ屏障功能是肠道疾病和IBD的关键致病因素。自噬(巨噬)是 一种细胞内降解系统，可提供不必要的或功能失调的细胞货物 在溶酶体内的双膜小泡(自噬小体)。新出现的证据表明，缺陷 自噬在IBD的易感性、病因和进展中起着重要作用。虽然临床数据 动物研究表明，IBD的肠道TJ屏障缺陷与肠道炎症之间存在直接联系 IBD患者和动物模型中自噬在肠上皮TJ屏障调节中的作用 仍然不为人知。我们的初步研究表明，自噬在促进 肠道TJ屏障。具体地说，自噬通过降解毛孔来降低细胞旁TJ的渗透性 形成紧密连接蛋白claudin-2提高屏障保护跨膜TJ蛋白水平 蛋白质封闭素。自噬的诱导导致溶酶体靶向Claudin-2的选择性增加 并使膜上封闭素的保留率增加。因此，我们的中心假设是 自噬选择性地调节TJ膜蛋白组成以诱导肠道的增强 TJ障碍。我们将以(1)描述细胞内小泡的具体目标来解释我们的假说 自噬诱导肠上皮细胞TJ屏障增强的转运机制；(2)阐明 细胞内信号在肠道TJ屏障自噬调节中的机制作用； 自噬增强小鼠肠道TJ屏障功能的保护作用 IBD模型的建立。我们将使用创新的技术工具，如体内siRNA技术，体内全长小鼠 结肠灌注、活体肠道TJ蛋白转运和高含量定量成像研究OUR 明确的目标。这项提议将提供新的见解，让人们了解自噬在 肠道屏障的动态平衡和弥合科学知识的差距，这对治疗将是重要的 防治IBD的努力。

Project Summary The long-term goal of our laboratory is to elucidate the molecular basis for intestinal homeostasis and its dysregulation in intestinal inflammation, and to develop novel approaches for prevention and therapy of inflammatory bowel diseases (IBD). The apically located inter-cellular tight junctions (TJ) within the intestinal epithelium act as a paracellular barrier and prevent permeation of noxious luminal antigens. Loss of intestinal TJ barrier function is a key pathogenic factor in intestinal disorders and IBD. Autophagy (macroautophagy) is an intracellular degradation system that delivers unnecessary or dysfunctional cellular cargo sequestered inside double-membrane vesicles (autophagosomes) to the lysosome. Emerging evidence shows that defects in autophagy play an important role in the susceptibility, etiology, and progression of IBD. Although clinical data and animal studies show a direct link between defective intestinal TJ barrier and intestinal inflammation in IBD patients and animal models of IBD, the role of autophagy in the regulation of intestinal epithelial TJ barrier remains unknown. Our preliminary studies indicated that autophagy plays a key role in the enhancement of intestinal TJ barrier. Specifically, autophagy reduces paracellular TJ permeability by degradation of the pore forming tight junction protein claudin-2 and increasing protein levels of barrier protective transmembrane TJ protein occludin. Induction of autophagy causes a selective increase in lysosomal targeting of claudin-2 from the membrane and causes an increase in membrane retention of occludin. Thus, our central hypothesis is that autophagy selectively modulates TJ membrane protein composition to induce an enhancement of the intestinal TJ barrier. We will address our hypothesis with the specific aims of (1) To delineate the intracellular vesicular trafficking mechanisms in autophagy-induced enhancement of intestinal epithelial TJ barrier; (2) To elucidate the mechanistic role of intracellular signaling in autophagy regulation of intestinal TJ barrier; and (3) To delineate the protective role of autophagy-mediated enhancement of intestinal TJ barrier function in murine models of IBD. We will use innovative technical tools such as in vivo siRNA technology, in vivo full length mice colon perfusion, in vivo intestinal TJ protein trafficking, and high content quantitative imaging to study our specific aims. This proposal will provide novel insights into the crucial role that autophagy plays in the homeostasis of intestinal barrier and bridge the gap in scientific knowledge that will be important for therapeutic efforts against IBD.