TNF-alpha Regulation of Intestinal Paracellular Transport

描述(申请人提供)：肠上皮细胞间的细胞旁转运或细胞旁渗透受紧密连接的调节。克罗恩病(CD)患者的肠道紧密连接(TJ)屏障有缺陷，表现为细胞旁通透性增加。肠道TJ屏障的缺陷是CD的一个重要致病因素，它允许有毒管腔抗原在细胞旁的渗透增加，从而导致肠道炎症。肿瘤坏死因子-1(TNF-1)是一种促炎细胞因子，已被证明在CD的肠道炎症过程中起核心作用。肿瘤坏死因子-1的一个重要的促炎作用是引起肠道TJ屏障的功能性开放，导致有害管腔抗原在细胞旁的渗透增加。肿瘤坏死因子-1诱导的肠道细胞旁通透性增加可能是导致CD的肠道TJ屏障缺陷和其他肠道炎症状态的重要机制。这项拨款提案的主要目的是阐明介导肿瘤坏死因子-1诱导的肠道TJ通透性增加的细胞和分子机制，并确定潜在的治疗靶点，以防止肿瘤坏死因子-1诱导的TJ屏障缺陷和随后的肠道炎症发展。为了实现这些目标，我们打算使用体外(由过滤生长的Caco-2肠道上皮细胞组成)和体内(小鼠肠道灌流系统)模型系统。我们的初步研究表明，肿瘤坏死因子-1诱导的肠上皮细胞TJ通透性增加部分是通过ERK1/2和p38信号通路诱导肌球蛋白轻链激酶(MLCK)基因活性增加和microRNA诱导occludin基因表达下调而实现的。在前期研究的基础上，我们提出了一个新的假说，即肿瘤坏死因子-1诱导的肠道TJ通透性增加部分是通过ERK1/2和p38信号通路诱导MLCK基因的激活和microRNA表达的调节而实现的。本研究的具体目的是：1)阐明介导肿瘤坏死因子-1诱导的MLCK基因活性和肠道TJ通透性增加的细胞内和分子过程；2)阐明介导肿瘤坏死因子-1下调occludin基因和蛋白表达的细胞和分子机制；以及3)阐明肿瘤坏死因子-1诱导的在体肠道通透性增加的机制，并确定靶向保留TJ屏障功能在肿瘤坏死因子-1诱导的肠炎症中的治疗意义。与公共卫生相关：克罗恩病患者有肠道渗漏，其特征是肠道对肠腔内有害抗原的渗透性增加。在这项拨款申请中提出的研究试图为导致克罗恩病肠道渗漏的细胞过程提供新的见解。这些研究还试图确定潜在的治疗目标和策略，以诱导克罗恩病渗漏肠道的治疗重新收紧或正常化。

DESCRIPTION (provided by applicant): Intestinal paracellular transport or paracellular permeation in-between intestinal epithelial cells is regulated by tight junctions. Patients with Crohn's disease (CD) have a defective intestinal tight junction (TJ) barrier, manifested by an increase in paracellular permeability. The defective intestinal TJ barrier is an important pathogenic factor of CD that allows increased paracellular permeation of toxic luminal antigens, leading to intestinal inflammation. Tumor necrosis factor-1 (TNF-1), a pro-inflammatory cytokine, has been shown to play a central role in the intestinal inflammation process of CD. An important pro-inflammatory action of TNF-1 is to cause a functional opening of the intestinal TJ barrier, leading to an increase in paracellular permeation of noxious luminal antigens. The TNF-1 induced increase in intestinal paracellular permeability has been postulated to be an important mechanism contributing to the intestinal TJ barrier defect in CD and other inflammatory conditions of the gut. The broad objectives of this grant proposal are to elucidate the cellular and molecular mechanisms that mediate the TNF-1 induced increase in intestinal TJ permeability and to determine the potential therapeutic targets to prevent the TNF-1 induced defect in TJ barrier and subsequent development of intestinal inflammation. To achieve these objectives, we intend to use both in-vitro (consisting of filter-grown Caco-2 intestinal epithelial cells) and in-vivo (mouse intestinal perfusion system) model systems. Our preliminary studies suggested that the TNF-1 induced increase in intestinal epithelial TJ permeability was regulated in part by ERK 1/2 and p38 signaling cascade induced increase in myosin light chain kinase (MLCK) gene activity and microRNA induced down-regulation of occludin gene expression. Based on our preliminary studies, we advance a novel hypothesis that the TNF-1 induced increase in intestinal TJ permeability is mediated in part by ERK1/2 and p38 signaling cascade induced activation of MLCK gene and modulation of microRNA expression. The proposed specific aims are to: 1) elucidate the intracellular and the molecular processes that mediate the TNF-1 induced increase in MLCK gene activity and intestinal TJ permeability; 2) delineate the cellular and molecular mechanisms that mediate the TNF-1 induced down- regulation of occludin gene and protein expression; and 3) delineate the mechanisms involved in TNF-1 induced increase in intestinal permeability in-vivo and determine the therapeutic implications of targeted preservation of TJ barrier function in TNF-1 induced intestinal inflammation. PUBLIC HEALTH RELEVANCE: Patients with Crohn's disease have a leaky gut, characterized by an increase in intestinal permeability to harmful antigens in the intestinal lumen. The studies proposed in this grant application seek to provide novel insight into the cellular processes that lead to the leaky gut of Crohn's disease. These studies also seek to identify potential therapeutic targets and strategies to induce therapeutic re-tightening or normalization of leaky gut in Crohn's disease.