TNF-alpha Regulation of Intestinal Paracellular Transport

描述（由申请人提供）：肠上皮细胞之间的肠细胞旁转运或细胞旁渗透是由紧密连接调节的。克罗恩病（CD）患者有缺陷的肠紧密连接（TJ）屏障，表现为细胞旁通透性增加。有缺陷的肠道TJ屏障是CD的一个重要致病因素，它允许毒性腔抗原增加细胞旁渗透，导致肠道炎症。肿瘤坏死因子-1 （Tumor necrosis factor-1, TNF-1）是一种促炎细胞因子，已被证明在CD的肠道炎症过程中发挥核心作用。TNF-1的一个重要促炎作用是引起肠TJ屏障的功能性打开，导致有害腔内抗原的细胞旁渗透增加。TNF-1诱导的肠旁细胞通透性增加被认为是导致CD和其他肠道炎症条件下肠TJ屏障缺陷的重要机制。本资助计划的主要目标是阐明介导TNF-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TNF-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（由过滤器培养的Caco-2肠上皮细胞组成）和体内（小鼠肠灌注系统）模型系统。我们的初步研究表明，TNF-1诱导的肠上皮TJ通透性增加部分受ERK 1/2和p38信号级联诱导的肌球蛋白轻链激酶（MLCK）基因活性增加和microRNA诱导的occludin基因表达下调的调控。基于我们的初步研究，我们提出了一个新的假设，即TNF-1诱导的肠道TJ通透性增加部分是由ERK1/2和p38信号级联诱导的MLCK基因激活和microRNA表达的调节介导的。提出的具体目标是：1)阐明介导TNF-1诱导的MLCK基因活性和肠TJ通透性增加的细胞内和分子过程；2)阐明TNF-1诱导occludin基因和蛋白表达下调的细胞和分子机制；3)阐明TNF-1诱导体内肠道通透性增加的机制，并确定靶向保护TJ屏障功能对TNF-1诱导的肠道炎症的治疗意义。公共卫生相关性：克罗恩病患者有漏肠，其特征是肠道对肠腔中有害抗原的渗透性增加。本基金申请中提出的研究旨在为导致克罗恩病漏肠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靶点和策略，以诱导克罗恩病中漏肠的治疗性再收紧或正常化。

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.