Mechanisms and pathways of trans-tight junction conductance

描述(申请人提供)：炎症性肠病(IBD)和其他肠道疾病患者的肠道屏障功能降低。形成主要的细胞旁屏障的紧密连接的变化，通过增加细胞旁离子和分子的通量而导致屏障的丧失。已发表的报告和我的初步数据评估了不同炎性细胞因子在IBD患者固有层中的作用，支持至少两种紧密连接屏障调节机制由炎性细胞因子激活的假设。肿瘤坏死因子通过封闭蛋白内化机制诱导紧密连接功能障碍，该机制改变紧密连接的大小选择性，并允许增加大分子通量。相反，IL-13激活一条功能不同的途径，涉及Claudin蛋白的表达，该途径影响离子选择性，但不增加大分子通量。尽管在理解细胞因子诱导的屏障调节过程中取得了进展，但定义大小或离子选择性变化的分子事件仍不清楚。这在一定程度上代表了屏障函数的传统时间和空间平均测量所构成的技术障碍。我假设紧密连接屏障在局部亚微米水平上是高度动态的，细胞旁通量的调节是通过调节涉及一类以上紧密连接孔或其他传导途径的开放和关闭“事件”而发生的。由于紧密连接跨越两个细胞，它不适用于分子和生物物理分析和特定药理调节剂的开发，例如那些存在于跨膜离子通道的调节剂。为了消除现有方法和分子理解上的这一差距，我开发了一种新的方法来分析局部亚微米级的紧密结势垒函数，使用高分辨率的单电极膜片钳技术。虽然这种方法被广泛应用于跨膜离子通道和转运体的研究，但还没有被应用于紧密连接功能的研究。我使用这种方法的初步记录显示了以前未被识别的屏障动力学，我假设这种行为是作为选择性通透性屏障的正常紧密连接功能的基础，也是理解疾病中屏障功能障碍的多个途径的分子机制和基础所必需的。这些局部测量将与传统的紧密连接功能分析(目标1)一起进行，以研究稳定状态下的紧密连接动力学(目标2)以及暴露于炎性细胞因子IL-13和肿瘤坏死因子(目标3)后的紧密连接动力学。这些数据将对人类健康产生重大的积极影响，提高对屏障功能和功能障碍的了解，可能为IBD、乳糜泻和感染性结肠炎等肠道疾病提供更好的治疗方法。此外，所获得的知识和开发的技术方法都将很容易适用于其他器官的紧密连接的研究，包括被膜、中枢神经系统、血管系统、肺、肾和皮肤，因此，可能会产生除胃肠道疾病以外的广泛影响。 公共卫生相关性：在炎症性肠病和其他肠道疾病中，叙述性肠道屏障功能降低。形成主要的细胞旁屏障的紧密连接的变化，通过增加细胞旁离子和分子的通量而导致屏障的丧失。屏障功能障碍涉及离子和大分子通量的不同机制，但这些不同途径的分子基础尚不清楚。概述的研究将使用紧密结势垒动力学的高分辨率膜片钳记录来研究这些机制。

DESCRIPTION (provided by applicant): Intestinal barrier function is reduced in inflammatory bowel disease (IBD) and other intestinal disorders. Alterations of tight junctions, which form the major paracellular barrier, contribute to barrier loss by allowing increased paracellular flux of ions and molecules. Published reports and my preliminary data, which assess the role of different inflammatory cytokines in the lamina propria of IBD patients, support the hypothesis that at least two mechanisms of tight junction barrier regulation are activated by inflammatory cytokines. TNF induces tight junction dysfunction via a mechanism involving occludin internalization that alters tight junction size selectivity and permits increased macromolecular flux. In contrast IL-13 activates a functionally distinct pathway involving expression of claudin proteins that affects ion selectivity but does not increase macromolecular flux. Despite advances in understanding the processes involved in cytokine-induced barrier regulation, the molecular events that define size- or ion-selective changes are unclear. This, in part, represents the technical obstacle posed by traditional time and spatially averaged measurements of barrier function. I hypothesize that the tight junction barrier is highly dynamic at the local, sub-micron level, and that regulation of paracellular flux occurs through modulation of opening and closing "events" involving more than one class of tight junction pore or other conductance pathway. Because the tight junction spans two cells, it has not been amenable to molecular and biophysical analyses and development of specific pharmacologic modulators, such as those that exist for transmembrane ion channels. In order to eliminate this gap in available methods and molecular understanding, I have developed a novel approach to analyze tight junction barrier function at the local sub-micron level, using a high resolution single electrode patch clamp technique. While this approach is extensively used in the study of transmembrane ion channels and transporters, it has not been applied to the study of tight junction function. My preliminary recordings using this approach show previously unrecognized barrier dynamics, and I hypothesize that this behavior underlies normal tight junction function as a selectively-permeable barrier and is also necessary for understanding the molecular mechanisms and basis for multiple pathways of barrier dysfunction in disease. These local measurements will be performed alongside traditional assays of tight junction function (Aim 1) to study tight junction dynamics at steady state (Aim 2) and after exposure to inflammatory cytokines IL-13 and TNF (Aim 3). The data will have significant positive effects on human health by improving the understanding of barrier function and dysfunction, which may provide better therapeutic approaches for intestinal disorders such as IBD, celiac disease, and infectious colitis. Furthermore, both the knowledge gained and the technical approaches developed will be easily adapted to the study of tight junctions in other organs, including integument, central nervous system, vasculature, lung, kidney, and skin, and, therefore, may have broad impact beyond gastrointestinal disease. PUBLIC HEALTH RELEVANCE: Narrative Intestinal barrier function is reduced in inflammatory bowel disease and other intestinal disorders. Alterations of tight junctions, which form the major paracellular barrier, contribute to barrier loss by allowing increased paracellular flux of ions and molecules. Barrier dysfunction involves separate mechanisms for ionic and macromolecular flux, but the molecular basis for these different pathways is unclear. The research outlined will study these mechanisms using high resolution patch clamp recordings of tight junction barrier dynamics.