EPITHELIAL INNATE RESPONSES IN CHRONIC SAMP ILEITIS

肠上皮代表主要的物理屏障，并且是抵抗摄入的毒素和细菌产物的第一道防线。此外，越来越多的证据表明，肠上皮细胞（IEC）积极参与肠道炎症。上皮功能的失调可破坏正常粘膜免疫的稳态并驱动对致病性疾病状态的适应性免疫应答。在上一个资助期间，我们证明了上皮表型改变和小肠通透性增加发生在SAMP 1/YitFc（SAMP）小鼠炎症发作之前。此外，用对照AKR骨髓重建的经辐射的SAMP小鼠的骨髓嵌合体（BMC）表现出： 显著回肠炎，表明上皮功能的先天缺陷可能代表SAMP小鼠中疾病易感性的主要来源。项目5的中心假设是肠上皮屏障功能的失调导致异常的先天免疫应答，其驱动致病性效应细胞的活化，导致与克罗恩病（CD）中观察到的类似的慢性肠道炎症。为了验证这一假设，我们将进行三个具体的目标：1）确定SAMP小鼠小肠通透性的增加是否是环境因素或遗传易感性的结果。上皮通透性的体内和体外分析将比较在无菌条件下饲养的SAMP小鼠与SPF饲养的SAMP小鼠。 小鼠我们还将确定遗传成分是否有助于增加小肠通透性的易感性，利用项目2产生的染色体组小鼠，其携带与SAMP背景下疾病严重程度相关的BL 76染色体间隔。最后，为了促进疾病易感基因的精细基因定位，将使用Affyphon芯片技术来比较来自同源和天然SAMP小鼠的分离的IEC的上皮衍生基因的相对表达。2)表征SAMP小鼠中构成顶端连接复合体（AJC）的上皮紧密连接（TJ）和粘附连接（AJ）蛋白的相对贡献，以及其表达的改变是否影响回肠炎。根据我们初步的 研究结果，我们将表征TJ蛋白ZO-1，claudins 1-4和occludin，和AJ蛋白E-钙粘蛋白和β-连环蛋白，使用实时RT-PCR，蛋白质印迹和免疫组织化学技术。此外，将评价具有含有AJC基因的BL/6染色体间隔的consomic小鼠的TJ和AJ蛋白的表达以及肠道炎症状态。3)评估造血细胞在回肠炎发展中的特殊作用。存在上皮屏障功能障碍。将对来自BMC的免疫细胞进行表型和功能表征，并测定特异性T/B细胞群过继转移疾病的能力。最后，我们将研究回肠炎和病原性适应性免疫反应的激活是否是微生物产物穿过渗漏的上皮屏障的不受控制的易位的结果。项目5的总体目标是阐明上皮和屏障功能障碍在慢性回肠炎发病机制中的确切作用，并促进基于肠上皮屏障操作的未来治疗策略的设计。

The intestinal epithelium represents a primary physical barrier, and is the first line of defense against ingested toxins and bacterial products. In addition, a growing body of evidence suggests that intestinal epithelial cells (IEC) actively participate in gut inflammation. Dysregulation of epithelial function can disrupt the homeostasis of normal mucosal immunity and drive adaptive immune responses to a pathogenic disease state. In the last funding period, we demonstrated that epithelial phenotypic alterations and increased small intestinal permeability occur early, before the onset of inflammation in SAMP1/YitFc (SAMP) mice. In addition, bone marrow chimeras (BMCs) of irradiated SAMP mice reconstituted with control AKR bone marrow exhibit significant ileitis, suggesting that an innate defect in epithelial function may represent the primary source of disease susceptibility in SAMP mice. The central hypothesis of Project 5 is that dysregulation of intestinal epithelial barrier function leads to aberrant innate immune responses that drive the activation of pathogenic effector cells, resulting in chronic intestinal inflammation similar to that observed in Crohn's disease (CD). To test this hypothesis, we will perform three specific aims: 1) Determine if the increase in small intestinal permeability of SAMP mice is a consequence of environmental factors or genetic predisposition. In vivo and in vitro analyses of epithelial permeability will compare SAMP mice raised under germ-free conditions to SPF-raised mice. We will also determine whether a genetic component contributes to the susceptibility of increased small intestinal permeability, utilizing consomic mice generated by Project 2 that carry BL76 chromosomal intervals associated with disease severity on the SAMP background. Finally, to facilitate fine gene mapping of disease susceptibility genes, Affymetrix chip technology will be used to compare the relative expression of epithelial-derived genes from isolated IEC of consomic and native SAMP mice. 2) Characterize the relative contribution of epithelial tight junction (TJ) and adherens junction (AJ) proteins comprising the apical junctional complex (AJC) in SAMP mice, and if alteration in their expression affects ileitis. Based on our preliminary findings, we will characterize the TJ proteins ZO-1, claudins 1-4 and occludin, and the AJ proteins E-cadherin and beta-catenin, using real-time RT-PCR, Western blotting and immunohistochemical techniques. In addition, consomic mice that possess BL/6 chromosomal intervals containing genes of the AJC will be evaluated for expression of TJ and AJ proteins, as well as the state of intestinal inflammation. 3) Evaluate the specific role of hematopoietic cells in the development of ileitis. in the presence of epithelial barrier dysfunction. Phenotypic and functional characterization will be performed on immune cells from BMCs, and the ability of specific T/B cell populations to adoptively transfer disease will be determined. Finally, we will investigate whether ileitis and activation of pathogenic adaptive immune responses are a consequence of uncontrolled translocation of microbial products across a leaky epithelial barrier. The overall goal of Project 5 is to elucidate the precise role of the epithelium and barrier dysfunction in the pathogenesis of chronic ileitis, and to facilitate the design of future therapeutic strategies based on manipulation of the intestinal epithelial barrier.