EPITHELIAL INNATE RESPONSES IN CHRONIC SAMP ILEITIS

肠道上皮是主要的物理屏障，是抵御摄入的毒素和细菌产品的第一道防线。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。上皮功能的失调可以破坏正常粘膜免疫的动态平衡，并驱动对致病状态的适应性免疫反应。在上一个资助期，我们证明了SAMP1/YitFc(SAMP)小鼠在炎症发生之前，就发生了上皮表型的改变和小肠通透性的增加。此外，与对照AKR骨髓重组的受照射SAMP小鼠的骨髓嵌合体(BMC)显示 严重的回肠炎，提示上皮功能的先天缺陷可能是SAMP小鼠疾病易感性的主要来源。项目5的中心假设是，肠上皮屏障功能失调导致异常的先天免疫反应，从而驱动致病效应细胞的激活，导致与克罗恩病(CD)类似的慢性肠道炎症。为了验证这一假设，我们将执行三个具体目标：1)确定SAMP小鼠小肠通透性的增加是环境因素还是遗传易感性的结果。体内和体外上皮通透性分析将比较无菌饲养的SAMP小鼠和SPF饲养的SAMP小鼠 老鼠。我们还将利用项目2产生的携带与SAMP背景下疾病严重程度相关的BL76染色体间隔的合体小鼠，确定基因成分是否导致小肠通透性增加的易感性。最后，为了便于疾病易感基因的精细基因定位，Affymetrix芯片技术将用于比较分离的家系和自然SAMP小鼠IEC的上皮来源基因的相对表达。2)鉴定SAMP小鼠顶端连接复合体(AJC)中上皮紧密连接(TJ)和黏附连接(AJ)蛋白的相对贡献，以及它们的表达变化是否影响回肠炎。根据我们的初步调查 我们将利用实时RT-PCR、Western blotting和免疫组织化学技术对TJ蛋白ZO-1、claudins 1-4和occludin以及AJ蛋白E-cadherin和β-catenin进行鉴定。此外，还将评估具有包含AJC基因的BL/6染色体间隔的共生体小鼠的TJ和AJ蛋白的表达，以及肠道炎症状态。3)探讨造血细胞在回肠炎发生发展中的特殊作用。在上皮屏障功能障碍的情况下。将对来自骨髓细胞的免疫细胞进行表型和功能鉴定，并将确定特定T/B细胞群体过继传播疾病的能力。最后，我们将调查回肠炎和致病性适应性免疫反应的激活是否是微生物产物不受控制地穿过渗漏的上皮屏障的结果。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，并促进基于对肠上皮屏障的操纵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.