INNATE AN ADAPTIVE IMMUNITY TO MICROBIAL FLAGELLINS IN IBD

IBD患者对微生物Flagellin的先天免疫和获得性免疫。宿主免疫异常 对肠道微生物群的反应是实验性IBD的基础，在人类IBD中也可能是如此。我们已经克隆并测序了一组微生物蛋白，这些蛋白在实验性结肠炎中作为免疫优势抗原。其中最大的一组是以前未知的厌氧共生菌群中的鞭毛蛋白，它们在多种IBD小鼠模型和一半的克罗恩病患者中被免疫学识别。使用鞭毛作为探针，发现易患结肠炎的C3H/HeJBir小鼠(C3H)存在明显的先天免疫缺陷，这种反应受C3H型结肠炎易感基因座CDcs1调节。矛盾的是，C3H小鼠的这种先天反应缺陷导致体内T细胞对鞭毛的反应增加。目的1将询问在易患结肠炎的C3H小鼠中，与耐受结肠炎的C57BI/6(B6)小鼠相比，以及在CDCS1基因同源的C3H和B6小鼠中，树突状细胞和巨噬细胞对微生物区系的先天免疫反应是否以及如何缺乏。首先将使用细菌鞭毛和其他TLR配体作为探针，然后用从小鼠身上分离的革兰氏阳性和革兰氏阴性肠道细菌进行挑战。将评估C3HvsB6树突状细胞向CD4T细胞递送微生物抗原的能力，以检验C3H树突状细胞对细菌的缺乏急性反应延长了向T细胞递送抗原的时间，从而导致C3H鼠的T细胞反应更强。此外，我们 会问鞭毛蛋白是否可以通过Toll样受体5(TLR5)为自己或其他微生物抗原充当佐剂。目标2将首次解决IBD中是否存在针对免疫优势的肠道微生物抗原的适应性免疫反应的明确、重复、非随机传播模式。根据我们的初步数据，需要检验的假设是，在实验性IBD中确实发生了重复的、分级的表位扩散模式，类似于在自身免疫性疾病中观察到的情况。我们将确定这种表位传播是否与疾病进展有关，是否需要TLR5，是否依赖白介素23，以及在传播级联早期识别的抗原免疫是否可以防止对稍后识别的抗原的反应，从而改善肠道炎症。目标3将定义T细胞在哪里、何时以及如何变得敏感 使用两个新的小鼠系，一个干扰素-Thy1.1报告小鼠和一个对CBIM细菌鞭毛蛋白起反应的TCR转基因小鼠，在易患结肠炎的宿主中检测微生物抗原。这些研究将为宿主免疫系统对微生物区系的反应机制以及对微生物区系的异常免疫反应如何导致慢性肠道炎症提供重要的新理解。将评估克罗恩病患者对这些抗原的先天和适应性反应。

Innate and Adaptive Immunity to Microbial Flagellins in IBD. Abnormalities of the host immune response to the intestinal microbiota is the basis of experimental IBD, and this is likely true in human IBD as well. We have cloned and sequenced a set of microbial proteins that serve as immunodominant antigens in experimental colitis. The largest group of these were previously unknown flagellins of the anaerobic commensal flora, which were recognized immunologically in multiple mouse models of IBD and in half of patients with Crohn's disease. Use of flagellins as probes has revealed an apparent deficiency in innate immunity in colitis-prone C3H/HeJBir mice (C3H), a response regulated by a C3H colitis-susceptibility gene locus on Chr 3 termed Cdcsl. Paradoxically this deficient innate response in C3H mice results in a increased T cell response to flagellins in vivo. Aim 1 will ask whether and how the dendritic cell and macrophage innate immune response to the microbiota is deficient in colitis-prone C3H compared to colitis-resistant C57BI/6 (B6) mice, and in C3H and B6 mice that are congenic for the Cdcs1 locus. Bacterial flagellins and other TLR ligands will be used as probes first, followed by challenge with gram-positive and gram-negative intestinal bacteria isolated from mice. The ability of C3H vs B6 dendritic cells to present microbial antigens to CD4 T cells will be assessed to test the hypothesis that the deficient acute C3H dendritic cell response to bacteria prolongs antigen presentation to T cells, thus resulting in a greater T cell responses in C3H mice. Further, we will ask whether flagellins can serve as adjuvants via Toll like receptor 5 (TLR5) for themselves or for other microbial antigens. Aim 2 will address for the first time whether there is a defined, repetitive, non-random pattern of spreading of the adaptive immune response to immunodominant enteric microbial antigens in IBD. Based on our preliminary data, the hypothesis to be tested is that a repetitive, hierarchical pattern of epitope spreading does occur in experimental IBD, similar to what has been observed in autoimmune diseases. We will determine if such epitope spreading is associated with disease progression, whether it requires TLR5, whether it is dependent on lnterleukin-23, and whether immunization with antigens recognized early in the spreading cascade can prevent reactivity to antigens recognized later and thus ameliorate intestinal inflammation. Aim 3 will define where, when, and how T cells become sensitized to microbial antigens in the colitis-prone host using two novel mouse lines, an interferon gamma-Thy1.1 reporter mouse and a TCR transgenic mouse reactive to CBiM bacterial flagellin. These studies will provide important new understanding of the mechanisms by which the host immune system responds to the microbiota and how an abnormal immune response to the microbiota results in chronic intestinal inflammation. Innate and adaptive response of Crohn's patients to these antigens will be assessed.