Enteric Neuronal Development as a Determinant of Intestinal Inflammation

描述（由申请人提供）：此申请是为了支持我在哥伦比亚大学的职业发展，由迈克尔D。Gershon（肠道神经生理学/发育）、Charalabos Pothoulakis（肠道炎症）和Lloyd Mayer（免疫学）。培训计划包括课程，指导研究和保护研究时间。本研究旨在分析肠神经系统（ENS）对肠道炎症和肠壁神经免疫相互作用的病理生理学的假定贡献。据报道，在患有炎症性肠病（IBD）或肠神经节瘤病的患者中，肠道发炎区域的肠神经元数量增加。在人类中，不可能确定神经元增生是否早于肠道炎症，是否由肠道炎症引起，或者是否导致肠道炎症的严重程度。我们使用，如 遗传模型，其中ENS是增生的（NSE-noggin小鼠）或发育不良的（Hand 2 +/-小鼠）的小鼠，以测试ENS增生是促炎性的假设。初步数据显示，TNBS-和DSS-诱导的结肠炎的严重程度的量度（存活、临床和组织学评分、编码促炎分子的基因的肠表达、嗜中性粒细胞弹性蛋白酶和p50 NF B的水平）在NSE-头蛋白中比在其野生型（WT）同窝小鼠中更高，而在Hand 2 +/-小鼠中比在其野生型（WT）同窝小鼠中更低。然而，在这两种小鼠中，在用二硝基氟苯诱发的耳中的迟发型超敏反应的严重程度（水肿、T细胞和中性粒细胞浸润以及IL 1、IFN和TNF的表达）的测量中发现与WT的差异。因此，转基因对炎症的影响仅限于肠道。这些观察结果与ENS增生导致肠道炎症严重程度并因此可能导致IBD发病的假设一致。我现在建议研究ENS影响肠道炎症的机制。我将确定ENS增生的促炎作用是否是由于（i）肠屏障功能，（ii）先天免疫和（iii）免疫调节的改变。将检查肠神经元在基线和炎症期间影响TLR 4和TLR 5信号传导的能力。我将确定ENS对上皮紧密连接完整性的影响， 基底层以及大分子穿过肠上皮的双向易位。对调节性T细胞（Treg）亚群（来自固有层和脾脏）的数量、位置和比例及其抑制淋巴细胞增殖的能力进行分析，以检验ENS增生降低Treg数量和/或功能的假设。肠道炎症发生在除IBD以外的肠道疾病中，包括坏死性小肠结肠炎、感染性腹泻和肠易激综合征; ENS可能导致其中任何一种或全部。因此，ENS和炎症效应物之间相互作用的知识有可能改变对许多肠道疾病的理解，并最终改变其治疗。

DESCRIPTION (provided by applicant): This application is to support my career development at Columbia University under the sponsorship of Michael D. Gershon (enteric neurophysiology/development), Charalabos Pothoulakis (intestinal inflammation) and Lloyd Mayer (immunology). The training plan includes courses, mentored research, and protected research time. My investigation is designed to analyze putative contributions of the enteric nervous system (ENS) to the pathophysiology of intestinal inflammation and neuroimmune interactions in the bowel wall. Increased numbers of enteric neurons have been reported in inflamed regions of the gut in patients with inflammatory bowel disease (IBD) or intestinal neurogangliomatosis. It is impossible to determine in humans whether neuronal hyperplasia predates intestinal inflammation, results from it, or contributes to its severity. We have used, as genetic models, mice in which the ENS is hyperplastic (NSE-noggin mice) or hypoplastic (Hand2+/- mice) to test the hypothesis that ENS hyperplasia is proinflammatory. Preliminary data show that measures of severity (survival, clinical and histological scores, intestinal expression of genes encoding proinflammatory molecules, levels of neutrophil elastase and p50 NF B) of TNBS- and DSS-induced colitis are higher in NSE- noggin and lower in Hand2+/- mice than in their wild-type (WT) littermates. In neither mouse, however, are differences from WT found in measures of the severity (edema, T cell and neutrophil infiltration, and expression of IL1 , IFN , and TNF ) of delayed type hypersensitivity evoked in the ears with dinitrofluorobenzene. Transgene effects on inflammation are thus limited to the bowel. These observations are consistent with the hypotheses that ENS hyperplasia contributes to the severity of intestinal inflammation and, potentially also therefore, to the pathogenesis of IBD. I now propose to investigate mechanisms by which the ENS affects intestinal inflammation. I will determine whether the proinflammatory effects of ENS hyperplasia are due to altered (i) intestinal barrier function, (ii) innate immunity, and (iii) immunoregulation. The ability of enterc neurons to affect TLR4 and TLR5 signaling at baseline and during inflammation will be examined. I will determine the effect of the ENS on the integrity of epithelial tight junctions and basal laminae as well as bidirectional translocation of macromolecules across the intestinal epithelium. Analyses of numbers, location, and proportions of regulatory T cell (Treg) subsets (from lamina propria and spleen) as well as their ability to inhibit lymphocyte proliferation will e employed to test hypotheses that ENS hyperplasia decreases Treg number and/or function. Intestinal inflammation occurs in intestinal disorders besides IBD, including necrotizing enterocolitis, infectious diarrhea, and irritable bowel syndrome; the ENS may contribute to any or all of them. Knowledge of interactions between the ENS and inflammatory effectors, therefore, has the potential to transform understanding and, ultimately, treatment of many intestinal disorders.