Role of stress-induced reduction in Lactobacillus reuteri on colonic inflammation

描述(申请人提供)：炎症性肠病(IBD，包括克罗恩病和溃疡性结肠炎)涉及由于多种遗传和环境因素导致的微生物区系和粘膜免疫系统之间的动态平衡相互作用。众所周知，增加IBD严重程度的一个主要环境因素是心理社会压力。然而，将压力与微生物区系和粘膜免疫系统之间的动态平衡关系破坏联系起来的生物学机制还不是很清楚。这一提议将检验这一新的假设，即肠道上皮细胞代表应激、肠道微生物区系的变化和病原体诱导的结肠炎之间的机制联系。我们的初步数据表明，将小鼠暴露于一种具有良好特征和广泛使用的社会应激源，称为社交破坏(SDR)，可以减少结肠中共生的罗氏乳杆菌的丰度。当口服小鼠结肠病原体罗氏柠檬酸杆菌时，暴露于SDR(因此具有较低水平的路氏杆菌)的小鼠的病原体诱导的结肠炎显著增加，表现为结肠组织病理学、趋化因子(例如CCL2)、细胞因子(例如TNF-β)、效应分子(例如iNOS)和巨噬细胞的浸润显著增加。重要的是，在应激源暴露期间，通过给小鼠喂食罗氏乳杆菌来防止应激源诱导的罗氏乳杆菌减少，消除了应激源对轮状芽胞杆菌诱导的结肠炎的影响。像它的人类同源物(即致病大肠杆菌)一样，轮状芽胞杆菌通过定植结肠上皮来诱导结肠炎。因此，使用齿状念珠菌是确定结肠上皮是否在应激、微生物区系和结肠炎恶化之间的联系中起关键作用的理想模型。这一提议将检验一种新的假设，即当路支杆菌因应激源暴露而减少时，结肠上皮细胞过度产生趋化因子(特别是CCL2)，从而增加炎性巨噬细胞向结肠的募集，最终加剧结肠炎。第一个目标将通过评估应激诱导的路氏杆菌对结肠上皮细胞产生CCL2的影响以及利用CCL2基因敲除小鼠来研究CCL2作为一种新的主要机制(途径)在应激对结肠炎症影响中的作用。在第二个目标中，我们将使用过继转移实验来测试外周炎性单核细胞(我们显示在应激小鼠的循环中增加)是否会因CCL2升高而流向结肠，在那里它们可以通过过量产生肿瘤坏死因子？？来加重结肠炎。和诱导型一氧化氮合酶。最后，在第三个目标中，我们将确定路氏杆菌影响结肠上皮细胞产生趋化因子的能力是否依赖于路氏杆菌在结肠的定植，和/或由于免疫调节因子的产生(S)。这一建议将确定新的机制(S)，可能导致新的治疗靶点的IBD的治疗。

DESCRIPTION (provided by applicant): The inflammatory bowel diseases (IBD, including Crohn's disease and ulcerative colitis) involve disrupted homeostatic interactions between the microbiota and the mucosal immune system as a result of multiple genetic and environmental factors. A major environmental factor that is well recognized to increase the severity of IBD is psychosocial stress. However, the biological mechanisms linking stress to disrupted homeostatic relationships between the microbiota and the mucosal immune system are not well understood. This proposal will test the novel hypothesis that intestinal epithelial cells represent a mechanistic link between stress, alterations of the gut microbiota, and pathogen-induced colitis. Our preliminary data demonstrate that exposing mice to a well characterized and widely used social stressor, called social disruption (SDR) reduces the abundance of commensal Lactobacillus reuteri in the colon. Upon oral challenge with the murine colonic pathogen Citrobacter rodentium, which induces colonic histopathology with similarities to human IBD, mice exposed to SDR (and thus having lower levels of L. reuteri) had a significant increase in pathogen-induced colitis as indicated by a significant increase in colonic histopathology, chemokines (e.g., CCL2), cytokines (e.g., TNF-?), effector molecules (e.g., iNOS), and macrophage infiltration. Importantly, preventing the stressor-induced reduction in L. reuteri by feeding L. reuteri to the mice during stressor exposure abrogated the effects of the stressor on C. rodentium-induced colitis. Like its human homologue (i.e., enteropathogenic E. coli), C. rodentium induces colonic inflammation by colonizing the colonic epithelium. Thus, the use of C. rodentium is an ideal model to determine whether the colonic epithelium is critical in the link between stress, the microbiota, and exacerbation of colitis. This proposal will test the novel hypothesis that when L. reuteri are decreased due to stressor exposure, colonic epithelial cells overproduce chemokines (particularly CCL2) that increase the recruitment of inflammatory macrophages to the colon where they ultimately exacerbate colitis. The first aim will examine the role of CCL2 as a novel primary mechanism (pathway) mediating the effects of stress on colonic inflammation, by assessing the effects of stress-induced alterations of L. reuteri on CCL2 production by colonic epithelial cells and by using CCL2 knockout mice. In the second aim, we will use adoptive transfer experiments to test whether peripheral inflammatory monocytes, which we show are increased in the circulation of stressed mice, traffic to the colon in response to the elevated CCL2, where they can exacerbate colitis through an overproduction of TNF-? and iNOS. Finally, in the third aim, we will determine whether the ability of L. reuteri to affect chemokine production by colonic epithelial cells is dependent upon L. reuteri colonizing the colon, and/or due to the production of immunomodulatory factor(s). This proposal will identify novel mechanism(s) that may lead to new therapeutic targets in the treatment of IBD.