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

描述（由申请人提供）：炎症性肠病（IBD，包括克罗恩病和溃疡性结肠炎）涉及微生物群和粘膜免疫系统之间的稳态相互作用被破坏，这是多种遗传和环境因素的结果。众所周知，增加IBD严重程度的一个主要环境因素是社会心理压力。然而，将应激与微生物群和粘膜免疫系统之间的稳态关系破坏联系起来的生物学机制尚不清楚。这一建议将验证肠上皮细胞在应激、肠道菌群改变和病原体诱导的结肠炎之间的机制联系的新假设。我们的初步数据表明，将小鼠暴露于一种特征明确且广泛使用的社会压力源，称为社会破坏（SDR），会减少结肠中共生罗伊氏乳杆菌的丰度。小鼠结肠病原体啮齿柠檬酸杆菌（Citrobacter rodentium）诱导的结肠组织病理学与人类IBD相似，在口腔攻击小鼠后，暴露于SDR（因此具有较低水平的罗伊氏乳杆菌）的小鼠的病原体诱导结肠炎显著增加，这表明结肠组织病理学、趋化因子（如CCL2）、细胞因子（如TNF-）、效应分子（如iNOS）和巨噬细胞浸润显著增加。重要的是，通过在应激源暴露期间给小鼠喂食罗伊氏乳杆菌来防止应激源诱导的罗伊氏乳杆菌减少，消除了应激源对C.啮齿动物诱导的结肠炎的影响。与其人类同源物（即肠致病性大肠杆菌）一样，啮齿c通过定殖结肠上皮诱导结肠炎症。因此，使用啮齿鼠是确定结肠上皮是否在应激、微生物群和结肠炎恶化之间的联系中起关键作用的理想模型。这一提议将验证一个新的假设，即当罗伊氏乳杆菌因应激源暴露而减少时，结肠上皮细胞会过度产生趋化因子（特别是CCL2），从而增加炎性巨噬细胞在结肠的募集，最终加剧结肠炎。第一个目的是通过评估应激诱导的罗伊氏乳杆菌改变对结肠上皮细胞产生CCL2的影响，并使用CCL2敲除小鼠，研究CCL2作为介导应激对结肠炎症影响的一种新的主要机制（途径）的作用。在第二个目标中，我们将使用过继性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）是否因CCL2升高而运输到结肠，在那里它们可以通过过量生产TNF-来加剧结肠炎。和进气阀打开。最后，在第三个目标中，我们将确定罗伊氏乳杆菌影响结肠上皮细胞趋化因子产生的能力是否依赖于罗伊氏乳杆菌定殖结肠，和/或由于免疫调节因子的产生。该提案将确定可能导致IBD治疗新靶点的新机制。

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. PUBLIC HEALTH RELEVANCE: The stress response exacerbates colitis, both in experimental animals and in patients with inflammatory bowel disease, but the biological mechanisms by which this occurs are not well understood. Our studies demonstrate that stressor-induced reduction in commensal Lactobacillus reuteri is involved with the observed increase in colonic histopathology in mice challenged with Citrobacter rodentium during exposure to a well characterized and widely used social stressor. This proposal will test the novel hypothesis that the stressor-induced reduction in L. reuteri leads to an overproduction of chemokines, particularly CCL2, and cytokines by colonic epithelial cells, ultimately resulting in the recruitment of TNF-?-producing inflammatory macrophages that exacerbate colitis.