Stat3 Activation in Bacteria-Induced Murine Th17 Predominant Colonic Inflammation

描述(申请人提供)：脆弱类杆菌是一种常见的人类共生菌，生活在结肠中。脆弱芽孢杆菌肠毒素(ETBF)类与炎症性腹泻、炎症性肠病和结直肠癌有关。最近的小鼠模型数据表明，ETBF的慢性定植导致了增殖和促炎反应，这可能是结肠肿瘤启动和促进的辅助因素，特别是在多发性肠道肿瘤(Min)模型中。信号转导和转录激活因子3(STATS)分别通过IL-6和IL-10的激活而具有促炎和抗炎作用。在克罗恩病等慢性肠炎患者的肠道T细胞中发现了磷酸化的STATs，这很可能抑制了T细胞的凋亡。它也存在于肿瘤中，在那里它作为肿瘤免疫抑制因子发挥作用。虽然它在慢性炎症和肿瘤中很重要，但它的作用还没有得到很好的研究。具体目的：确定ETBF定植对小鼠结肠癌形成中STATS和结肠炎相关小鼠炎症反应的影响。小鼠(C57BL/6和Min)游动到3-4个月龄将用于拟议的实验。C57BL/6小鼠将用于展示STATS在结肠炎相关模型炎症中的活性，而Min小鼠将用于分析STATS在结肠肿瘤形成中的活性。在麻醉下用抗生素预治疗后，小鼠将通过口服ETBF、无毒的脆弱芽孢杆菌或安慰剂进行接种。将密切监测小鼠的临床状态(体重、活动、直肠出血、腹泻)，并在接种后的不同时间点(1天至3-4个月)处死小鼠。结肠、盲肠和脾将被采集并用于病理。使用特定抗体的免疫荧光染色和电泳性迁移率改变分析(EMSA)也将用于证明磷酸化STATS的存在。此外，还将利用组织对特定位置(上皮、固有层)和细胞类型(淋巴细胞、巨噬细胞、中性粒细胞等)进行STATS活性的流式细胞仪分析。这项研究将在几个方面与公共卫生相关。它将进一步定义一种名为ETBF的细菌在炎症和增殖中的潜在作用和途径，这些炎症和增殖可能与结肠癌和炎症性肠病等疾病有关。此外，它将建立一种模式，在这种模式中，可以尝试进行治疗干预，以期治疗这种致命的流行疾病。

DESCRIPTION (provided by applicant): Bacteroides fragilis is a common human commensal residing in the colon. B. fragilis enterotoxigenic (ETBF) class has been associated with inflammatory diarrheal disease, inflammatory bowel disease, and colorectal cancer. Recent murine model data has shown that chronic colonization with ETBF leads to a proliferative and proinflammatory response which can act as a co-factor in colon tumor initiation and promotion specifically in the multiple intestinal neoplasia (Min) model. Signal transducer and activator of transcription 3 (StatS) has both pro- and anti-inflammatory properties through activation by IL-6 and IL-10, respectively. Phosphorylated StatS is found in intestinal T cells from patients with chronic gut inflammation such as Crohn's disease most likely inhibiting apoptosis of T cells. It also is present in tumors where it functions as a tumor immune suppressor. Though important in chronic inflammation and tumors, its role has not been well studied. Specific Aim: To determine the effect of ETBF colonization on StatS in colonic tumor formation in Min mice and inflammation in a colitis-associated murine model. Mice (C57BL/6 and Min) Swks to 3-4mo of age will be used in the proposed experiments. The C57BL/6 mice will be used to demonstrate StatS activity in inflammation of the colitis-associated model while the Min mice will be used to analyze StatS activity in colonic tumor formation. After pre-treatment with antibiotics, under anesthesia, mice will be inoculated via oral gavage with ETBF, non-toxic B. fragilis, or placebo. Mice will be closely monitored in terms of clinical status (weight, activity, rectal bleeding, diarrhea) and sacrificed at various time points post inoculation (1 day to 3-4 months). Colon, cecum, and spleen will be harvested and used for pathology. Immunofluorescent staining using specific antibodies and Electophoretic mobility shift assay (EMSA) will also be used to demonstrate presence of phosphorylated StatS. Also tissue will be utilized for flow cytometry analysis of specific locations (epithelium, lamina propria) and cell types (lymphocytes, macrophages, neutrophils, etc) for StatS activity. This research will be relevant to public health in several ways. It will further define the potential role and pathway of a bacterium, ETBF, in inflammation and proliferation that could relate to disorders such as colon cancer and Inflammatory Bowel Disease. Furthermore it will establish a model in which therapeutic interventions could be attempted in hopes of treating such deadly prevalent diseases.