芳香烃受体介导肠固有淋巴细胞ILC3/ILC1异常分化在克罗恩病中的机制研究

The abnormal differentiation of intestinal innate lymphoid cells ILC3 and ILC1 exist in autoimmune disease.ILC3 decreased and ILC1 increased in Crohn's disease (CD) patients, suggesting that CD patients have abnormal intestinal ILC3/ILC1 alteration, but the mechanism of abnormal alteration of ILC3/ILC1 keep unclear. Our previous study found that the expression of aryl hydrocarbon receptor (AhR) in the intestinal tissue in CD patients was decreased, and the number of ILC3 in the AhR knockout mice decreased while ILC1 increased. The intestinal inflammation in AhR knockout mice given TNBS was more severe than wild type mice. Thus, we propose that AhR may mediate abnormal differentiation of ILC3/ILC1, and the production of inflammatory cytokines, finally, lead to the formation of intestinal inflammation. This project intends to use cell differentiation experiment in vitro, AhR gene knockout in vivo to verify that AhR involved in the pathogenesis of CD and ILC3/ILC1 differentiation; over expression and silencing AhR, to observe the expressions of ILC3/ILC1 specific differentiation factor ROR gamma t/T-bet, furthermore, in vivo experiment, deletion of AhR , increased intestinal inflammation, on the contrary, the activation or recovery of AhR, increase the number of ILC3, improve the intestinal inflammation. Based on these experiments, we demonstrate that AhR mediated the differentiation of ILC3/ILC1, and elucidate the molecular mechanism in the pathogenesis of CD, which provide evidence for prevention and treatment of CD.

肠固有淋巴细胞ILC3与ILC1间异常分化介导自身免疫性疾病发生。克罗恩病（CD）患者肠组织ILC3减少而ILC1增加，导致ILC3/ILC1异常改变机制尚不清楚。我们前期研究发现：CD患者病变肠组织芳香烃受体（AhR）低表达；AhR基因敲除鼠肠组织ILC3减少，ILC1增加,TNBS造模后肠道炎症重于野生型鼠。故提出假说：AhR介导ILC3/ILC1异常分化，炎性因子产生，最终导致CD发病。本课题拟利用体外细胞诱导分化，AhR敲基因鼠等实验手段，获得AhR参与CD发病及其与ILC3/ILC1异常分化相关依据；过表达及沉默AhR，明确AhR调控ILC3/ILC1特异分化因子ROR-γt/T-bet表达，介导ILC3/ILC1分化；进一步在体实验验证纠正AhR缺失，ILC3增加而ILC1减少，肠道炎症改善，阐明在CD发病中AhR介导ILC3/ILC1异常分化的分子机制，为CD防治提供靶点。

项目背景：肠固有淋巴细胞ILC3与ILC1间异常分化介导自身免疫性疾病发生。克罗恩病（CD）患者肠组织ILC3减少而ILC1增加，导致ILC3/ILC1异常改变机制尚不清楚。我们前期研究发现：CD患者病变肠组织芳香烃受体（AhR）低表达；AhR基因敲除鼠TNBS造模后肠道炎症重于野生型鼠，肠组织ILC3减少，ILC1增加，故提出科学假说：AhR的表达及活化异常介导ILC3/ILC1异常分化，ILC1相关促炎性因子增多，最终导致CD发病。.主要研究内容：通过临床相关研究，获得AhR参与CD发病及其与ILC3/ILC1细胞失衡相关临床依据；利用AhR基因敲除鼠，观察AhR缺失对TNBS结肠炎以及ILC3/ILC1细胞分化平衡影响；给予AhR配体激动剂色氨酸，进一步确定AhR活化可介导ILC3/ILC1细胞分化平衡，论证AhR介导ILC3/ILC1分化平衡的调控机制，阐明CD发病中导致ILC3/ILC1失衡分子机制。.重要结果、关键数据及其科学意义：.1. WB检测AhR及活化标志物CYP1A1在CD肠组织中表达，发现AhR及CYP1A1在活动期CD病变肠组织低表达，提示AhR可能参与CD发病。.2.免疫荧光检测显示活动期CD肠组织中ILC1增加，ILC3减少。.3. 构建AhR敲基因小鼠，予TNBS造模，结果显示AhR缺失小鼠TNBS造模后肠道炎症明显重于野生鼠。免疫荧光检测肠组织ILC1/ILC3改变，发现：与野生型结肠炎模型鼠相比，AhR基因敲除鼠TNBS造模后肠组织中ILC1细胞比例升高，ILC3细胞比例减低。.4. 给予DSS肠炎鼠不同浓度色氨酸饮食（0、0.21%、0.4%、4% Trp），结果发现色氨酸缺失，肠道炎症加重，补充0.4%色氨酸，DSS结肠炎模型鼠肠道炎症明显减轻。.5.流式染色技术检测分析ILC1及ILC3细胞在不同浓度色氨酸饮食结肠炎小鼠肠组织中的改变，WB检测小鼠肠组织中AhR的表达及活化改变。结果：0.4%色氨酸饮食小鼠DSS造模后肠道AhR明显升高，ILC1比例降低，ILC3比例升高。.6. PCR检测肠组织中ILC1/ILC3相关炎性细胞因子表达，结果显示色氨酸抑制ILC1相关炎性细胞因子TNF-α、IL-1β、IL-17A、IFN-γ表达。.综合以上结果提示色氨酸活化AhR，影响ILC3/ILC1细胞分化平衡，减轻肠道炎性损伤过程。