梭菌通过树突状细胞调控肠道RORγt+Treg的发育在霍乱毒素打破耐受促进TH2中的作用和机制研究

Cholera toxin (CT) is a classic mucosal adjuvant that has been used for breaking oral tolerance and inducing food allergy, while the mechanism is unknown. In the latest reports, a new Treg subset in the intestinal lamina propria, RORγt+ Treg, was identified. The generation of these Treg cells, which can selectively inhibit the type 2 immune response, is dependent on the microbiota and Clostridium plays an important role in this process. In our preliminary experiments，we found that the amount of Clostridium in the small intestine and the percentage of RORγt+ Treg subsets in the lamina propria were both significantly reduced after CT treatment. These results suggest that decreased amount of Clostridium caused by CT treatment maybe play a role in promoting TH2 response, while the detailed mechanism is not clear. It has been demonstrated that the expression of αvβ8 in intestinal dendritic cell (DC) was critical for the induction of Foxp3+ Treg via activating TGF-β, although which Treg subset in the gut was affected is unknown. So, we hypothesized that the observed TH2 response in our system may be due to the decreased RORγt+ Treg cells, which might be caused by down-regulated expression of αvβ8 in DC by CT treatment-induced reduction of the amount of Clostridium in the small intestine. Therefore, in this study the cellular and molecular immunological methods, and knockout mice will be used to explore the reasons that caused the decline of the amount of Clostridium in the small intestine after CT treatment, and to examine whether the expression of αvβ8 in DC subsets was affected by Clostridium, which will help us to explain the reasons why CT treatment resulting the decrease of RORγt+ Tregs and induction of type 2 immune response eventually. This research is likely to shed light on the mechanism of food allergy and also may provide a possible target for the prevention and treatment of food allergy.

霍乱毒素（CT）是一种经典的打破耐受、诱导食物过敏的黏膜佐剂，作用机理不明。最新研究发现肠道中存在菌群依赖的、选择性抑制肠道TH2反应的RORγt+Treg亚群，梭菌对该亚群的发育发挥重要促进作用。前期发现CT处理后小肠中的梭菌数量显著下降，固有层中RORγt+Treg比例也显著降低，提示CT处理引起的梭菌减少可能促进了TH2反应，但机制不明。鉴于树突状细胞（DC）维持肠道耐受的重要作用，及预实验发现DC缺失αvβ8严重影响RORγt+Treg生成，推测CT促进TH2反应可能是由于梭菌数量的降低引起DC亚群 αvβ8表达下降，导致RORγt+Treg下降，对TH2的抑制减弱所致。本课题将采用细胞及分子免疫学方法探明CT引起小肠梭菌下降的原因，明确梭菌是否影响DC亚群对αvβ8的表达及机制，以期解释CT导致RORγt+Treg下降的原因，为阐明食物过敏机理做铺垫，为防治食物过敏提供理论基础。

最近文献报道肠道固有层存在Foxp3+RORγt+Treg亚群，具有抑制TH2免疫反应的功能，对其发育和稳态的维持机理目前还不十分清楚。TGF-β是重要的免疫调控因子，体内免疫细胞以及非免疫细胞都会产生TGF-β，但是都以非活化的形式分泌，TGF-β只有活化后才能发挥生物学功能。对活化环节的精准调控是TGF-β准确发挥其生物学功能的重要保障。整合素β8是能够活化TGF-β的黏附分子。DC与ILC3均是宿主重要的免疫细胞，对于肠道免疫稳态的维持发挥关键作用，它们都表达整合素β8。DC及ILC3表达的整合素β8在肠道免疫稳态中发挥何种作用是亟待回答的重要科学问题。为此，课题组首先利用Zbtb46creItgb8f/f小鼠发现，DC表达的整合素β8能够活化TGF-β，诱导RORγt+Treg以及RORγt+TH17的分化发育，并抑制TH2免疫反应；课题组还发现RORγt+ILC3缺失整合素β8后，RORγt+Treg的数量和比例显著下降，机体出现与年龄相关的自身免疫性疾病、TH2显著上升、IgA分泌显著增加、口服耐受被打破等现象。进一步的机理研究发现，ILC3通过整合素β8调控定居分子在RORγt+Treg的表达来调控该细胞在肠道固有层的定居，从而发挥维持肠道免疫稳态的功能；霍乱毒素（CT）是一种经典的打破耐受、诱导肠道食物过敏反应的黏膜佐剂，但作用机理不明。研究发现CT可以通过抑制肠道梭菌数量，导致肠道RORγt+Treg的比例下降并诱导TH2免疫反应。进一步的研究发现，肠道上皮的MyD88参与CT打破口服耐受。由于RORγt+Treg的比例下降，最终导致肠道免疫耐受被打破。总的来说，该课题首次揭示了DC与ILC3都以表达整合素β8的形式，分别调控RORγt+Treg的分化发育和肠道定居的两个不同方面，共同维持肠道稳态的免疫学新机制，并初步探明CT打破口服耐受的机制。这不但加深了人们对TGF-β生物学的理解，也拓展了对肠道免疫稳态维持机制的认识。

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