枯草芽孢杆菌刺激猪肠上皮细胞分泌IL-33活化黏膜树突状细胞的机制研究

Bacillus subtilis, as an oral immunity enhancer, can induce efficient intestinal mucosal immunity. But the widespread epithelial barrier prevents the activation of DCs induced by Bacillus subtilis directly. Bacillus subtilis may activate DCs indirectly by inducing the "signal" secretion of intestinal epithelial cells. IL-33 is a kind of cytokine with immune function. IL-33 maybe the key cytokines activated mucosal DCs under Bacillus subtilis inducing. The following experiments will be performed. First, the ability of Bacillus subtilis activating DCs will be detected by in vitro intestinal epithelial cells/DCs indirect-culture model. Second, the cytokines secreted by intestinal epithelial cells will be selected through Array and the expression level of IL-33 will be detected by ELISA. Third, to verify the function of IL-33 during the process, IL-33 gene knockout intestinal epithelial cells and IL-33 receptor blocker sST2 will be used. Finally, discuss the mechanism of IL-33 from intestinal epithelial cells after Bacillus subtilis inducing through TLRs blocker/siRNA/over-expression. This study will objectively elucidate the novel mechanism of Bacillus subtilis enhanced mucosal immunity, and lay a foundation for the development of efficient oral mucosal vaccine.

枯草芽孢杆菌作为口服免疫增强剂，诱导高效的肠道黏膜免疫。但广泛存在的黏膜上皮屏障阻碍了枯草芽孢杆菌对黏膜下树突状细胞（DCs）的直接活化，枯草芽孢杆菌可能通过诱导肠上皮细胞分泌“信号”间接活化DCs。IL-33是可以由上皮细胞分泌具有免疫调节功能的细胞因子，推测枯草芽孢杆菌可能刺激肠道上皮细胞分泌IL-33活化黏膜下DCs。因此开展如下研究：（1）运用猪肠上皮细胞/DCs非接触共培养模型，研究枯草芽孢杆菌对DCs的间接活化；（2）验证枯草芽孢杆菌诱导猪肠上皮细胞产生IL-33的水平；（3）缺失IL-33基因和阻断IL-33受体，反向验证（2）中枯草芽孢杆菌对DCs的间接活化作用；（4）通过阻断、干扰和过表达猪肠上皮细胞TLRs，探究枯草芽孢杆菌促进猪肠上皮细胞分泌IL-33机制。本研究将进一步阐明枯草芽孢杆菌增强黏膜免疫的机理，为预防肠道感染性疾病的高效口服黏膜疫苗的研制奠定基础。

枯草芽孢杆菌能诱导高效的黏膜免疫反应。肠道树突状细胞（DCs）虽然能伸出跨上皮树突摄取肠腔中的枯草芽孢杆菌，但是，枯草芽孢杆菌进入肠道后主要接触的是肠道上皮细胞。IL-33是一种由上皮细胞分泌的具有免疫调节功能的细胞因子。为了探讨枯草芽孢杆菌能否通过刺激肠道上皮细胞分泌IL-33活化DCs，进而增强黏膜免疫反应。本项目首先运用荧光定量PCR和ELISA的方法确定了枯草芽孢杆菌能够刺激小鼠肠上皮细胞分泌IL-33。其次，通过间接培养发现，枯草芽孢杆菌刺激细胞的培养上清能上调小鼠骨髓源树突状细胞成熟表型；并且运用RNA干扰技术反向证明了IL-33在活化树突状细胞中的作用。随后，在枯草芽孢杆菌刺激小鼠肠上皮细胞分泌IL-33的机制研究中发现，枯草芽孢杆菌能够促进IL-33前体蛋白的切割和IL-33成熟肽的分泌，初步解析了枯草芽孢杆菌刺激肠上皮细胞分泌IL-33的机制。最后，通过小鼠肠道结扎术和口服免疫等实验进一步证明，枯草芽孢杆菌能够提高小鼠肠道中IL-33的mRNA和蛋白水平，并且对小鼠的固有免疫和特异性免疫有增强作用。本研究说明枯草芽孢杆菌刺激肠上皮细胞产生的IL-33在诱导黏膜免疫中具有重要作用，揭示了枯草芽孢杆菌增强肠道黏膜免疫的新机制。