IL-27调控LL-37/CRAMP表达在艰难梭菌肠道感染中的免疫保护作用及机制研究

Clostridium difficile is a leading cause of nosocomial infection and antibiotic-associated diarrhea. The immunity mediated by intestinal epithelial cells plays an important role in combating Clostridium difficile infection. However, its molecular and immunoregulatory mechanisms remain to be elucidated. IL-27 is a recently described immunoregulatory cytokine. Our preliminary studies have demonstrated that the mortality rate of IL-27R-deficient mice was significantly higher than that of wild-type mice upon Clostridium difficile infection. Furthermore, the expression of antimicrobial peptide cathelicidin LL-37/CRAMP was significantly down-regulated in IL-27R-deficient intestinal epithelial cells. Therefore, we hypothesize that IL-27 stimulates the expression of cathelicidin LL-37/CRAMP in intestinal epithelial cells, which mediates intestinal immunity against Clostridium difficile infection. In this study, we will evaluate the clinical significance of IL-27 expression in a large cohort of patients with Clostridium difficile infection. Furthermore, we will use various gene knockout mice and primary intestinal epithelial cells to investigate the role of IL-27 in regulating cathelicidin LL-37/CRAMP expression from intestinal epithelial cells in combating Clostridium difficile infection and its potential intracellular signaling pathways by use of flow cytometry analysis, antibody-blocking technology, cDNA microarray,siRNA and EMSA, etc. Our work will provide new strategies for the diagnosis and treatment of Clostridium difficile infection.

艰难梭菌是院内感染和抗生素相关腹泻最重要的病原菌。肠上皮细胞在抵抗艰难梭菌感染中发挥关键作用，但分子免疫机制不详。最近发现IL-27是一种重要的免疫调节因子。预实验显示IL-27特异性受体缺陷小鼠在艰难梭菌感染时，死亡率显著上升，且肠上皮细胞内cathelicidin分子CRAMP的表达显著下调。由此提出“IL-27诱导肠上皮细胞表达cathelicidin分子LL-37/CRAMP，介导肠道免疫，抵抗艰难梭菌感染”的科学假说。本项目拟用大宗临床样本明确IL-27表达在艰难梭菌感染发病中的临床意义；并以基因敲除小鼠和原代肠上皮细胞等为研究模型，通过流式细胞分析、抗体阻断、表达谱芯片、siRNA和EMSA等技术，在分子、细胞和动物水平上揭示“IL-27诱导肠上皮细胞表达LL-37/CRAMP而抵抗艰难梭菌感染”的作用及分子信号机制，以期为艰难梭菌感染的诊治研究找到重要线索。

艰难梭菌是院内感染和抗生素相关腹泻最重要的病原菌。肠上皮细胞在抵抗艰难梭菌感染中发挥关键作用，但分子免疫机制不详。本项研究中，我们首先通过体外实验发现IL-27可诱导人原代肠上皮细胞表达人组织蛋白酶抑制素（cathelicidin）抗菌肽（LL-37），且JAK、PI3K和p38MAPK信号通路调控IL-27诱导人肠上皮细胞表达LL-37；进一步在体内动物模型中，发现在艰难梭菌感染条件下，IL-27受体缺陷会导致小鼠体内表达鼠组织蛋白酶抑制素相关抗菌肽（CRAMP，鼠CRAMP为人LL-37的同系物）；IL-27受体缺陷会导致小鼠在艰难梭菌感染条件下细菌清除率降低，死亡率上升；而给予小鼠鼠CRAMP会使得IL-27受体缺陷小鼠抵抗艰难梭菌感染的能力显著增强；在临床艰难梭菌感染确诊患者的血清和粪便中，我们发现IL-27浓度与LL-37含量呈正相关。上述研究表明IL-27可通过诱导LL-37/CRAMP表达来抵抗艰难梭菌感染，为艰难梭菌感染的治疗提供了宿主导向治疗的免疫靶标。上述研究发表在Gut Microbes（2021 Jan-Dec;13:1968258，影响因子为10.2），“白介素-27在治疗艰难梭菌感染中的应用”也已经获得中国授权发明专利（专利号：ZL201710007655.5），有望未来在临床转化。

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