IL-33通过ST2改变MDSC功能参与MCMV肺炎

IL-33 involves MCMV-induced pneumonia through binding MDSC ST2 receptor

目的:探讨白细胞介素-33(interleukin-33,IL-33)对鼠巨细胞病毒(murine cytomegalovirus,MCMV)肺炎的影响。方法:通过分子生物学的方法构建IL-33原核表达载体纯化并鉴定纯化产物;通过聚合酶链式反应(polymerase chain reaction,PCR)法鉴定IL-33受体ST2^-/-小鼠的基因型;通过鼻腔感染MCMV建立小鼠肺炎模型,并通过病理学评价肺炎模型及与IL-33的相关性;野生型及ST2^-/-小鼠尾静脉注射外源性IL-33蛋白后,酶联免疫吸附法(enzyme-linked immunoabsorbent assay,ELISA)检测小鼠肺中IL-33蛋白变化情况,病理学比较肺炎模型的严重程度,空斑实验比较肺部病毒载量程度;流式细胞术检测2种小鼠骨髓原性的抑制性细胞(myeloid-derived suppressor cell,MDSC)细胞的ST2表达情况及MDSC细胞在肺中的比例;实时定量聚合酶链式反应(Real-time polymerase chain reaction,Real-time PCR)检测肺中MDSC发挥抑制功能相关的的精氨酸酶-1(Arginase-1)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和Th2型细胞因子。结果:成功构建IL-33原核表达载体并通过SDS-PAGE和Western Blot方法验证;PCR成功鉴定并区分野生型及ST2^-/-型小鼠;通过病理学检测成功建立鼻腔感染MCMV肺炎模型,肺炎模型组IL-33 mRNA和蛋白含量显著高于对照组;野生型小鼠尾静脉注射IL-33蛋白后,第7天肺炎病理学表现最严重,肺病毒载量最高;ST2^-/-小鼠尾静脉注射IL-33蛋白后,与野生型小鼠相比,肺炎病理学表现严重程度及病毒载量显著降低;外源加入IL-33蛋白后,Arginase-1、iNOS及Th2型细胞因子显著升高。结论:IL-33可以通过活化肺中MDSC细胞促进MCMV肺炎。

Objective:To explore the interleukin-33(IL-33)effect on murine cytomegalovirus(MCMV)pneumonia.Methods:Construction of the IL-33 prokaryotic expression system and identification on the purified IL-33 protein by molecular biology method.The genotype of ST2^-/-(IL-33 receptor)mice was identified by polymerase chain reaction(PCR).The mice pneumonia model was established by intranasal administration with MCMV,and evaluated the relationship between MCMV pneumonia and pulmonary IL-33(both mRNA and protein)by pathohistological score system(H&E staining).Exogenous IL-33 protein was administrated via tail vein in both wild type and ST2^-/-mice,then enzyme-linked immunoabsorbent assay(ELISA)was used to detect the dynamic of pulmonary IL-33 protein between 2 mice group;pathohistological evaluation(H&E staining)was employed to observe the pulmonary pathology changes between 2 mice group;plaque assay was conducted to determine pulmonary viral loading between 2 mice group.The proportion of pulmonary,myeloid-derived suppressor cell(MDSC)and its surface ST2 expression were determined in 2 mice groups by flow cytometry.Immunosuppression related Arginase-1,inducible nitric oxide synthase(iNOS)and Th2 cytokines were determined by Real-time polymerase chain reaction(Real-time PCR).Results:The IL-33 protein was purified and identified by SDS-PAGE and Western Blot.The genotype of wild type,ST2^-/-and heterozygote was differentiated by PCR.The mouse model of MCMV pneumonia via intranasally infection was successful establishment that was confirmed by pathohistological evaluation(H&E staining).The IL-33 level(mRNA and protein)of MCMV pneumonia group was significantly higher than that of control group.In wild type mice,exogenous IL-33 protein injection via vein statistically aggravated MCMV pneumonia and the highest severity of MCMV pneumonia and highest pulmonary viral loading at day 7 post infection.However,compared with wild type mice,exogenous IL-33 protein i.v.administration resulted in a significantly decreased severity of MCMV pneumonia,meanwhile the pulmonary viral loading was greatly lower in ST2^-/-mice at day 7 post-infection.The prominent increased immunosuppressive Arginase-1,iNOS and Th2 cytokines expression on pulmonary MDSC were observed by exogenous IL-33 protein injection via vein.Conclusion:IL-33 aggravated MCMV-induced pneumonia via activated pulmonary MDSC.