摘要
【背景】副猪嗜血杆菌可引起多种炎性反应和较高死亡率,其致炎机制目前尚不清楚。【目的】探究副猪嗜血杆菌外膜囊泡(outermembranevesicles,OMVs)诱导RAW264.7细胞caspase-11及NLRP3炎性体活化的功能,以及caspase-11在OMVs活化炎性体诱导炎性因子表达过程中的关键作用。【方法】副猪嗜血杆菌OMVs感染RAW264.7细胞,收集6、12和24 h细胞,RT-PCR检测caspase-11、NLRP3、ASC和caspase-1 mRNA的表达;收集感染后48 h细胞,Western blotting检测caspase-11、NLRP3、ASC和caspase-1蛋白表达。收集感染后6、12、24、48和72 h细胞上清,ELISA检测interleukin(IL)-1β和IL-18表达水平;不同浓度OMVs(0、2.5、10、25、50和100μg/mL)感染RAW264.7细胞24 h,ELISA检测上清中IL-1β和IL-18水平。构建caspase-11沉默RAW264.7细胞株,收集OMVs感染后12、24和48 h细胞培养上清检测IL-1β和IL-18水平。【结果】Caspase-11mRNA转录水平在OMVs感染6、12和24h均极显著高于对照组(P<0.01);NLRP3 mRNA转录水平在感染后6、24 h极显著高于对照组(P<0.01);ASC mRNA转录水平在感染后12、24 h显著低于对照组(P<0.05);caspase-1 mRNA转录水平在感染后6、12和24 h显著高于对照组(P<0.05)。Westernblotting结果显示,与空白对照组相比,OMVs组caspase-11、NLRP3、ASC和caspase-1蛋白表达均明显升高。OMVs感染RAW264.7细胞后12、24、48和72 h,IL-1β表达量极显著高于对照组(P<0.01),而且呈现时间依赖性升高,IL-18表达水平在感染后6、12、24、48和72 h显著高于对照组(P<0.05);不同浓度OMVs感染细胞时,OMVs对细胞的致炎作用呈现剂量依赖性增强。Caspase-11沉默后,IL-1β水平在感染后12、24和48h显著低于未沉默组;Caspase-11沉默组IL-18表达量在感染后24、48h均显著低于未沉默组(P<0.05)。【结论】副猪嗜血杆菌OMVs在副猪嗜血杆菌诱导的炎症反应中发挥重要作用,OMVs可诱导RAW264.7细胞caspase-11介导的非经典NLRP3炎性体信号通路活化。
[Background]Haemophilus parasuis can cause a variety of inflammatory reactions and high mortality,while the inflammatory mechanism remains unclear.[Objective]To study the activation of caspase-11 and NOD-like receptor family pyrin domain containing protein 3(NLRP3)inflammasome in RAW264.7 cells by H.parasuis outer membrane vesicles(OMVs)and the key role of caspase-11 in the OMVs-induced expression of inflammatory cytokines.[Methods]The RAW264.7 cells were infected with H.parasuis OMVs and collected 6,12,and 24 h post infection.RT-PCR was employed to determine the mRNA levels of caspase-11,NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),and caspase-1.Western blotting was employed to determine the protein levels of caspase-11,NLRP3,ASC,and caspase-148 h post infection.Enzyme-linked immunosorbent assay(ELISA)was employed to determine the levels of interleukin-1β(IL-1β)and IL-18 in the cell supernatants 6,12,24,48,and 72 h post infection.After the RAW264.7 cells were stimulated with different concentrations of OMVs(0,2.5,10,25,50,and 100μg/mL)for 24 h,the cell supernatants were collected for the measurement of IL-1βand IL-18 levels by ELISA.The RAW264.7cells with the silencing of caspase-11 were established and then infected with OMVs,and the supernatants were collected for the measurement of IL-1βand IL-18 levels 12,24 and 48 h post infection.[Results]The mRNA level of caspase-11 was up-regulated in the RAW264.7 cells 6,12 and 24 h post infection with OMVs(P<0.01).The mRNA level of NLRP3 was higher than that in the control group 6 and 24 h post infection(P<0.01).The mRNA level of ASC was significantly lower than that in the control group 12 and 24 h post infection(P<0.05).The mRNA level of caspase-1 was significantly higher than that in the control group 6,12,and 24 h post infection(P<0.05).Western blotting showed that the expression levels of caspase-11,NLRP3,ASC,and caspase-1 were up-regulated after infection with OMVs.The level of IL-1βelevated in a time-dependent manner after the cells were stimulated with OMVs for 12,24,48 and 72 h and was higher than that in the control group(P<0.01),and the level of IL-18 was higher than that in the control group at the time points of 6,12,24,48 and 72 h(P<0.05).When the cells were stimulated with different concentrations of OMVs,the inflammatory effect increased in a dose-dependent manner.The level of IL-1βin the caspase-11-silenced cells stimulated with OMVs was lower than that in the non-silenced group at the time points of 12,24 and 48 h(P<0.05),and the level of IL-18 showed the same trend as that of IL-1βat the time points of 24 and 48 h(P<0.05).[Conclusion]The OMVs of H.parasuis play an important role in the inflammatory response induced by H.parasuis.OMVs can induce the activation of non-canonical NLRP3 inflammasome signaling pathway mediated by caspase-11 in RAW264.7 cells.
作者
李艳
杨君
翟少伦
楚品品
卞志标
张昆丽
李春玲
LI Yan;YANG Jun;ZHAI Shaolun;CHU Pinpin;BIAN Zhibiao;ZHANG Kunli;LI Chunling(Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,Guangdong,China;Key Laboratory of Livestock Disease Prevention of Guangdong Province,Guangzhou 510640,Guangdong,China;Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province,Ministry of Agriculture and Rural Affairs,Guangzhou 510640,Guangdong,China;Maoming Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology,Maoming 525000,Guangdong,China)
出处
《微生物学通报》
CAS
CSCD
北大核心
2022年第12期5171-5183,共13页
Microbiology China
基金
国家自然科学基金(31772776)
广东省自然科学基金(2020A1515010475)
广东省科技专项资金(江科[2021]183号)
广东省现代农业产业技术体系创新团队项目(2021KJ119)
广东省农业科学院学科团队建设计划(XTXM202202,XT202208)
国家重点研发计划(2016YFD0500709)。
