副猪嗜血杆菌外膜囊泡激活caspase-11和NLRP3炎性体诱导炎性细胞因子IL-1β和IL-18分泌

Outer membrane vesicles from Haemophilus parasuis activate caspase-11 and NLRP3 inflammasome to induce the secretion of IL-1β and IL-18

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(outer membrane vesicles，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24h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72h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24h，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48h细胞培养上清检测IL-1β和IL-18水平。【结果】Caspase-11 mRNA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、24h极显著高于对照组(P<0.01)；ASC mRNA转录水平在感染后12、24h显著低于对照组(P<0.05)；caspase-1 mRNA转录水平在感染后6、12和24h显著高于对照组(P<0.05)。Western blotting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72h，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72h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。

关键词:副猪嗜血杆菌;外膜囊泡;caspase-11;NLRP3炎性体;IL-1β;IL-18

Abstract:

[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-1 48 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 100 μg/mL) for 24 h, the cell supernatants were collected for the measurement of IL-1β and IL-18 levels by ELISA. The RAW264.7 cells with the silencing of caspase-11 were established and then infected with OMVs, and the supernatants were collected for the measurement of IL-1β and 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-1β elevated 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-1β in 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-1β at 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.

Key words:Haemophilus parasuis;outer membrane vesicles;caspase-11;NLRP3 inflammasome;IL-1β;IL-18