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

    LI Yan

    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
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  • YANG Jun

    YANG Jun

    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
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  • ZHAI Shaolun

    ZHAI Shaolun

    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
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  • CHU Pinpin

    CHU Pinpin

    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
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  • BIAN Zhibiao

    BIAN Zhibiao

    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
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  • ZHANG Kunli

    ZHANG Kunli

    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
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  • LI Chunling

    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
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    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.

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LI Yan, YANG Jun, ZHAI Shaolun, CHU Pinpin, BIAN Zhibiao, ZHANG Kunli, LI Chunling. Outer membrane vesicles from Haemophilus parasuis activate caspase-11 and NLRP3 inflammasome to induce the secretion of IL-1β and IL-18[J]. Microbiology China, 2022, 49(12): 5171-5183

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History
  • Received:July 13,2022
  • Revised:September 21,2022
  • Online: December 06,2022
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