european crystal network workshop

    Hydrogen sulfide inhibits NLRP3 inflammasome activation induced by monosodium urate crystals

     

    Mariela Castelblanco 1, Jerome Lugrin 2, Driss Ehirchiou 2, Isao Ishii 3, Sonia Nasi 1, Alexander So 1, Fabio Martinon 2, Nathalie Busso 1

    1 Service of Rheumatology, DAL, Lausanne University Hospital (CHUV), University of Lausanne, Switzerland, 2 Department of Biochemistry, University of Lausanne, Epalinges, Switzerland, 3 Showa Pharmaceutical University, Tokyo, Japan

     

    Background: Monosodium urate (MSU) crystal is the aetiological agent in the onset of the acute inflammatory condition gout by triggering NLRP3 inflammasome activation and interleukin (IL)-1β secretion. The NLRP3 inflammasome is a multi-protein complex formed by the sensor NLRP3, the adaptor protein ASC and caspase-1. NLRP3 inflammasome activation requires xanthine oxidase activity, and mitochondrial functions (such as ROS production). Upon ASC oligomerization, caspase-1 becomes active and cleaves the pro-inflammatory cytokines IL-1β into its active secreted form. Hydrogen sulfide (H2S), a gasotransmitter involved in various pathophysiological processes including inflammation, can be generated by H2S donors. H2S is also produced in cells by three enzymes; among them, cystathionine γ-lyase (CSE) is the major one in bone marrow-derived macrophages (BMDM). We investigated here the effects of exogenous and endogenous H2S on NLRP3 inflammasome activation in vitro and in vivo.

    Methods: Primed bone-marrow derived macrophages (BMDM) from wild-type (WT) or CSE-deficient mice as well as human macrophages (THP1 cell line and primary macrophages) were stimulated with 500 µg/ml MSU crystals for 6h, in the presence or absence of sodium thiosulfate (STS) or GYY4137 (GYY), two H2S donors. IL-1β levels were determined by Western-blot and ELISA; caspase-1 levels by Western-blot and specific bioluminescent assay; xanthine oxidase (XO) activity by pterin assay; and mitochondrial ROS production by MitoSOX fluorescence. In vivo, peritonitis was induced by i.p. injection of 1 mg MSU crystals to mice that was pretreated with GYY 50 mg/kg or vehicle alone 30 min before. At 6 h later, IL-1β, IL-6 and MCP-1 levels in peritoneal washes were measured by ELISA.

    Results: Both STS and GYY administration inhibited MSU crystal-induced IL-1β secretion from murine and human macrophages in dose-dependent manners. They also inhibited XO activity, mitochondrial ROS generation, ASC oligomerization, and caspase-1 activation in both macrophages. Accordingly, in murine macrophages deficient for CSE, IL-1β secretion, together with XO activity and caspase-1 activity, were significantly increased compared to wild-type BMDM. Finally, in the MSU–induced peritonitis models, the GYY pretreatment significantly decreased IL-6 and MCP-1 in peritoneal washes, whereas IL-1β level was decreased but did not reach significance.

    Conclusion: Exogenous H2S as well as endogenous (CSE-produced) H2S inhibited NLRP3-inflammasome activation. H2S donors have anti-inflammatory effects in the MSU-induced peritonitis model in vivo. Our study provides a novel anti-inflammatory therapeutics using H2S-releasing compounds against crystal-associated diseases such as gout.

     

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