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.