Abstract
A variety of stimuli, including monosodium urate (MSU) crystals, activate the NLRP3 inflammasome, and this activation involves several molecular mechanisms including xanthine oxidase (XO) up-regulation and mitochondrial dysfunction. Upon oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 becomes active and cleaves the proinflammatory cytokine IL-1β into its active secreted form. Hydrogen sulfide (H2S), a gasotransmitter mainly produced by cystathionine γ-lyase (CSE) in macrophages, could modulate inflammation. Here, we sought to investigate the effects of exogenous and endogenous H2S on NLRP3 inflammasome activation in vitro and in vivo Primed bone marrow-derived macrophages (BMDM) isolated from wildtype (wt) or CSE-deficient mice and human macrophages (THP1 cells and primary macrophages), were stimulated with MSU crystals in the presence or absence of a H2S donor, sodium thiosulfate (STS) or GYY4137 (GYY). In murine and human macrophages in vitro, both STS and GYY inhibited MSU crystal-induced IL-1β secretion in a dose-dependent manner. Moreover, the H2S donors inhibited MSU crystal-induced XO/caspase-1 activities, mitochondrial reactive oxygen species (ROS) generation, and ASC oligomerization. Accordingly, IL-1β secretion and XO/caspase-1 activities were higher in CSE-deficient BMDMs than in wt BMDMs. For in vivo studies, we experimentally induced peritonitis by intraperitoneal injection of MSU crystals into mice. GYY pretreatment ameliorated inflammation, evidenced by decreased IL-6/monocyte chemoattractant protein-1 (MCP-1) released into peritoneal lavages. Taken together, our results suggest that both exogenous (via H2S donors) and endogenous (via CSE) H2S production may represent approaches for managing, for example, acute gout or other inflammation conditions.
Highlights
A variety of stimuli, including monosodium urate (MSU) crystals, activate the NLRP3 inflammasome, and this activation involves several molecular mechanisms including xanthine oxidase (XO) up-regulation and mitochondrial dysfunction
We previously demonstrated that xanthine oxidase (XO)-derived mitochondrial reactive oxygen species (ROS) are the trigger for IL-1 release, and that XO blockade results in impaired IL-1/caspase-1 secretion [17]
As we showed before that H2S donors inhibited both MSU-induced XO activity and mitochondrial ROS generation, we hypothesized that, lowering endogenous H2S levels could lead to opposite effects
Summary
H2S donors inhibit NLRP3 inflammasome activation in MSU or nigericin-stimulated macrophages. In primed murine and human macrophages (BMDM and THP1 cells, respectively), GYY inhibited MSU-induced IL-1 secretion in dose-dependent manners (Fig. 1, A and C). The RT-PCR Ct values of SLC13a4 are 26.9 for THP1, whereas Ͼ40 (i.e. undetectable) for BMDM Both GYY and STS inhibited MSU-induced IL-1 secretion in primary human macrophages (Fig. 1D). In line with the IL-1 ELISA results, H2S donors inhibited both caspase-1 processing and IL-1 maturation that were induced by NRLRP3 agonists (MSU crystals and nigericin), as evidenced by reduced active p20 caspase-1 and active p17 IL-1 levels in cell supernatants, respectively (Fig. 1E). GYY was effective for such protein level changes in all these cell types, whereas STS only in human macrophages (THP1 and primary cells, Fig. 1E). The inhibitory effects of H2S donors could not be attributed to their effects on cell death or proliferation
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