Background: Hydrogen sulfide (H ₂ S) is a gasotransmitter involved in various pathophysiological processes, including inflammation. Physiological and pharmacological levels of H ₂ S can be reached, both in vitro and in vivo , using H ₂ S donors. H ₂ S is produced in cells by three enzymes, one of which is cystathionine gamma lyase (CSE). NLRP3 inflammasome is a multiprotein complex that can be activated by monosodium urate (MSU) crystals, the etiological factor in gout. When the inflammasome is activated, ASC oligomerizes, leading to the activation of caspase-1, which cleaves pro-IL-1β into its active, secreted form. We previously demonstrated that both exogenous and endogenous H ₂ S inhibit NLRP3 inflammasome activation in macrophages in vitro and in murine peritoneal fluids in vivo [1].

Objectives: To test if positive allosteric activators of CSE (CSE-PAMs) could inhibit NLRP3 inflammasome activation by MSU crystals.

Methods: A CSE-PAM (SAN523) was identified and tested for its ability to enhance H₂S production by recombinant or cellular CSE. H ₂ S production was measured using the H ₂ S specific fluorescent probe AzMc or the lead acetate method. Supernatants from primed murine bone-marrow derived macrophages (BMDM) and human macrophage-like THP1 cells, stimulated with 500 µg/ml MSU crystals for 6h, in the presence or absence of SAN523 (0.1-50 µM) were analyzed by IL-1β Western-Blot (WB) and ELISA. In vivo , peritonitis was induced in mice by i.p. injection of 1 mg MSU crystals, with SAN523 treatment (5 mg/kg) or vehicle (DMSO) administered i.v. 15 minutes prior to MSU injection. THP1 cells, stimulated with 500 µg/ml MSU crystals for 1h, in the presence or absence of SAN523 (50 µM). Lysates were analyzed by the maleimide assay for detection of protein persulfidation.

Results: SAN523 stimulated in a dose-dependent way H ₂ S production by human recombinant CSE and by THP1 cells. In addition, SAN-523 reduced ASC oligomerization (% ASC speck positive cells over total cells: MSU = 11.5; MSU+SAN523 = 3.5, p = 0.037) and inhibited in a dose-dependent way IL-1β secretion in primed THP1 macrophages stimulated with MSU crystals (IC50 = 1µM, Figure 1A). A similar decrease in IL-1β secretion was observed in primed BMDMs. Inhibition of IL-1β secretion by SAN523, in both human and murine cell supernatants, was confirmed by WB. In the MSU-induced peritonitis model, pretreatment with SAN523 significantly reduced inflammatory cell recruitment (millions of cells: MSU = 5.3; MSU+SAN523 = 3.7, p = 0.012, Figure 1B) and IL-1β levels in peritoneal fluid (IL-1β in pg/ml: MSU = 30.27; MSU+SAN523 = 2.27, p = 0.009, Figure 1C), as well as other inflammatory cytokines and chemokines such as IL-6, IL-1α, Cxcl1. Finally, we identified that SAN523 acts, at least partly, via induction of protein persulfidation.

Figure 1.

A) Secretion of IL-1β (pg/ml) by THP1 cells determined by ELISA; experiment performed in triplicates. B) Inflammatory cell recruitment in murine peritoneal lavage n=4. C) Secretion of IL-1β (pg/ml) in murine peritoneal lavage n=4.

Conclusion: SAN523 enhances H ₂ S production leading to reduced NLRP3 inflammasome activation in vitro and in the MSU-induced peritonitis model in vivo . This new compound can be of interest as a novel drug for acute gout and other IL-1β mediated inflammatory diseases.

REFERENCES: [1] M. Castelblanco, J. Lugrin, D. Ehirchiou, S. Nasi, I. Ishii, A. So, F. Martinon, N. Busso, Hydrogen sulfide inhibits NLRP3 inflammasome activation and reduces cytokine production both in vitro and in a mouse model of inflammation, J Biol Chem 293(7) (2018) 2546-2557.

Acknowledgements: NIL.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.