Induction of hydrogen sulfide production and protein persulfidation in activated human and mouse macrophages.

Inflammation plays an essential role in host defense and homeostasis but its dysregulation contributes to some of the commonest human diseases, including cancer, cardiovascular disease, and neurodegenerative disorders. Hydrogen sulfide (H2S) is an endogenously produced signaling molecule that regulates various physiological functions including inflammation. H2S signaling is largely mediated by protein persulfidation (Cys-SSH), a posttranslational modification of cysteine thiols (Cys-SH). The molecular mechanisms by which H2S regulates the inflammatory response remain poorly defined.

Here, we analyzed H2S generation and protein persulfidation in human or mouse macrophages stimulated ± lipopolysaccharide (LPS) plus interferon-γ (LPS/IFN-γ), representing classical pro-inflammatory conditions. Imaging with fluorescent probes revealed that LPS/IFN-γ stimulation triggered the production of H2S and polysulfur species. By employing the biotin thiol assay (BTA) we found that LPS/IFN-γ stimulation of differentiated THP-1 macrophages caused a substantial elevation in global protein persulfidation. Similarly, we observed increased protein persulfidation in J774 mouse macrophages stimulated with LPS/IFN-γ. Inhibitor studies indicated that cystathionine γ-lyase (CSE)-mediated H2S generation plays a major role in driving protein persulfidation in activated macrophages. We conducted a large-scale proteomic analysis of persulfidation by coupling the BTA assay to mass spectrometry (MS) analysis. The MS analyses identified hundreds of proteins whose persulfidation was increased upon LPS/IFN-γ stimulation in both human and mouse macrophages. These target proteins are involved in various cellular processes including protein synthesis and degradation, mRNA splicing, carbon metabolism, and phagocytosis. Taken together, these data support the notion that H2S may regulate macrophage function and inflammatory response via protein persulfidation.