Long-chain S-acylation is a key modulator during the macrophage inflammatory response

Long-chain S-acylation is a reversible lipid modification critical for regulating protein localization, stability, and signaling, yet its role in macrophage-mediated inflammation remains incompletely understood. Here, we combine stable isotope labeling by amino acids in cell culture (SILAC) with site-specific acyl-biotin exchange (ssABE) to generate a comprehensive map of the long-chain S-acylation landscape in THP-1 macrophages polarized to M0 and M(LPS+ IFNɣ) states. Our quantitative proteomics reveal polarization-specific S-acylation patterns and uncover numerous inflammation-related modification sites, including novel S-acyl peptidoforms, which are distinct S-acylated variants of the same peptide such as WARS (C305/C309), highlighting their potential relevance in macrophage activation. Pharmacological inhibition of S-acylation with the broad-spectrum inhibitor 2-bromopalmitate suppresses secretion of key pro-inflammatory chemokines (CXCL9, CXCL10, CCL4) and disrupts IDO1-mediated tryptophan catabolism, while Palmostatin B mainly stabilizes S-acylation on GPCR signaling proteins. Together, these findings position long-chain S-acylation as a key regulatory mechanism during macrophage activation, and a promising target for therapeutic intervention in inflammatory disease.