The interaction of O-GlcNAc-modified NLRX1 and IKK-α modulates IL-1β expression in M1 macrophages.

NOD-like receptor (NLR)X1 (NLRX1) is a negative regulator of inflammation by inhibiting nuclear factor-κB (NF-κB) signaling and downstream pro-inflammatory factors. However, its post-translational modification and how it participates in regulating the inflammatory responses in macrophages are still unclear. Here, we found that NLRX1 was modified with O-linked N-acetylglucosamine (O-GlcNAc). The interaction and co-localization between NLRX1 and O-GlcNAc transferase (OGT) was validated by co-immunoprecipitation and confocal microscopy analysis, and the nucleotide-binding domain (NBD) region of NLRX1 was required for its interaction with OGT. NLRX1 protein increased significantly after treatment with a high dose of OGT inhibitor OSMI-1. Elevated O-GlcNAcylation level promoted NLRX1 ubiquitination and decreased NLRX1 stability proved by ubiquitination and cycloheximide (CHX) chase experiments, and enhanced the interaction between NLRX1 and inhibitor of nuclear factor kappaB kinase-α (IKK-α), thus reducing the expression of inflammatory cytokine IL-1β in M1 macrophages. Together, our results indicate that the interaction between NLRX1 and O-GlcNAcylation coordinates and modulates the inflammatory process in macrophages.