Relationships between protein O-GlcNAcylation, oxidative stress and inflammatory processes in the macrophage

O-GlcNAcylation is a dynamic post-translational modification of proteins consisting of the attachment of N-Acetyglucosamine (GlcNAc) to serine/threonine residues. This modification is only mediated by a single pair of enzymes: OGT which adds GlcNAc on the protein, and OGA, which hydrolyzes and removes the GlcNAc moiety. This modification occurs in nuclear, cytosolic and mitochondrial compartments. Through modulation of protein stability, enzymatic activity, subcellular localization, and protein–protein interactions, O-GlcNAcylation regulates numerous cellular physiological and pathological processes.

Previous work from our laboratory demonstrated that macrophage-specific deletion of OGT increases the effect of LPS on NOS2 expression and production of pro-inflammatory cytokines, suggesting that O-GlcNAcylation restricts excessive inflammation.

In the present work, through the use of pharmacological inhibition of OGT and of Ogt-deficient macrophages, we observed that O-GlcNAcylation deficiency increased mitochondrial ROS production, oxidative stress markers and pro-inflammatory gene expression upon LPS stimulation. The hyper-inflammatory profile was partially corrected by the mitochondrial-targeted ROS scavenger MitoTEMPO, indicating a role for mitochondrial ROS in the observed effects. In agreement with this notion, the expression of the mitochondrial oxidative stress marker SOD2 significantly correlates with inflammatory genes expression in Ogt-KO macrophages. Additionally, we also find that LPS induced protein O-GlcNAcylation in mitochondrial compartment, confirming the potential role of this modification in regulating mitochondrial ROS homeostasis upon LPS stimulation.

Altogether, our findings reveal a novel role for O-GlcNAcylation in controlling macrophage inflammatory responses through regulation of mitochondrial ROS homeostasis. Our work highlights mitochondria as critical sites where metabolic and inflammatory pathways converge via O-GlcNAc signaling, offering new insights into the regulation of innate immune activation under stress conditions.

Keywords:

O-GlcNAcylation, Post-translational modification, Macrophage, Pro-inflammatory pathways, Mitochondrial functions, Reactive Oxygen Species, Oxidative stress response