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Effects of augmented O‐GlcNAcylation on activation and differentiation of macrophages

Macrophages (MØs) are classified as classically‐(M1) or alternatively‐(M2) activated, based on their exposure to different fate‐determining mediators. The post‐translational modification of proteins by O‐GlcNAcylation (O‐GlcNAc) is highly dynamic and modulates cell‐signaling processes. Acute increases in O‐GlcNAc levels reduce the release of pro‐inflammatory mediators and regulate inflammatory processes by decreasing e.g. NF‐kB activation. This study tested the hypothesis that increased O‐GlcNAc levels favor polarization of MØs to the anti‐inflammatory/M2 phenotype. Macrophages obtained from bone marrow of male BALB/c mice were incubated with vehicle, glucosamine, the O‐GlcNAcase inhibitors PugNAc (100 µM) and Thiamet‐G (TMG, 1µM), LPS + IFN‐g (1mg/ml + 200ng/ml) for M1 or interleukin‐4 (IL‐4, 50ng/ml) for M2 polarization. Expression of polarization markers (F4/80 and CD206) was assessed by flow cytometry. Cellular viability was not affected by these compounds, as measured by MTT reduction assay. Incubation of MØs with TMG, glucosamine and PugNAc produced a time‐dependent increase in O‐GlcNAc levels, determined by western blot. Incubation of undifferentiated MØs with TMG, as well as with LPS (1mg/ml), for 24h significantly increased IL‐1b release. In M1‐differentiated MØs, stimulation for 24h with TMG further increased IL‐1b and IL‐6 mRNA expression. In M2‐differentiated MØs, TMG did not change arginase I expression. These preliminary results suggest that increases in O‐GlcNAcylation in 24h contribute to a pro‐inflammatory phenotype in macrophages. Our studies suggest that the O‐GlcNAc pathway as a potential therapeutic target in diseases associated inflammatory responses.