Elevated O-GlcNAcylation Enhances Pro-Inflammatory Th17 Function by Altering the Lipid Microenvironment

Abstract

Chronic inflammation is a feature of obesity and enhances the risk of atherosclerosis, cancer, diabetes, and autoimmunity. Specifically, pro-inflammatory Th17 and Th1 CD4+ effector T cells are increased in metabolic diseases. However, a clear molecular mechanism linking metabolic changes with pro-inflammatory T cells is lacking. We hypothesize that elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification of nuclear and cytoplasmic proteins, promotes pro-inflammatory CD4+ T cell function. Since production of O-GlcNAc involves input from carbohydrate, amino acid, fatty acid, and nucleic acid metabolism, the modification acts as a general sensor of a cell’s nutritional status. To investigate the role of O-GlcNAc in CD4+ T cell function, we treated CD4+ T cells with Thiamet-G (TMG, an inhibitor of the enzyme that removes O-GlcNAc (O-GlcNAcase, OGA)) before activation of the cells without addition of polarizing cytokines. After culturing for four days and then re-stimulating the cells for 24 hours, we found that these non-polarized CD4+ T cells produced significantly more pro-inflammatory IL-17A by both protein and transcript levels. Since IL-17A is critically involved in exacerbating obesity-related pathology, such as atherosclerosis and insulin resistance, we next looked for an effect of O-GlcNAcylation in metabolic disease. To investigate the role of O-GlcNAc in a setting of metabolic disease, we analyzed O-GlcNAc levels in CD4+ T cells from mice fed a high fat and cholesterol “Western” diet. Naïve CD4+ T cells from obese mice have elevated O-GlcNAc levels compared to cells from mice fed standard chow. When polarized to a Th17 lineage, cells from obese mice secrete more IL-17A, the eponymous Th17 cytokine. Importantly, when naïve CD4+ T cells from lean and obese mice are polarized to a Th17 lineage in the presence of TMG, the cells from lean mice secrete levels of IL-17 comparable to those from obese mice. Moreover, IL-17A secretion was exacerbated in cells from obese mice. Transcript levels of IL-17 are similarly elevated in both lean and obese mice Th17 cells treated with TMG. Importantly, these results are recapitulated in human CD4+ T cells treated with TMG, suggesting a similar mechanism may operate in both mice and humans. RORγt (retinoic acid-related orphan receptor gamma) acts as the Th17 master transcription factor, orchestrating the Th17 differentiation program. While protein and transcript levels of RORγt did not change in the presence of TMG, chromatin immunoprecipitation demonstrated enhanced RORγt association with the IL-17 promoter and an enhancer, CNS2 (conserved noncoding sequence 2) with TMG treatment. Uniquely among the CD4+ effector T cell master transcription factors, RORγt is regulated by lipid ligands that promote or repress its activity. We performed a broad lipidomics analysis on T cells and discovered that sterols and saturated fatty acids, known activating ligands of RORγt, were increased with TMG treatment. An investigation of potential mechanisms underlying these changes in lipid content in T cells revealed that the rate-limiting enzyme in fatty acid synthesis, ACC1, is O-GlcNAcylated in the carboxyltransferase and central region domains, which are important for catalytic conversion of acetyl CoA to malonyl CoA. Collectively, our data suggest that elevated O-GlcNAc levels increase pro-inflammatory IL-17 secretion from Th17 cells through alteration of the lipidome and retention of RORγt at the IL-17 promoter complex. These studies provide a potential molecular link between nutritional excess and Th17 differentiation and function. Further study of these molecular mechanisms will provide insight into the link between O-GlcNAcylation and inflammatory disease associated with obesity and other metabolic disorders.