CD4+ T cells can differentiate into distinct effector subsets upon activation. Changes in immunometabolism impact this important fate decision due to different bioenergetic requirements of CD4+ T cell subsets. The short chain fatty acid butyrate is a metabolite generated by the intestinal microbiota as a by-product of dietary fibre fermentation. It has been shown to be metabolised by many cell types. Although the effects of butyrate under Treg and Th17-polarising conditions have been intensively studied, its impact on Th0 cell differentiation and function in the absence of polarising conditions is poorly understood. We aimed to investigate how butyrate impacts a key function of CD4+ T cells: their capacity to differentiate into distinct subsets. Therefore, we treated CD4+ T cells with butyrate in vitro to characterise its effects on CD4+ T cell differentiation, function and metabolism. Additionally, we adoptively transferred butyrate-treated cells and challenged recipient mice with HSV-1.
We demonstrated that butyrate promotes activated CD4+ T cell mitochondrial respiration, but does not influence glycolysis. This metabolic change was accompanied by a significantly elevated T-bet expression, indicating greater polarisation into Th1 cells even under non-polarising conditions. Moreover, Eomes co-expression suggested differentiation into CD4+ cytotoxic lymphocytes – an underappreciated but important subset that directly eliminates target cells. Cytotoxicity was hence tested using in vitro killing assays, in which butyrate induced a 75% increase in CD4+ T cell-mediated direct killing of B16 melanoma cells. This correlated with significantly upregulated expressions of IFN-g, granzyme B, perforin, and Fas ligand. Greater polarisation of butyrate-treated CD4+ T cells into Th1 cells was maintained long-term after adoptive transfer and HSV-1 infection in vivo. Butyrate may therefore prime CD4+ T cells to become more effective killers. These findings highlight the importance of metabolite availability, such as butyrate, in the microenvironment for pro-inflammatory responses towards infection and cancer.