Alcohol Dysregulates CD4 T Cell Mitochondrial Homeostasis

Abstract

CD4 T cell differentiation to pro‐inflammatory and immunosuppressive subsets requires distinct metabolic pathways. Pro‐inflammatory CD4 subsets rely on glycolysis, while immunosuppressive (Treg cells) subsets, require functional mitochondria for their differentiation and function. Previous studies have shown that binge alcohol (ethanol, EtOH) administration increased Tbet‐expressing (Th1) and decreased FOXP3‐expressing (Treg) CD4 T cells in the colons of mice. We tested the hypothesis that EtOH dysregulates normal CD4 T cell differentiation, after stimulation, by impairing mitochondrial homeostasis. Human naïve CD4 T cells were isolated from buffy coats from blood bank donors (N = 6) using MACS sorting. Cells were stimulated using anti‐CD3‐coated dishes in the presence of anti‐CD28 and IL‐12, and exposed to EtOH (0 and 50 mM) for 3 days. Mitochondrial content was measured with Mitotracker Deep Red. Gene expression indicative of: autophagosome formation (ATG5, ATG7, ATG13, MAP1LC3B, BECN1, BNIP3L, ULK1), mitophagy (PINK1, PRKN),mitochondrial fusion (MFN1, MFN2, OPA1), mitochondrial fission (MFFand FIS1), and mitochondrial biogenesis (PPARC1A, PPARC1B, TFAM) was determined by RT2 profiler arrays. EtOH‐treated CD4 T cells had increased mitochondrial content (p = 0.0008) with Tregs accounting for the greatest increase in mitochondria (p = 0.04). There was a main effect of stimulation (p < 0.05) to increase ATG5, ATG13, MAP1LC3B, BECN1, BNIP3L, ULK1, MFF, PPARC1B, and TFAM, and a main effect of EtOH (p < 0.05) to increase PINK1 and decrease ATG7. There was a main effect of both EtOH and stimulation (p < 0.05) to increase MFN2, and OPA1.Taken together, these results indicate that EtOH increases mitochondrial content in Treg cells and dysregulates mitochondrial gene expression important for mitochondrial repair and mitophagy. These EtOH‐mediated alterations in gene expression could result in an inability of CD4 T cells to maintain mitochondrial homeostasis and remove damaged mitochondria that is required for normal differentiation and function of anti‐inflammatory Treg cells.