Supplementary Material for: Alcohol-Induced Immune Dysregulation in the Colon Is Diurnally Variable

Introduction: Alcohol increases the risk of colon cancer. Colonic inflammation mediates the effects of alcohol on colon carcinogenesis. Circadian rhythm disruption enhances the alcohol’s effect on colonic inflammation and cancer. Objective: Here, we investigate the diurnal variation of lymphocyte infiltration in the colonic mucosa in response to alcohol. Methods: Sixty C57BL6/J mice were fed a chow diet, and gavaged with alcohol at a specific time once per day for 3 consecutive days. Immunohistochemistry and immunofluorescence staining were used to quantify total, effector, and regulatory T cells in the colon. Student’s t test, one-way ANOVA, and two-way ANOVA were used to determine significance. Results: Following the alcohol binge, the composition of immune T cell subsets in the mouse colon was time-dependent. Alcohol did not alter the total number of CD3+ T cells. However, upon alcohol treatment, T-bet+ T helper 1 (Th1) cells appeared to dominate the T cell population following a reduction in Foxp3+ regulatory T cell (Treg) numbers. Depletion of Tregs was time-dependent, and their numbers were dramatically reduced when alcohol was administered during the rest phase. A reduction in Tregs significantly increased the Th1/Treg ratio, resulting in a more proinflammatory milieu. Conclusions: Alcohol enhanced the proinflammatory profile in the colon mucosa, as demonstrated by a higher T-bet+/Foxp3+ ratio, especially during the rest phase.These findings may partly account for the interaction of circadian rhythm disruption with alcohol in colon inflammation and cancer.