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

<b><i>Introduction:</i></b> 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. <b><i>Objective:</i></b> Here, we investigate the diurnal variation of lymphocyte infiltration in the colonic mucosa in response to alcohol. <b><i>Methods:</i></b> 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 <i>t</i> test, one-way ANOVA, and two-way ANOVA were used to determine significance. <b><i>Results:</i></b> 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. <b><i>Conclusions:</i></b> Alcohol enhanced the proinflammatory profile in the colon mucosa, as demonstrated by a higher T-bet⁺/Foxp3⁺ ratio, especially during the rest phase<b><i>.</i></b>These findings may partly account for the interaction of circadian rhythm disruption with alcohol in colon inflammation and cancer.