Acute alcohol treatment alters macrophage heterogeneity by interfering with the polarization process

Abstract Alcohol abuse disorder increases the risk of a series of diseases. However, there is limited evidence to indicate how acute alcohol exposure alters immune and inflammatory responses. Macrophages are phagocytic cells with diverse functions. The heterogeneity of macrophages leads to a challenge of clear and precise identification of the functional status and mechanism by which acute alcohol alters macrophage function. To address this issue, we first establish an optimized eleven-parameter flow cytometry panel based on RAW 264.7 cells for identifying macrophage subtypes (Naïve, M1, M2a, and M2c). Then three sets of experiments have been conducted: 1) polarization stimuli were applied first, then alcohol was added to the cultures, 2) alcohol was added to the cells first then polarization stimuli, 3) alcohol and polarization stimuli were added simultaneously. A total of 32 cytokines and chemokines involved in inflammation and immunity were measured. Based on these results, we developed a mathematical expression of the M1-M2 continuum with the principal component analysis that can quantify the degree to which all the markers are consistent with an M1 phenotype or one of the M2 phenotypes, which provides a concise indicator to evaluate macrophage status. Results were consistent with previous studies in our lab using mouse peritoneal macrophages in vitro, and the results indicate that the effects of acute alcohol exposure differ depending on macrophage polarization status, and this may explain some of the effects of alcohol on macrophages that we and others have reported in vivo in a mouse model for binge drinking. The authors were supported by funding from an NIH Center of Biomedical Research Excellence (P20 GM103646-09). The authors were supported by funding from an NIH Center of Biomedical Research Excellence (P20 GM103646-09).