We identified four distinct states of microglial cells that did not adhere closely to the classical M1/M2 (inflammatory/anti-inflammatory) distinction66, although some of these cell clusters were enriched for markers of inflammation Indeed, recent studies are beginning to question the biological accuracy of the widely-cited M1/M2 classification.67 One subcluster was distinguished by high CD83 expression, a gene implicated in regulating both inflammatory and anti-inflammatory processes.39 An increased proportion of microglia showed an inflammatory gene expression profile in those with AUD. Chronic alcohol exposure has been shown to cause microglial activation in mice, leading to neuroinflammation.68 Our analyses found that expression of ZBTB16, a negative regulator of inflammation,44 was a key regulator of gene module 1, and ZBTB16 expression was decreased in those with AUD. Thus, the increased inflammatory response in microglia in AUD could be due in part to decreased ZBTB16 activity. ZBTB16 is known to counteract microglial M1 activation,45 and its knockout in mice caused increased microglia and autism-like and schizophrenia-like behaviors,46 but it had not been implicated in AUD.
