Involvement of microglial α7AChR in binge alcohol modulation of gut dysbiosis - Binge alcohol drinking is very common among the young people. Excessive alcohol consumption affects innate immune signaling in the gut and the brain by altering gene expression and molecular pathways which lead to alcohol use disorder (AUD). The binge alcohol-modulated gut-brain axis, retrogradely, is associated with neuroinflammation and microglial activation. On the other hand, binge alcohol, anterogradely, may cause neuroinflammation-mediated gut dysbiosis, via the “microglia-gut axis”. Targeting the neuroimmune system is a new avenue for developing and repurposing effective pharmacotherapies and it is clinically relevant and highly significant. The nicotinic alpha7 acetylcholine receptor (7AChR), which is expressed in microglia regulates neuroinflammation through cholinergic anti-inflammatory pathway. Our published data and other study showed that lipopolysaccharide (LPS)-induced inflammation releases pro-inflammatory cytokines and upregulates non- functional 7AChR (futile) in macrophages and microglia. Stimulation of futile 7AChR with GTS-21 (7AChR agonist), decreases LPS-induced TNF release in macrophages, and knockdown of 7AChR with 7AChR siRNA in LPS-induced macrophages abolish GTS-21 beneficial effects. Our preliminary studies demonstrated that mice given binge ethanol (EtOH) suffer with gut dysbiosis and continuously exhibit significant differences in -diversity along with an increase in commensal proportions of Prevotellaceae family. Taken these premises together, we hypothesize that binge EtOH exposure causes neuroinflammation and gut dysbiosis due to the release of proinflammatory cytokines, which lead to microglial activation and increased expression of microglial 7AChR (futile), and bioinformatic analysis of these factors could establish a functional relationship among them. In in vivo, disruption of microglia and microglial 7AChR utilizing PLX-5622 (microglia depletion reagent) and microglial 7AChR conditional knockout (7AChRcKO) mice, respectively, will elucidate the discrete roles of microglia and microglial 7AChR after binge EtOH exposure. The pharmacological intervention of GTS-21 in vivo will reverse EtOH-induced effects on microglia activation, neuroinflammation and gut dysbiosis involving 7AChR. To test these hypotheses, we have posited two specific aims. Aim 1 is to characterize activation of microglia, neuroinflammation and gut dysbiosis following binge EtOH exposure using C57BL/6J (B6) mice and bioinformatics tools. Aim 2 is to elucidate the involvement of microglial 7AChR and role of microglia in alcohol- modulation of neuroinflammation and gut dysbiosis by utilizing B6 and 7AChRcKO mice using PLX-5622 and GTS-21. Our innovative project with high clinical relevance, might have high risk, yet very likely high reward. Successful completion of this R21 project shall shift the paradigm from observing the gut-brain axis to understanding and pharmacologically targeting the microglia-gut axis following binge exposure to EtOH and would allow us to develop a full length R01 project to target the neuroimmune signaling in developing and repurposing effective pharmacotherapies for alcohol drinkers.
