Ethanol modulates human microglial inflammatory cytokines secretion in response to Spike protein

In the central nervous system (CNS), microglia regulate immune responses and mediate neuroinflammation. Alcohol abuse can promote neuroinflammation and neurodegeneration, and alcohol consumption increased during the COVID-19 pandemic as a coping strategy for anxiety and depression associated with social isolation. SARS-CoV-2 can also trigger neuroinflammation and glial reactivity through interactions of its Spike protein with ACE2 and co-receptors such as TMPRSS2. However, the effect of alcohol exposure on Spike-induced microglial reactivity remains unclear. In this study, human microglial cultures were exposed to different ethanol concentrations and/or subsequently challenged with Spike protein. Immunofluorescence showed that 170 mM ethanol did not alter ACE2, TMPRSS2, or TLR4 levels, whereas 340 mM ethanol increased ACE2 compared with control. Treatment with 170 mM ethanol followed by Spike did not change microglial reactivity markers, including CD11b, C3, Arginase-1, and iNOS. However, ethanol + Spike produced an apparent increase in NF-κB protein levels, which was not observed with ethanol or Spike alone by Western blotting. A multiplex cytokine array identified 73 of 80 cytokines and chemokines, including GRO-α, IL-2, IL-6, IL-8, M-CSF, TIMP-1, IGFBP-2, and IGF-1, with higher levels in conditioned medium from ethanol + Spike-treated microglia than in ethanol-only, Spike-only, or control conditions. These findings suggest that ethanol alone may not induce a clear inflammatory phenotype in microglia, but it can alter secreted cytokine and chemokine profiles and may prime cells for a stronger inflammatory response after subsequent exposure to Spike protein.