Abstract
BackgroundMicroglial activation contributes to the neuropathology associated with chronic alcohol exposure and withdrawal, including the expression of inflammatory and anti-inflammatory genes. In the current study, we examined the transcriptome of primary rat microglial cells following incubation with alcohol alone, or alcohol together with a robust inflammatory stimulus.MethodsPrimary microglia were prepared from mixed rat glial cultures. Cells were incubated with 75 mM ethanol alone or with proinflammatory cytokines (“TII”: IL1β, IFNγ, and TNFα). Isolated mRNA was used for RNAseq analysis and qPCR. Effects of alcohol on phagocytosis were determined by uptake of oligomeric amyloid beta.ResultsAlcohol induced nitrite production in control cells and increased nitrite production in cells co-treated with TII. RNAseq analysis of microglia exposed for 24 h to alcohol identified 312 differentially expressed mRNAs (“Alc-DEs”), with changes confirmed by qPCR analysis. Gene ontology analysis identified phagosome as one of the highest-ranking KEGG pathways including transcripts regulating phagocytosis. Alcohol also increased several complement-related mRNAs that have roles in phagocytosis, including C1qa, b, and c; C3; and C3aR1. RNAseq analysis identified over 3000 differentially expressed mRNAs in microglia following overnight incubation with TII; and comparison to the group of Alc-DEs revealed 87 mRNAs modulated by alcohol but not by TII, including C1qa, b, and c. Consistent with observed changes in phagocytosis-related mRNAs, the uptake of amyloid beta1–42, by primary microglia, was reduced by alcohol.ConclusionsOur results define alterations that occur to microglial gene expression following alcohol exposure and suggest that alcohol effects on phagocytosis could contribute to the development of Alzheimer’s disease.
Highlights
Microglial activation contributes to the neuropathology associated with chronic alcohol exposure and withdrawal, including the expression of inflammatory and anti-inflammatory genes
Alcohol increases microglial inflammatory activation Enriched (> 95%) primary microglia were prepared by the shake-off method from postnatal day 2 rat cortical mixed glial cultures, incubated with 75 mM ethanol, with or without Mixture of IL1β (TII) to induce inflammatory activation as assessed by measurement of nitrite levels in the culture media
Alcohol-dependent changes in microglial mRNA RNA sequencing (RNAseq) analysis was used to identify mRNAs regulated by exposure to alcohol
Summary
Microglial activation contributes to the neuropathology associated with chronic alcohol exposure and withdrawal, including the expression of inflammatory and anti-inflammatory genes. Microglial activation occurs in a variety of neurological conditions and diseases, and increasing number of studies show that microglial activation contributes to the neuroinflammation associated with chronic alcohol exposure and withdrawal. While pathway analysis identified many microglial associated functions, only a single study has directly examined the transcriptome of microglia acutely isolated from cortex of alcohol-fed mice [24]. Over 400 transcripts were identified in the microglial that were not present in pre-frontal cortical RNA [22], suggesting that cell type enrichment is necessary to fully characterize the effects of alcohol on the microglial transcriptome
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