Transcriptome analysis of alcohol-treated microglia reveals downregulation of beta amyloid phagocytosis - PubMed (original) (raw)

Transcriptome analysis of alcohol-treated microglia reveals downregulation of beta amyloid phagocytosis

Sergey Kalinin et al. J Neuroinflammation. 2018.

Abstract

Background: Microglial 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.

Methods: Primary 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.

Results: Alcohol 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.

Conclusions: Our 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.

Keywords: Alcohol; Alzheimer’s disease; Amyloid; Complement; Microglia; Phagocytosis.

PubMed Disclaimer

Conflict of interest statement

Ethics approval

All experiments were conducted with approval of the University of Illinois Institutional Animal Care and Use Committee (protocol number 15-129).

Competing interests

The authors declare that they have no competing interests

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

Fig. 1

Alcohol increases microglial inflammatory activation. Primary rat microglia were incubated overnight with nothing (control), with TII to induce an inflammatory response, with 75 mM ethanol (Alc), or with TII and ethanol (Alc and TII). The next day, nitrite production was measured using Griess reagent. The data is the mean ± sem of n = 6–15 replicates done in 3 independent studies. *P < 0.05; ***P < 0.0001; one-way ANOVA, Tukey’s post hoc analysis

Fig. 2

Fig. 2

Validation of RNAseq results. Reverse transcriptase quantitative PCR (qPCR) was used to measure mRNA levels of the indicated Alc-DEs. qPCR was carried out in samples from microglia incubated for 24 h with nothing (control, n = 3) or 75 mM ethanol (n = 3), and results normalized to values measured for β-actin in the same samples. The _y_-axis shows the ratio of the average mRNA level measured in the ethanol versus the control samples (filled bars) and is plotted next to the fold-difference calculated from RNAseq data (open bars). For qPCR results, *P < 0.05; #P < 0.10 control vs alcohol. For the RNAseq data, all DEs were found significantly different using Deseq2

Fig. 3

Fig. 3

Overlap of identified DEs. A Venn diagram illustrating overlap of Alc-DEs (n = 312; orange, regions 1, 2, 4, and 5), TII-DEs (n = 3082; green, regions 2, 3, 5, and 6) and AlcTII-DEs (n = 3552; blue, regions 4, 5, 6, and 7). The full list of the DEs in each group is provided in Additional file 1: Tables S1-S4

Fig. 4

Fig. 4

Alcohol reduces amyloid beta phagocytosis in rat microglia. Primary rat microglia were incubated overnight with a nothing (control), b 75 mM ethanol, c TII, or d 75 mM ethanol and TII. The next day, the cells were assayed by flow cytometry for phagocytosis of FAM-labeled oligomeric Aβ1–42. e Average internalized FAM fluorescence per cell. Data is from 3 different batches of microglial cells totaling 9197 (controls), 8976 (alcohol), 24,588 (TII), and 28,773 (Alc * TII) events. *P < 0.005 versus control cells, one-way ANOVA, Tukey’s post hoc analysis

References

    1. Robinson G, Most D, Ferguson LB, Mayfield J, Harris RA, Blednov YA. Neuroimmune pathways in alcohol consumption: evidence from behavioral and genetic studies in rodents and humans. Int Rev Neurobiol. 2014;118:13–39. doi: 10.1016/B978-0-12-801284-0.00002-6. - DOI - PMC - PubMed
    1. Warden A, Erickson E, Robinson G, Harris RA, Mayfield RD. The neuroimmune transcriptome and alcohol dependence: potential for targeted therapies. Pharmacogenomics. 2016;17:2081–2096. doi: 10.2217/pgs-2016-0062. - DOI - PMC - PubMed
    1. Crews FT, Lawrimore CJ, Walter TJ, Coleman LG., Jr The role of neuroimmune signaling in alcoholism. Neuropharmacology. 2017;122:56–73. doi: 10.1016/j.neuropharm.2017.01.031. - DOI - PMC - PubMed
    1. Agrawal RG, Hewetson A, George CM, Syapin PJ, Bergeson SE. Minocycline reduces ethanol drinking. Brain Behav Immun. 2011;25(Suppl 1):S165–S169. doi: 10.1016/j.bbi.2011.03.002. - DOI - PMC - PubMed
    1. Wu Y, Lousberg EL, Moldenhauer LM, Hayball JD, Robertson SA, Coller JK, Watkins LR, Somogyi AA, Hutchinson MR. Attenuation of microglial and IL-1 signaling protects mice from acute alcohol-induced sedation and/or motor impairment. Brain Behav Immun. 2011;25(Suppl 1):S155–S164. doi: 10.1016/j.bbi.2011.01.012. - DOI - PubMed

MeSH terms

Substances

LinkOut - more resources