Alcohol Consumption Modulates Host Defense in Rhesus Macaques by Altering Gene Expression in Circulating Leukocytes

Abstract

Several lines of evidence indicate that chronic alcohol use disorder leads to increased susceptibility to several viral and bacterial infections, whereas moderate alcohol consumption decreases the incidence of colds and improves immune responses to some pathogens. In line with these observations, we recently showed that heavy ethanol intake (average blood ethanol concentrations > 80 mg/dl) suppressed, whereas moderate alcohol consumption (blood ethanol concentrations < 50 mg/dl) enhanced, T and B cell responses to modified vaccinia Ankara vaccination in a nonhuman primate model of voluntary ethanol consumption. To uncover the molecular basis for impaired immunity with heavy alcohol consumption and enhanced immune response with moderate alcohol consumption, we performed a transcriptome analysis using PBMCs isolated on day 7 post-modified vaccinia Ankara vaccination, the earliest time point at which we detected differences in T cell and Ab responses. Overall, chronic heavy alcohol consumption reduced the expression of immune genes involved in response to infection and wound healing and increased the expression of genes associated with the development of lung inflammatory disease and cancer. In contrast, chronic moderate alcohol consumption upregulated the expression of genes involved in immune response and reduced the expression of genes involved in cancer. To uncover mechanisms underlying the alterations in PBMC transcriptomes, we profiled the expression of microRNAs within the same samples. Chronic heavy ethanol consumption altered the levels of several microRNAs involved in cancer and immunity and known to regulate the expression of mRNAs differentially expressed in our data set.

Figure 1. Peripheral blood mononuclear cell (PBMC) gene expression

(A) Bar graph showing the number of downregulated and upregulated genes for each comparison. (B) Venn diagram depicting the overlap of the annotated DEGs between controls (C), moderate drinkers (M), and heavy drinkers (H) on day 7 after booster vaccination (C7, M7 and H7 respectively). (C) Venn diagram depicting the overlap between genes that are downregulated with heavy alcohol consumption compared to controls and moderate drinkers. (D) Venn diagram depicting the overlap between genes that are upregulated with heavy alcohol consumption compared to controls and moderate drinkers. (E, F) Fold change of (E) downregulated and (F) upregulated genes with heavy drinking.

Figure 2. Chronic heavy alcohol consumption down-regulates genes that promote wound healing and contribute to obstructive lung diseases compared to controls and moderate drinkers

(A) Bar graph displaying the 10 most significant GO terms associated with the 170 genes downregulated with heavy ethanol consumption (H7) compared to controls and moderate drinkers (C7 and M7). Line represents the −log(p value) associated with each GO term. (B) Heatmap of DEGs between H7 and C7 in the “response to wounding” GO term. (C) The 10 most significant diseases by biomarkers associated with the 170 genes downregulated with heavy ethanol consumption (H7) compared to controls and moderate drinkers (C7 and M7). (D) Heatmap of DEGs between H7 and C7 that mapped to “lung diseases-obstructive” category. (E) Network of DEGs that mapped to “obstructive lung diseases” and show direct interactions.

Figure 3. Alcohol abuse uniquely down-regulates additional genes that promote wound healing and contribute to obstructive lung diseases

(A) Bar graph displaying the 10 most significant GO terms to which the 186 DEGs downregulated with heavy ethanol consumption (H7) compared to controls only (C7). Line represents the −log(p value) for each GO term. (B) Heatmap of DEGs between H7 and C7 mapping to the GO term “response to wounding”. (C) Bar graph displaying the 10 most significant Diseases by Biomarker to which the 186 DEGs downregulated with heavy drinking (H7) compared to controls (C7) only. Line represents the −log (p value) for each disease category. (D) Heatmap of the DEGs between H7 and C7 mapping to the “lung diseases-obstructive” disease category. (E) Heat map of the 21 DEGs between H7 and C7 mapping to “immune system diseases”.

Figure 4. Heavy alcohol consumption downregulates genes associated with regulation of the immune system

(A) Bar graph displaying the top 10 significant GO terms associated with the 122 genes downregulated with heavy ethanol consumption (H7) compared to moderate drinkers (M7) only. Line represents the −log(p value) of each GO term. (B) Network image of DEGs that mapped to the GO term “immune system processes” and directly interact with one another. (C) Heatmap of the DEGs between H7 and M7 that mapped to the “immune system processes” GO term.

Figure 5. Alcohol abuse upregulates genes that interfere with wound healing and contribute to cancer

Heatmaps of the DEGs upregulated with heavy ethanol consumption (H7) compared to: (A) both controls (C7) and moderate (M7) drinkers; (B) controls (C7) only and mapped to the GO term “response to wounding”; and (C) moderate (M7) drinkers only and mapped to the GO term “neuro-ectodermal tumors”.

Figure 6. Moderate ethanol consumption modulates genes associated with immune response

(A) Heatmap of the DEGs uniquely activated with moderate drinking (M7) compared to controls (C7). (B) Heatmap of the DEGs uniquely repressed with moderate drinking (M7) compared to controls (C7).

Figure 7. Heavy ethanol consumption changes expression of several microRNA

Heatmaps of the (A) upregulated and (B) downregulated microRNAs with heavy drinking (H7) compared to controls (C7). (C) Network image of a subset of the differentially expressed microRNAs and their mRNA targets that were both differentially expressed in our study.