Transcriptome Profiling Reveals Disruption of Innate Immunity in Chronic Heavy Ethanol Consuming Female Rhesus Macaques

<div><p>It is well established that heavy ethanol consumption interferes with the immune system and inflammatory processes, resulting in increased risk for infectious and chronic diseases. However, these processes have yet to be systematically studied in a dose and sex-dependent manner. In this study, we investigated the impact of chronic heavy ethanol consumption on gene expression using RNA-seq in peripheral blood mononuclear cells isolated from female rhesus macaques with daily consumption of 4% ethanol available 22hr/day for 12 months resulting in average ethanol consumption of 4.3 g/kg/day (considered heavy drinking). Differential gene expression analysis was performed using edgeR and gene enrichment analysis using MetaCore™. We identified 1106 differentially expressed genes, meeting the criterion of ≥ two-fold change and p-value ≤ 0.05 in expression (445 up- and 661 down-regulated). Pathway analysis of the 879 genes with characterized identifiers showed that the most enriched gene ontology processes were “response to wounding”, “blood coagulation”, “immune system process”, and “regulation of signaling”. Changes in gene expression were seen despite the lack of differences in the frequency of any major immune cell subtype between ethanol and controls, suggesting that heavy ethanol consumption modulates gene expression at the cellular level rather than altering the distribution of peripheral blood mononuclear cells. Collectively, these observations provide mechanisms to explain the higher incidence of infection, delay in wound healing, and increase in cardiovascular disease seen in subjects with Alcohol use disorder.</p></div

Chronic heavy ethanol consumption results in robust changes in gene expression within PBMC.

<p>(A) Volcano plot of global gene expression changes with red specks denoting genes with significant fold changes in gene expression, with gene names annotated for those with fold change ≥ 32. (B) Bar graph depicting the 8 most significant Gene Ontology (GO) terms enriched among all differentially expressed genes (DEGs), (C) Venn diagram depicting the overlap of genes enriched for four major GO terms—Signaling, Blood Coagulation, Wounding and Immune System Process. (D) Heatmap of the 27 differentially expressed that belong to all four GO processes—red depicts higher expression and grey, lower expression.</p

Chronic heavy ethanol consumption results in up-regulation of genes involved in blood coagulation and wound-healing.

<p>(A) Network of DEGs with direct interactions that mapped to “Wound healing”. (B) Heatmap of the 40 DEGs with a fold-change ≥ four-fold (30 up-regulated and 10 down-regulated) involved in “Blood Coagulation”. (C) Heatmap of the 18 DEGs that mapped to “Wound healing” but did not map to “Blood Coagulation”.</p

Chronic heavy ethanol consumption changes the expression of genes involved in heart diseases and cancer.

<p>(A) Bar graph depicting 8 disease terms enriched among the up-regulated genes. The line graph in both figures represents negative log (FDR) of the enriched term. (B) Heatmap of up-regulated genes involved in cardiovascular diseases. (C) Bar graph depicting 8 disease terms enriched among the down-regulated genes. (D) Heatmap of the down-regulated genes involved in cancer.</p

Chronic heavy ethanol consumption results in changes in expression of epigenetic regulators.

<p>(A) Functional profiles of the 128 down-regulated genes mapping to ‘Regulation of Gene Expression’ (B) Bar graph of expression levels (RPKM) of genes involved in chromatin remodeling (**—FDR of 5% and *—FDR of 10%). (C) Bar graph of 5 most significantly up- and down-regulated transcription factor networks. Green bars indicate up-regulated network and blue bars indicate down-regulated networks. Each bar is linked to a group of target genes (orange–up-regulated and grey down-regulated) that are differentially expressed in heavy drinkers.</p

Summary of down-regulated microRNAs and their up-regulated targets in drinkers.

<p>Summary of down-regulated microRNAs and their up-regulated targets in drinkers.</p

Chronic heavy ethanol consumption results in dysregulation of genes involved in innate immunity and immune system development.

<p>(A) Heatmap of DEGs that map to “Defense Response/Innate Immune Response” (B) Heatmap of DEGs that map to “Immune System Development”/“Myeloid Cell differentiation”.</p

Assessment of autophagy in alveolar macrophages from adult RM.

<p>A. Western analysis was performed on cell lysates from resting control (C) or autophagic (Rap) alveolar macrophages using antibody against LC-3 and actin. The LC-3 antibody reacts with both free cytosolic LC3-I (18 kDa) and membrane associated LC3-II (16 kDa). Levels of LC3-II were normalized to actin by densitometry and the ratio of LC3-II/actin given below the blot. A representative image from a single rhesus macaque (RM) sample is shown. B. Immunofluorescence microscopy was performed on control and rapamycin-treated alveolar macrophages using primary antibody against LC-3. C. The number of cells possessing LC-3+ puncta (top) and the average number of LC3+ vacuoles in LC-3+ cells (bottom) were quantified (n = 6 NHP samples, n>50 macrophages in each condition). The difference between the number of cells possessing LC-3+ puncta was significantly different between control and autophagic cells (***, <i>p</i><0.001; **, <i>p</i><0.01; ANOVA).</p

Autophagic Killing Effects against <i>Mycobacterium tuberculosis</i> by Alveolar Macrophages from Young and Aged Rhesus Macaques

<div><p>Non-human primates, notably rhesus macaques (<i>Macaca mulatta</i>, RM), provide a robust experimental model to investigate the immune response to and effective control of <i>Mycobacterium tuberculosis</i> infections. Changes in the function of immune cells and immunosenescence may contribute to the increased susceptibility of the elderly to tuberculosis. The goal of this study was to examine the impact of age on <i>M. tuberculosis</i> host-pathogen interactions following infection of primary alveolar macrophages derived from young and aged rhesus macaques. Of specific interest to us was whether the mycobactericidal capacity of autophagic macrophages was reduced in older animals since decreased autophagosome formation and autophagolysosomal fusion has been observed in other cells types of aged animals. Our data demonstrate that alveolar macrophages from old RM are as competent as those from young animals for autophagic clearance of <i>M. tuberculosis</i> infection and controlling mycobacterial replication. While our data do not reveal significant differences between alveolar macrophage responses to <i>M. tuberculosis</i> by young and old animals, these studies are the first to functionally characterize autophagic clearance of <i>M. tuberculosis</i> by alveolar macrophages from RM.</p></div

Bactericidal capacity of autophagic macrophages from adult RM.

<p>A. Alveolar macrophages were infected at an MOI of 5∶1 with <i>M. tuberculosis</i> CDC1551. Bacterial colony forming units (cfu) were determined following control treatment, 4 h treatment with 50 µg/mL rapamycin (rap) to induce autophagy, and 4 h treatment with 50 µg/mL rapamycin and 10 mM 3-methyladenine (3-MA) to block autophagy. Viability is expressed as % survival relative to the number of viable bacteria in untreated resting control macrophages. Each symbol represents the average of three triplicate infections for each condition using RM sample. The average and standard deviation of all samples are shown. The difference between bacterial survival in control and autophagic macrophages was significant (**, <i>p</i><0.01; ANOVA). B. Immunofluorescence microscopy was performed on control and autophagic alveolar macrophages infected with fluorescently labeled <i>M. tuberculosis</i> CDC1551 (green). Primary antibodies against either the autophagosomal marker LC3 (top) or the lysosomal marker LAMP (bottom) and DyeLight 594-conjugated secondary antibody were used. Arrows indicate a representative mycobacterium co-localized with LAMP-1. C. The number of <i>M. tuberculosis</i> that co-localize with LAMP-1 were quantified. The difference between control and autophagic cells was not significantly different (p = 0.0586; ANOVA; n = 6 NHP samples).</p