Chronic ethanol consumption modulates growth factor release, mucosal cytokine production, and microRNA expression in nonhuman primates

Abstract

Background: Chronic alcohol consumption has been associated with enhanced susceptibility to both systemic and mucosal infections. However, the exact mechanisms underlying this enhanced susceptibility remain incompletely understood.

Methods: Using a nonhuman primate model of ethanol (EtOH) self-administration, we examined the impact of chronic alcohol exposure on immune homeostasis, cytokine, and growth factor production in peripheral blood, lung, and intestinal mucosa following 12 months of chronic EtOH exposure.

Results: EtOH exposure inhibited activation-induced production of growth factors hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), and vascular-endothelial growth factor (VEGF) by peripheral blood mononuclear cells (PBMC). Moreover, EtOH significantly reduced the frequency of colonic Th1 and Th17 cells in a dose-dependent manner. In contrast, we did not observe differences in lymphocyte frequency or soluble factor production in the lung of EtOH-consuming animals. To uncover mechanisms underlying reduced growth factor and Th1/Th17 cytokine production, we compared expression levels of microRNAs in PBMC and intestinal mucosa. Our analysis revealed EtOH-dependent up-regulation of distinct microRNAs in affected tissues (miR-181a and miR-221 in PBMC; miR-155 in colon). Moreover, we were able to detect reduced expression of the transcription factors STAT3 and ARNT, which regulate expression of VEGF, G-CSF, and HGF and contain targets for these microRNAs. To confirm and extend these observations, PBMC were transfected with either mimics or antagomirs of miR-181 and miR-221, and protein levels of the transcription factors and growth factors were determined. Transfection of microRNA mimics led to a reduction in both STAT3/ARNT as well as VEGF/HGF/G-CSF levels. The opposite outcome was observed when microRNA antagomirs were transfected.

Conclusions: Chronic EtOH consumption significantly disrupts both peripheral and mucosal immune homeostasis, and this dysregulation may be mediated by changes in microRNA expression.

Keywords: Ethanol; Immunity; MicroRNA; Nonhuman Primate; Self-Administration.

Figure 1. Chronic ethanol self-administration reduces PBMC growth factor production

PBMC were collected from controls (closed circles, n = 13) or drinkers (open circles, n = 14) and stimulated o/n in the presence of PMA/ionomycin. Cytokine (A), chemokine (B) and growth factor (C) production were determined in supernatants by multiplex bead array. Horizontal lines represent geometric mean, each symbol represents an individual animal. * = p < 0.05, *** = p < 0.001.

Figure 2. Lung cytokine responses and T cell phenotype are ethanol-independent

Bronchoalveolar lavage (BAL) and Tracheo bronchical lymph node (TBLN) cells were collected from controls (closed circles, n = 7) or drinkers that self-administered ethanol for 12 months (closed circles, n = 13–14/group). (A–B) The frequency of TNFa, IL-2, IFNg or IL-17 producing CD4 (A) or CD8 (B) T cells was determined by flow cytometry following 6hr stimulation of BAL cells with PMA/ionomycin. (C–D) The frequency of central memory (CM) and effector memory (EM) CD4 and CD8 T cells was determined by flow cytometry. (E) The ratio of CD4/CD8 T cell frequency is shown. Horizontal lines represent group mean, each symbol represents an individual animal.

Figure 3. T cell cytokine production in intestinal tissues of rhesus macaques following chronic ethanol self-administration

Lymphocytes were isolated from MLN, duodenum, jejunum, ileum and colon from control animals (closed circles, n = 7) or animals self-administering ethanol for 12 months (open circles, n = 10–14/group). Cells were stimulated for 6hr with PMA/ionomycin. The frequency of TNFα+ and IFNγ+IL-17+ CD4 and CD8 T cells was determined by flow cytometry. Within intestinal tissue, cytokine production by intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) is shown, with the exception of colonic IEL. Horizontal bars represent group means, each symbol represents an individual animal. * = p < 0.05.

Figure 4. Increasing ethanol consumption is negatively associated with colonic CD4 T cell cytokine production

Lymphocytes were isolated from colonic lamina propria of controls (n = 7, closed circles) or drinkers (n = 12, open circles) and stimulated for 6hr with PMA/ionomycin. The frequency of IL-2+, TNFα+, IFNγ+, IL-17+ and IFNγ+IL-17+ CD4 T cells was determined and plotted against each animal’s mean daily ethanol intake (g/kg/day) during 12 month ethanol self-administration. Correlations between frequency of cytokine producing cells and ethanol intake were determined by Spearman’s rank correlation. Two drinkers (ID# 26234 and 26235) were omitted due to insufficient cell yield for analysis.

Figure 5. Chronic ethanol consumption significantly upregulates expression of microRNAs miR-181a and miR-221 within PBMC and microRNA 155 within colon biopsy

The expression level of microRNAs 181a-5p, 146a-5p, 155-5p, 221-3p, 17-3p, 17-5p, 21-5p and 29a was determined in PBMC (A) and colon tissue (B) from controls (n = 14, closed circles) or drinkers (n = 14, open circles) by RT-qPCR. Expression is shown relative to housekeeping gene U6 (AU = arbitrary units). Horizontal lines represent group geometric means, each symbol an individual animal. * = p < 0.05.

Figure 6. Chronic ethanol consumption significantly downregulates expression of transcription factors ARNT and STAT3 in PBMC

(A) The expression level of transcription factors that regulate growth factor production and are predicted targets of microRNAs miR-181a and miR-221 was determined by RT-qPCR in PBMC from controls (n = 11, closed circles) or drinkers (n = 9, open circles). Expression is shown relative to housekeeping gene glutathione synthetase. Horizontal bars represent group geometric means, each symbol represents an individual animal. (B) The expression levels of STAT3 and ARNT protein levels was determined in PBMC from controls (n=4) or drinkers (n=4). (C) Quantification of the immunoblot intensity. * = p < 0.05.

Figure 7. miR-181 and miR-221 modulates transcription factors STAT3 and ARNT and growth factors VEGF, HGF and G-CSF protein expression

(A–C) Western blot analysis of STAT3 and ARNT expression from PMA/ionomycin stimulated PBMC cells transfected with non-targeting control, miR-181 and miR-221 mimics (A), or with non-targeting control, miR-181 and miR-221 antagomirs (B). Immunoblot intensity is summarized in panel (C). (D–F) Western blot analysis of VEGF and HGF expression from PMA/ionomycin stimulated PBMC cells transfected with non-targeting control, miR-181 and miR-221 mimics (D), or with non-targeting control, miR-181 and miR-221 antagomirs (E). Immunoblot intensity is summarized in panel (F). (G) Concentration of G-CSF in tissue culture supernatant. One of three independent experiments is illustrated.