Altered expression of cytokine signaling pathway genes in peripheral blood cells of alcohol dependent subjects: preliminary findings

Abstract

Background: Preclinical and clinical studies have implicated changes in cytokine and innate immune gene-expression in both the development of and end-organ damage resulting from alcohol dependence. However, these changes have not been systematically assessed on the basis of alcohol consumption in human subjects.

Methods: Illumina Sentrix Beadchip (Human-6v2) microarrays were used to measure levels of gene-expression in peripheral blood in 3 groups of subjects: those with alcohol dependence (AD, n = 12), heavy drinkers (HD; defined as regular alcohol use over the past year of at least 8 standard drinks/wk for women and at least 15 standard drinks/wk for men, n = 13), and moderate drinkers (MD; defined as up to 7 standard drinks/wk for women and 14 standard drinks/wk for men, n = 17).

Results: Four hundred and thirty-six genes were differentially expressed among the 3 groups of subjects (false discovery rate corrected p-value < 0.05). Two hundred and ninety-one genes differed between AD and MD subjects, 240 differed between AD and HD subjects, but only 6 differed between HD and MD subjects. Pathway analysis using DAVID and GeneGO Metacore(®) software showed that the most affected pathways were those related to T-cell receptor and Janus kinase-Signal transducer and activator of transcription (JAK-Stat) signaling.

Conclusions: These results suggest the transition from heavy alcohol use to dependence is accompanied by changes in the expression of genes involved in regulation of the innate immune response. Such changes may underlie some of the previously described changes in immune function associated with chronic alcohol abuse. Early detection of these changes may allow individuals at high risk for dependence to be identified.

Figure 1. Venn diagram of overlapping gene-expression differences observed among Alcohol dependent (AD), Heavy Drinker (HD) and Moderate Drinker (SD) subjects

Changes included in the diagram met criteria for statistical significance in the 3-group comparison after adjustment for multiple testing (FDR adjusted p-value <0.05) and in addition differed significantly between the two groups listed (fold change ≥ 1.3, nominal p-value <0.05). The red circle depicts differences between AD and MD subjects (291 genes); the green circle, differences between AD and HD subjects (240 genes), and the blue circle, differences between HD and MD subjects (6 genes).

Figure 2. Cytokine-Related Gene-Expression Changes in AD subjects

The upper level of the diagram depicts signaling molecules at the cell surface, while the lower level depicts resulting changes in gene-transcription and apoptosis-related pathways. IL-15 signals through a receptor complex consisting of 3 subunits: the IL-15Rα, IL-2Rβ, and the common cytokine receptor γ chain, γc. IL-21 signals through a complex consisting of the IL-21 receptor (IL21R) and γc. IFNα and related proteins signal through the interferon receptor (IFNAR), which consists of 2 proteins, IFNAR1 and IFNAR2. TNFα signals through two cell surface receptors, TNFR-1 and TNFR-2. Interleukins, interferons, and TNFα can all signal to the nucleus by way of the JAK-Stat pathway depicted on the left hand-side of the figure. In addition, TNFα can activate both an anti-apoptotic pathway involving activation of the NF-kappaB (central part of figure) and a pro-apoptotic pathway leading to caspase activation (right hand side of figure). Proteins shown in green were up-regulated at the mRNA level in AD subjects compared to HD and MD subjects, while those shown in red were down-regulated. Additional information on each of these pathways is included in the text.