Transgenerational inheritance of fetal alcohol exposure adverse effects on immune gene interferon-ϒ

Abstract

Background: Alcohol exposures in utero have been shown to alter immune system functions in the offspring which persists into adulthood. However, it is not apparent why the in utero alcohol effect on the immune system persists into adulthood of fetal alcohol-exposed offspring. The objective of this study was to determine the long-term effects of fetal alcohol exposure on the production of interferon-ϒ (IFN-ϒ), a cytokine known to regulate both innate and adaptive immunity.

Methods: Isogenic Fisher 344 rats were bred to produce pregnant dams, which were fed with a liquid diet containing 6.7% alcohol between gestation days 7 and 21 and pair-fed with an isocaloric liquid diet or fed ad libitum with rat chow; their male and female offspring were used for the study. F1-F3 generation rats were used when they were 2 to 3 months old. Fetal alcohol exposure effects on the Ifn-ɣ gene was determined by measuring the gene promoter methylation and mRNA and protein expression in the spleen. Additionally, transgenerational studies were conducted to evaluate the germline-transmitted effects of fetal alcohol exposure on the Ifn-ɣ gene.

Results: Fetal alcohol exposure reduced the expression of Ifn-ɣ mRNA and IFN-ϒ protein while it increased the proximal promoter methylation of the Ifn-ɣ gene in both male and female offspring during the adult period. Transgenerational studies revealed that the reduced levels of Ifn-ɣ expression and increased levels of its promoter methylation persisted only in F2 and F3 generation males derived from the male germ line.

Conclusion: Overall, these findings provide the evidence that fetal alcohol exposures produce an epigenetic mark on the Ifn-ɣ gene that passes through multiple generations via the male germ line. These data provide the first evidence that the male germ line transmits fetal alcohol exposure's adverse effects on the immune system.

Keywords: Epigenetic; Fetal alcohol; Interferon-ϒ; Transgenerational transmission.

Fig. 1

Effects of fetal alcohol exposure on Ifn-ɣ gene in the spleen of F1 adult offspring. Changes in the levels of Ifn-ɣ mRNA (a, b), Ifn-ɣ promoter DNA methylation at various CpGs (c–e), and IFN-ɣ protein (f, g) in the spleen of male (♂) and female (♀) fetal alcohol-exposed (AF) and control (AD and PF) rat offspring. a and bIfn-ɣ mRNA levels were measured by quantitative RT-PCR. Data are mean + SEM (n = 8) and were analyzed using one-way ANOVA with the Newman-Keuls post-hoc test; ** P < 0.01, *** P < 0.001 between AF and controls. c A schematic diagram of rat IFN-ϒ promoter CpG island identified by the Urogene MethPrimer web tool (http://www.urogene.org/methprimer). d and e. Ifn-ɣ promoter CpG methylation changes were measured by pyrosequencing analysis. Four different CpGs of the IFN-ϒ promoter region—CpG1 (-322), CpG2 (-313), CpG3 (-305), and CpG4 (-285) were analyzed. Data are mean + SEM (n = 8) and were analyzed using two-way ANOVA with the Bonferroni post-hoc test; * P < 0.05, ** P < 0.01, and *** P < 0.001 between AF and controls of the same CpG. f and g IFN-ϒ protein levels in spleen samples were measured by western blotting. Representative blots for IFN-ϒ and β-actin are shown in the upper panel and quantification measurements were represented as a histogram in the lower panel. Data are mean + SEM (n = 6) and were analyzed using one-way ANOVA with the Newman-Keuls post-hoc test; * P < 0.05, ** P < 0.01, and *** P < 0.001 between AF and controls. F statistics and P values of data shown in figures were presented in Supplementary Table 1

Fig. 2

Transgenerational changes in Ifn-ɣ mRNA expression in the spleen after alcohol feeding in pregnant female rats. A schematic diagram to indicate how F2 and F3 male germ line (AFM) and female germ line (AFF) of fetal alcohol-exposed (a) and male germ line (PFM) and female germ line (PFF) of pair-fed control offspring (b) were generated. Ifn- mRNA levels in the spleen tissues of F2 (c, d) and F3 (e, f) male and female rat offspring from different germ lines were measured by quantitative RT-PCR. Data are mean + SEM (n = 8) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; ** P < 0.01, *** P < 0.001 between AFM and controls (AD, PFM). F statistics and P values of data shown in figures were presented in Supplementary Table 1

Fig. 3

Transgenerational changes in Ifn-e methylation in the spleen after alcohol feeding in pregnant female rats. IFN-pleen after alcohol feeding inwere determined by pyrosequencing in the spleen of F2 male (a), F2 female (b), F3 male (c), and F3 female (d) rat offspring from different germ lines as defined in Fig. 2a. Four different CpGs of the IFN- different CpGs o—CpG1 (-322), CpG2 (-313), CpG3 (-305), and CpG4 (-285) were analyzed. Data are mean + SEM (n = 7–8) and were analyzed using two-way ANOVA with the Bonferroni post-hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001 between AFM vs. controls (AD, PFM). F statistics and P values of data shown in figures were presented in Supplementary Table 1

Fig. 4

Transgenerational changes in IFN- enprotein levels in the spleen after alcohol feeding in pregnant female rats. IFN-ϒ protein levels in the spleens of F2 and F3 male (a, c) and female (b, d) rat offspring from different germ lines as defined in Fig. 2a. IFN-ɣ protein levels in the spleens were measured by western blotting. Representative blots for IFN-ϒ, β-actin were shown in the upper panel and quantification data normalized with β-actin were represented as a histogram in the lower panel for each figure. Data are mean + SEM (n = 6–7) and were analyzed using one-way ANOVA with the Newman-Keuls post-hoc test. * P < 0.05, ** P < 0.01 between AFM vs. AD or PFM. F statistics and P values of data shown in figures were presented in Supplementary Table 1