Fig. 5: IRF3 is a transcription factor which can regulate the expression of genes associated with alcohol craving and the activation of IRF3 is associated with ethanol-induced endoplasmic reticulum (ER) stress signaling which can be attenuated by anti-craving drugs.

A ATAC-seq tag density was correlated positively with IRF3 binding density in the promoter regions of the genes associated with alcohol craving as shown in Fig. 1B. The dot plot represents basal ATAC peak quantification (y axis) and IRF3 ChIP-seq fold change over input (x axis). B Representative regional plot in the CTSL locus showing open chromatin tracks in iPSC-derived astrocytes in response to EtOH (25 mM), acamprosate (5 µM) or naltrexone (30 nM) treatment of iPSC-derived astrocytes and IRF3 ChIP-seq (GSE91752) [37]. Note that the ATAC-seq tracks represent ATAC-seq tag density, and the publically available IRF3 ChIP-seq track represents IRF3 binding fold change over control. C ChIP assays showing the effect of IRF3 binding to the promoter regions of genes that were associated with alcohol craving intensity in response to EtOH (25 mM), acamprosate (5 µM) or naltrexone (30 nM) treatment of iPSC-derived astrocytes (n = 3). Percentage of ChIP DNA/input was determined by qPCR. Data are represented as % input, (enrichment relative to IgG control) = % input (IRF3 antibody) - % input (IgG). One-way ANOVA was used for data analysis. *P < 0.05 vs vehicle. Three independent experiments were performed. D Cartoon model for the activation of IRF3 and endoplasmic reticulum (ER) stress induced by ethanol. ER stress genes, ie GRP78 and XBP-1, can be induced by ethanol. STING phosphorylation can recruit IRF3, which itself can be phosphorylated by TBK1. This process will enhance IRF3 translocation and activate the transcription of downstream genes. Specifically, phospho-TBK1—a kinase required for IRF3 phosphorylation, and STING—an adaptor protein that resides in the ER membrane--were also induced by ethanol. However, exposure to anti-craving drugs i.e. acamprosate or naltrexone, could attenuate ER stress signaling. IRF3 protein directly interacted with STING, TBK1, and GRP78. As a result, IRF3 might play a role in ethanol-induced ER stress through the phosphorylation of IRF3. This, in turn, could facilitate the translocation of IRF3 to the nucleus as a transcription factor which plays a role in the regulation of gene expression. E mRNA expression of ER stress genes in response to exposure to ethanol, acamprosate or naltrexone in iPSC-derived astrocytes as determined by RNA-seq (n = 6). *FDR < 0.05. F Protein expression of GRP78, XBP-1s, p-IRF3, IRF3, TBK1, pTBK1, STING, and p-STING in iPSC-derived astrocytes in response to exposure to ethanol, acamprosate or naltrexone was determined by Western blot analysis (n = 4). Alpha-tubulin and vinculin were used as loading controls. Images are representative of iPSC-derived astrocytes from AUD patients (n = 4). G Immunoprecipitation was used to determine whether IRF3 protein could interact with STING, TBK1 and GRP78 in iPSC-derived astrocytes. Whole cell lysates from 1×10⁷ iPSC-derived astrocytes were immunoprecipitated with anti-IRF3 (1:50) antibodies or anti-IgG antibodies. Pull down protein samples were immunoblotted and probed with antibodies against IRF3, STING, TBK1 and GRP78 (D). Pulldowns are representative of iPSC-derived astrocytes from four AUD subjects.