The G9a methyltransferase inhibitor BIX01294 inhibits IFN-α production and regulates DNMT1 in virus-activated pDC

Abstract

Abstract Plasmacytoid dendritic cells (pDC) are innate immune cells that represent 0.2–0.5% of human PBMC and produce IFN-α upon TLR7 or -9 activation by virus to stimulate innate and adaptive responses. Because of its potency and propensity to induce autoimmunity, IFN-α production must be tightly controlled. We investigated the role of histone 3 lysine 9 di-methylation (H3K9me2) and DNA Methyltransferase 1 (DNMT1) in IFN-α gene silencing in virus-activated pDC. DNMT1 recruits histone deacetylases, which are known to regulate cytokines. H3K9me2 is methylated by G9a methyltransferase, which suppresses IFN-α in fibroblasts. However, in virus-activated pDC, H3K9me2 may induce IFN-α. PBMCs were isolated from healthy donors and stimulated with IAV, HIV, or HSV for 24-hrs. We surface stained for pDC markers CD123 and HLA-DR, and CD11c, then intracellular stained for DNMT1. We also treated pDC with G9a methyltransferase inhibitor, BIX01294, and measured IFN-α and DNMT1 6-hr post stimulation with HSV and IAV. qRT-PCR was performed to measure DNMT1, demethylases, and G9a mRNA in purified pDC. In IAV and HSV stimulated pDC, DNMT1 was significantly upregulated at 24-hrs compared to 8-hr stimulated and unstimulated controls. IFN-α production and DNMT1 expression in IAV-activated pDC, but not in HSV, was significantly inhibited by BIX01294. Furthermore, G9a methyltransferase mRNA was elevated in IAV and HIV-1 purified pDC, but absent in HSV-stimulated cells. The elevation of DNMT1 may assist with limiting IFN-α production in pDC at 24-hrs to prevent excessive immune activation. Further, the reduction of DNMT1 in IAV, but not HSV-activated pDC upon treatment with BIX01294 implies a virus or TLR pathway (TLR7 vs -9) specific role of DNMT1.