Mechanisms Regulating Type I Interferon Signaling in Human Keratinocytes Triggered by Exogenous DNA

Abstract

Activation of STING DNA sensing pathway triggers the production of type I interferons (IFNs) in keratinocytes. In lupus keratinocytes, this pathway becomes overactive, leading to sustained, STING dependent type I IFN production through an autocrine loop, skewed towards females. Thus, strategies to inhibit STING and its downstream events including type I IFN signaling are an active research area to identify therapies for lupus. While a pathogenic role for type I IFNs and STING signaling in autoimmune skin diseases is well established, the key regulators of IFN signaling, the interplay between STING and type I IFN signaling pathways, and the mechanisms underlying the female bias in IFN activity remain to be fully understood. Here we show that HERC6, an IFN-induced E3 ubiquitin ligase is induced in human keratinocytes through the epidermal type I IFN; IFN-κ. Knockdown of HERC6 in human keratinocytes led to increased activation of interferon-stimulated genes (ISGs) when treated with a double-stranded (ds)DNA STING activator cGAMP, but not in response to the RNA-sensing TLR3 agonist. In the absence of HERC6, keratinocytes display sustained STING signaling following cGAMP stimulation due to changes in the activity of LATS2, a key kinase involved in Hippo signaling, resulting in more robust IFN responses, particularly in female keratinocytes. This enhanced female-biased immune responses with loss of HERC6 is dependent on VGLL3, a sex-biased autoimmune regulator. These findings highlight HERC6 as a novel negative regulator of ISG expression specific to dsDNA sensing and establish it as a regulator of female-biased immune responses through modulation of Hippo signaling pathway. In contrast to an autoimmune response, IFNs and STING activity in keratinocytes provide defense responses upon sensing exogenous DNA. This is evident by the fact that keratinocytes have poor DNA transfection efficiency, hindering CRISPR-Cas9 mediated genetic knockouts (KO) generation. The mechanism involving transfection resistance in keratinocytes is not well characterized. We show that CRISPR plasmid transfection activates STING, resulting in production of type I IFN through induction of IFNκ, ISG expression, and the cytidine deaminase, APOBEC3G, to decrease the plasmid stability. KOs generated in keratinocytes show persistent IFNκ suppression and DNMT3B-mediated hypermethylation in the IFNK gene promoter. Inhibiting type I IFN signaling using baricitinib before transfection resulted in increased transfection efficiency, normal IFNκ activity, and ISG expression without hypermethylation in the IFNK promoter region. This data suggests that CRISPR-based gene editing modifies antiviral IFN responses, which can be prevented by blocking the IFN signaling. This study has implications for gene therapy in treating inherited skin disorders using CRISPR technology. Together, these data identify the complex immune signaling regulation in keratinocytes upon sensing exogenous DNA. Our investigation into the cross-regulation of STING-Hippo signaling has broadened our understanding of the downstream IFN signaling, identified a novel role for HERC6, and elucidated a way to generate genetic KOs in keratinocytes.