Comparative Analysis of Six IRF Family Members in Alveolar Epithelial Cell-Intrinsic Antiviral Responses.

Host cell-intrinsic antiviral responses are largely mediated by pattern-recognition receptor (PRR) signaling and the interferon (IFN) system. The IFN regulatory factor (IRF) family of transcription factors takes up a central role in transcriptional regulation of antiviral innate immunity. IRF3 and IRF7 are known to be key players downstream of PRRs mediating the induction of type I and III IFNs. IFN signaling then requires IRF9 for the expression of the full array of interferon stimulated genes (ISGs) ultimately defining the antiviral state of the cell. Other members of the IRF family clearly play a role in mediating or modulating IFN responses, such as IRF1, IRF2 or IRF5, however their relative contribution to mounting a functional antiviral response is much less understood. In this study, we systematically and comparatively assessed the impact of six members of the IRF family on antiviral signaling in alveolar epithelial cells. We generated functional knockouts of IRF1, -2, -3, -5, -7, and -9 in A549 cells, and measured their impact on the expression of IFNs and further cytokines, ISGs and other IRFs, as well as on viral replication. Our results confirmed the vital importance of IRF3 and IRF9 in establishing an antiviral state, whereas IRF1, 5 and 7 were largely dispensable. The previously described inhibitory activity of IRF2 could not be observed in our experimental system.

Brief Report: Blockade of TANK-Binding Kinase 1/IKKɛ Inhibits Mutant Stimulator of Interferon Genes (STING)-Mediated Inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

OBJECTIVE: Gain-of-function mutations in TMEM173, encoding the stimulator of interferon (IFN) genes (STING) protein, underlie a novel type I interferonopathy that is minimally responsive to conventional immunosuppressive therapies and associated with high frequency of childhood morbidity and mortality. STING gain-of-function causes constitutive oversecretion of IFN. This study was undertaken to determine the effects of a TANK-binding kinase 1 (TBK-1)/IKKɛ inhibitor (BX795) on secretion and signaling of IFN in primary peripheral blood mononuclear cells (PBMCs) from patients with mutations in STING. METHODS: PBMCs from 4 patients with STING-associated disease were treated with BX795. The effect of BX795 on IFN pathways was assessed by Western blotting and an IFNß reporter assay, as well as by quantification of IFNα in cell lysates, staining for STAT-1 phosphorylation, and measurement of IFN-stimulated gene (ISG) messenger RNA (mRNA) expression. RESULTS: Treatment of PBMCs with BX795 inhibited the phosphorylation of IFN regulatory factor 3 and IFNß promoter activity induced in HEK 293T cells by cyclic GMP-AMP or by genetic activation of STING. In vitro exposure to BX795 inhibited IFNα production in PBMCs of patients with STING-associated disease without affecting cell survival. In addition, BX795 decreased STAT-1 phosphorylation and ISG mRNA expression independent of IFNα blockade. CONCLUSION: These findings demonstrate the effect of BX795 on reducing type I IFN production and IFN signaling in cells from patients with gain-of-function mutations in STING. A combined inhibition of TBK-1 and IKKɛ therefore holds potential for the treatment of patients carrying STING mutations, and may also be relevant in other type I interferonopathies.