Background/Purpose: Increased Type I IFN activation plays a key role in Scleroderma (SSc), Systemic Lupus Erythematosus and Inflammatory Myositis. Deubiquitylase (DUB) BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by selectively cleaving K63-linked polyubiquitin chains on Type I interferon (IFN) receptors (IFNAR1) and by modulating its degradation. We tested the ability of BRISC inhibitors to reduce IFN signalling in 15 distinct cellular models and human peripheral blood mononuclear cells (PBMC) from SSc patients.

Methods: THP1-Dual^TM cells, dermal fibroblasts and PBMCs from healthy donors and SSc patients were cultured with BRISC inhibitors and scaffold controls at titrated doses for 16 hours with and without IFN2A or LPS. Tofacitinib was used as Jak/Stat inhibition control. THP1 supernatant was analysed for the IRF pathway activity (IFN stimulated response elements (ISRE)) using QUANTI-Luc™ 4 Lucia/Gaussia. IFNAR1 and tetherin (BST2) surface levels were assessed by FACS analysis and transcriptome was assessed by RNAseq. PBMCs were assayed for interferon-stimulated gene (ISG) expression by human IFN I RT2 Profiler PCR Array and by ELISA for interferon inducible CXCL10 protein secretion. Biomap analysis of 12 human primary cell-based systems was used to assess BRISC inhibition in distinct cellular models. Dermal fibroblast protein lysates were analysed by Western Blot.

Results: BRISC inhibitors significantly reduced surface levels of IFNAR1 in THP1 cells post stimulation with recombinant IFN2A (0.59 fold P=0.0155) or LPS (0.49 fold P=0.0062) compared to Tofacitinib or scaffold control. Accordingly, BRISC inhibitors significantly suppressed IFN-induced ISRE compared to control (0.69 fold P=0.002), without neutering the signalling pathway to the extent of tofacitinib (0.32 fold P=0.0002). RNAseq analysis of THP1 cells showed a consistent set of results indicating a significant reduction in expression of IFI44, OAS2, IFIT1, IFIT2, IFIT3, IFIT5, IFIH1, STAT1, IFI16, and DDX58 following IFN2A stimulation. This effect was also associated with a reduction in the surface level of BST2 (Tetherin) a known surface marker of Type I IFN activation, more effectively than tofacitinib (0.62 fold P< 0.0001). BRISC inhibition significantly reduced the IFN2A-induced expression of 13/35 ISG in healthy PBMCs (P< 0.05).Consistent with these findings, treatment of unstimulated SSc PBMCs with BRISC inhibitors suppressed ISG composite score (0.77 fold P=0.0025) compared to control, and significantly reduced CXCL10 secretion (0.29 fold P=0.0028). Accordingly, IFN2A-treated dermal fibroblasts showed reduced activated STAT1 when BRISC was inhibited. Biomap analysis indicated a strong reduction in IL-17a expression in T and B cell models, as well as reduction in collagen and SMA expression in TGF-b stimulated fibroblasts.

Conclusion: We show that modulation of IFNAR1 deubiquitination by novel BRISC compounds can dampen type I IFN response and myofibroblast stimulation in vitro and ex vivo. This indicates a novel therapeutic avenue to target Type I IFN driven diseases by taking advantage of receptor degradative pathways.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/deubiquitylase-dub-brcc36-isopeptidase-complex-brisc-inhibitors-prevent-ifnar1-deubiquitination-to-restore-natural-type-i-ifn-response-in-autoimmunity/