10779/rcsi.10783637.v1 Siobhán Smith Siobhán Smith Joan Nı Gabhann Joan Nı Gabhann Rowan Higgs Rowan Higgs Kevin Stacey Kevin Stacey Marie Wahren-Herlenius Marie Wahren-Herlenius Alexander Espinosa Alexander Espinosa Maria Grazia Totaro Maria Grazia Totaro Antonio Sica Antonio Sica Elizabeth Ball Elizabeth Ball Aubrey Bell Aubrey Bell James Johnston James Johnston Peter Browne Peter Browne Lorraine O'Neill Lorraine O'Neill Grainne Kearns Grainne Kearns Caroline A. Jefferies Caroline A. Jefferies Enhanced interferon regulatory factor 3 binding to the interleukin-23p19 promoter correlates with enhanced interleukin-23 expression in systemic lupus erythematosus. Royal College of Surgeons in Ireland 2019 Animals Bone Marrow Cells Chromatin Immunoprecipitation Disease Models Animal Female Gene Expression Regulation Humans Interferon Regulatory Factor-3 Interleukin-23 Subunit p19 Lupus Erythematosus Systemic Macrophages Male Mice Inbred C57BL Knockout Monocytes Poly I-C Protein Array Analysis Protein Binding Protein-Serine-Threonine Kinases Toll-Like Receptor 3 Clinical Pharmacology and Therapeutics 2019-11-22 16:25:17 Journal contribution https://repository.rcsi.com/articles/journal_contribution/Enhanced_interferon_regulatory_factor_3_binding_to_the_interleukin-23p19_promoter_correlates_with_enhanced_interleukin-23_expression_in_systemic_lupus_erythematosus_/10783637 <p>OBJECTIVE: To examine the role of interferon regulatory factor 3 (IRF-3) in the regulation of interleukin-23 (IL-23) production in patients with systemic lupus erythematosus (SLE).</p> <p>METHODS: Bone marrow-derived macrophages were isolated from both wild-type and IRF3(-/-) C57BL/6 mice. These cells were stimulated with the Toll-like receptor 3 (TLR-3) agonist poly(I-C), and IL-23p19 cytokine levels were analyzed by enzyme-linked immunosorbent assay. IRF-3 binding to the IL-23p19 gene promoter region in monocytes from patients with SLE and healthy control subjects was analyzed by chromatin immunoprecipitation (ChIP) assay. Luciferase reporter gene assays were performed to identify key drivers of IL-23p19 promoter activity. TANK-binding kinase 1 (TBK-1) protein levels were determined by Western blotting.</p> <p>RESULTS: ChIP assays demonstrated that IRF-3 was stably bound to the human IL-23p19 promoter in monocytes; this association increased following TLR-3 stimulation. Patients with SLE demonstrated increased levels of IRF-3 bound to the IL-23p19 promoter compared with control subjects, which correlated with enhanced IL-23p19 production in monocytes from patients with SLE. Investigations of the TLR-3-driven responses in monocytes from patients with SLE revealed that TBK-1, which is critical for regulating IRF-3 activity, was hyperactivated in both resting and TLR-3-stimulated cells.</p> <p>CONCLUSION: Our results demonstrate for the first time that patients with SLE display enhanced IL-23p19 expression as a result of hyperactivation of TBK-1, resulting in increased binding of IRF-3 to the promoter. These findings provide novel insights into the molecular pathogenesis of SLE and the potential role for TLR-3 in driving this response.</p>