Background Rheumatoid arthritis (RA) is the most common inflammatory chronic joint disorder. Interleukin-1 (IL-1) family members play a key part in the pathogenesis of RA. Among the IL-1 cytokine family members, IL-18 is a proinflammatory cytokine, which modulates Th1 development and induces angiogenesis. We previously described regulation of TNF-α-induced-IL-18 bioactivity by blocking the ERK pathway. Here, we focused on modulation of TNF-α-induced-IL-18 bioactivity by reduction of caspase-1 expression.
Methods Caspase-1 expression in RA synovial fibroblasts treated with TNF-α was assessed by qRT-PCR and Western blot. The critical pathways for TNF-α-induced caspase-1 expression were determined by using chemical inhibitors: pyrrolidine dithiocarbamate (PDTC; a nuclear factor kappa-light-chain-enhancer of activated B cells [NFκB] inhibitor; 200μM), MAPK inhibitors (ERK1/2, PD98059; p38, SB202190; or JNK2, SP600125; 10μM), or AG-490 (a JAK2 inhibitor; 10 μM) followed by TNF-α stimulation. Caspase-1 expression was determined by qRT-PCR and Western blot. Immunofluorescence (IF) staining was performed to check IL-18 production induced by TNF-α with or without preinhibition of ERK1/2 or JAK2 by using antibody recognized immature and mature IL-18. IL-18 level was also assessed by ELISA in cell lysates and concentrated culture supernatants. IL-18 functional activity was assessed using an IL-18 bioactivity assay using KG-1 cells and culture supernatants.
Results TNF-α induced RA synovial fibroblast caspase-1 expression at the mRNA and protein levels in a time-dependant manner (P<0.05; n≥6 patients).
Blocking the JAK2 pathway reduced TNF-α-induced-caspase-1 expression at the transcriptional and protein level by approximately 60% and 40%, respectively (P<0.05; n≥4). Blocking NFκB, ERK1/2, JNK or p38 pathways had no effect on TNF-α-induced-caspase-1 mRNA expression. We then confirmed by IF that TNF-α-induced IL-18 and investigated roles of ERK1/2 and JAK2 pathways. Blocking the ERK1/2 pathway dramatically decreased IL-18 expression induced by TNF-α. However, when blocking the JAK2 pathway, TNF-α induced intracytoplasmic granular IL-18 expression, suggesting a defect of caspase-1. Blocking JAK2 pathway increased intracellular TNF-α-induced IL-18 level, but reduced extracellular TNF-α-induced IL-18 level in concentrated supernatant assessed by ELISA. Finally, blocking the JAK2 pathway, we observed a reduction of IL-18 bioactivity by 52% in RA synovial fibroblasts.
Conclusions These results show a unique way to block TNF-α-induced-IL-18 bioactivity by blocking capase-1. These data provide a novel role for the JAK2 pathway in RA patients and emphasize the use of JAK inhibitors as a new therapeutic option in the management of RA.
Disclosure of Interest None Declared