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AB0084 Novel regulation of TNF-α-induced-IL-18 bioactivity in rheumatoid arthritis synovial fibroblasts by reducing caspase-1 via JAK2 inhibition
  1. H. Marotte1,
  2. T. Fedorova1,
  3. A.J. Pinney1,
  4. B. Lewis1,
  5. A.E. Koch2
  1. 1University of Michigan Medical School
  2. 2Veteran’s Administration and University of Michigan Medical School, Ann Arbor, United States

Abstract

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

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