Background Fibroblast-like synoviocytes (FLS) have recently emerged as attractive therapeutic targets in rheumatoid arthritis (RA). FLS, during the chronic course of synovitis in RA, are a major source of inflammatory cytokines, chemokines, complement, growth factors and tissue destructive enzymes. Thus, RA FLS mediate synovial recruitment, retention, organization, activation and survival of inflammatory cells, enhance synovial neoangiogenesis, and induce osteoclastogenesis and cartilage degradation.
Objectives In the current study we test the hypothesis that chronic exposure of FLS to TNFα augments inflammatory responses to secondary stimuli (priming effect).
Methods FLS obtained from RA patients were chronically exposed to TNFα (3 days) and then were stimulated with interferons (IFN). Expression of IFN-target genes was measured by real-time quantitative reverse transcription-polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA). Total STAT1 protein and IFN-mediated STAT1 activation were evaluated by Western blotting. Total histone levels, histone acetylation, NF-κB p65 and RNA polymerase II (pol II) recruitment were measured at the promoter of CXCL10/IP-10 by chromatin immunoprecipitation assay.
Results Chronic pre-exposure to TNFα sensitizes RA FLS to the proinflammatory effects of suboptimal levels of IFN (Fig. 1). We observed enhanced magnitude and extended kinetics of CXCL10/IP-10, CXCL9/MIG and CXCL11/ITAC production upon subsequent IFN stimulation. This phenotype was retained over a period of days even after the removal of TNFα. This finding suggests that FLS gain and retain an “inflammatory memory” upon chronic exposure to TNFα. We have tested the hypothesis that, upon chronic exposure of FLS to TNFα, an open chromatin state is established at CXCL10/IP-10 promoter. Chromatin accessibility can be increased by depletion of histones/nucleosomes, which exposes DNA to transcription factors, or by histone acetylation, which weakens DNA-histone interactions. Chronic TNFα decreased histone levels, increased acetylation of the remaining histones (Fig. 2), and heightened recruitment of NF-κB p65 and pol II at the open chromatin of CXCL10/IP-10 promoter. In parallel, an increase in intracellular STAT1 reservoir led to amplification of IFN-induced STAT1 activation (Fig. 3).
Conclusions Our study reveals a novel pathogenic function of TNFα, namely prolonged and gene-specific priming of FLS for enhanced transcription of inflammatory chemokines due to removal of the chromatin barrier, sustained activation of NF-κB, and amplification of STAT1 activation downstream of IFNs. These data also suggest that FLS gain an “inflammatory memory” upon chronic exposure to TNFα. An attractive molecular explanation for this “inflammatory memory” could be the induction by chronic TNFα of molecules and chromatin modifications that are not rapidly reversed or are turned over with slow kinetics. Finally, our study provides a molecular explanation for the production of CXCL10/IP-10, CXCL9/MIG and CXCL11/ITAC by FLS during the course of synovial inflammation, where TNFα and IFNs are co-expressed.
Disclosure of Interest : None declared