Background In macrophages, repeated stimulation of Toll-like receptor (TLR) 4 leads to a tolerant state of the cell which protects inflamed tissues from damage. We have recently shown that rheumatoid arthritis (RA) synovial fibroblasts (SF) lack these protective mechanisms and maintain the secretion of inflammatory cytokines and matrix degrading metalloproteinases (MMPs) after repeated LPS stimulation.
Objectives To investigate mechanisms behind LPS tolerizable and non-tolerizable effects in RASF and macrophages.
Methods RASF, and in vitro differentiated peripheral blood derived macrophages from healthy donors were treated with LPS (100 ng/ml) for 24h and then re-stimulated with low doses of LPS (10 ng/ml) for another 24h. The expression levels of interleukins (IL), chemokines, MMPs, retionoic acid-inducible gene 1 (RIG1), melanoma differentiation associated protein-5 (MDA5) and 2'-5'-oligoadenylate synthetase (OAS1) were analyzed by multiplex immunoassays on the Luminex®200 platform, ELISAs or quantitative Real-time PCR. Nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) promoter activities in RASF (n=4) were evaluated by Dual-Luciferase reporter assays after repeated stimulation with LPS. The levels of histone 3 (H3) and H4 acetylation (H3ac, H4ac) as well as H3 lysine 4 trimethylation (H3K4me3) in promotor regions of tolerizable (OAS1, RIG1) and non-tolerizable (IL6, IL8) genes in SF (n=6–7) and macrophages (n=5) were analyzed by Chromatin Immune precipitation (ChIP).
Results Multiplex protein platform and ELISAs revealed that most of the IL and chemokines were tolerizable in macrophages and non-tolerizable in supernatants from RASF after repeated LPS stimulation. A lack of tolerizable effects of RASF was also found for MMP1 and MMP3, whereas the anti-viral response genes OAS1, RIG1 and MDA5 were tolerizable in RASF and macrophages. Reporter gene activities for NF-κB and AP-1 were similar in single and double stimulated RASF, excluding potential differences in the activation of these transcription factors as underlying mechanisms for tolerizable/non-tolerizable effects in RASF. The levels of the activating histone marks H3ac and H3K4me3 were decreased by LPS double compared to single stimulation of SF and macrophages in promoter regions of OAS1 and RIG1. These chromatin marks were not changed or even increased in promoter regions of IL6, IL8 and MMP1 in RASF. Interestingly, AcH3 levels in an IL6 (-525/-349) and an IL8 (-80/-25) promoter region were increased in RASF (IL6: single LPS: 1.38; double LPS: 1.5; IL8: single LPS: 0.17 double LPS: 0.24) but decreased in macrophages (IL6: single LPS: 1.55; double LPS: 1.14; IL8: single LPS: 0.61 double LPS: 0.51).
Conclusions Epigenetic modifications on gene promoters contribute to differences in tolerization between RASF and macrophages. Since many pro-inflammatory cytokines and MMPs are non-tolerizable genes in RASF, epigenetic enzymes that maintain the aggressive phenotype of RASF in persistent inflammation are future therapeutic targets.
Acknowledgement IMI-BT Cure (Pfizer), IAR Epalinges, euroTEAM
Disclosure of Interest K. Klein. Grant/research support from: IMI-BT Cure (Pfizer), IAR Epalinges, euroTEAM, R. Gay: None declared, C. Kolling: None declared, L.-L. Lin Shareholder of: Pfizer, Employee of: Pfizer, N. Bostanci: None declared, G. Belibasakis: None declared, B. Michel: None declared, S. Gay Grant/research support from: IMI-BT Cure (Pfizer), IAR Epalinges, euroTEAM, C. Ospelt Grant/research support from: IMI-BT Cure (Pfizer), IAR Epalinges, euroTEAM