Background/Purpose:

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 also after repeated LPS stimulation. The objective was to investigate mechanisms behind tolerizable and non-tolerizable effects in RASF.

Methods:

RASF, osteoarthtitis (OA) SF, and in vitro differentiated peripheral blood derived macrophages from healthy donors and RA patients were treated with LPS (100 ng/ml) for 24h and then re-stimulated with LPS (10 ng/ml) for another 24h. The expression levels of interleukin (IL) 6, IL8, chemokine (C-X-C mofif) ligand 10 (CXCL10), matrix metalloproteinases (MMP) 1 and MMP3, as well as retionoic acid-inducible gene 1 (RIG1) and 2`-5`-oligoadenylate synthetase (OAS1) were analyzed by quantitative Real-time PCR or ELISA. Nuclear factor-қB (NF-қB) 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, CXCL10) and non-tolerizable (IL6, IL8, MMP1, MMP3) genes in SF (n=3) were analyzed by Chromatin Immune precipitation (ChIP).

Results:

As expected, the expression of IL6 decreased in double-stimulated (886 ± 596 pg/ml) compared to single-stimulated (2368 ± 315 pg/ml; p<0.01) macrophages from healthy donors (n=4) and RA patients (n=6, p=0.06). On the other hand, RASF (n=10) and OASF maintained their production of IL6 after repeated TLR4 stimulation (RASF: single stimulation: 13.2 ± 5.8 ng/ml, double stimulation: 12.4 ± 7.1 ng/ml). A lack of tolerizable effects of RASF was also found for MMP1 and MMP3, whereas the interferon-responsive genes OAS1, RIG1 and CXCL10 were tolerizable. RASF (n=5) secreted 531 ± 385 pg/ml CXCL10 after a single LPS stimulation and 111 ± 97 pg/ml CXCL10 after double stimulation (p<0.05). Reporter gene activities for NF-қB 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. Interestingly, the levels of the activating histone marks H4ac and H3K4me3 were decreased by LPS double compared to single stimulation of SF in different promoter regions of OAS1 and CXCL10, whereas these chromatin marks were not changed or even increased in promoter regions of IL6, IL8, MMP1 and MMP3. On the other hand, changes in the levels of H3ac after LPS double stimulation did not distinguish tolerizable from non-tolerizable genes.

Conclusion:

Epigenetic modifications on target gene promoters are likely to contribute to differences in tolerization between RASF and macrophages. Since many pro-inflammatory cytokines and MMPs are non-tolerizable genes in RASF, we conclude that the lack of tolerization in these cells keeps RASF aggressive in persistent inflammation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/epigenetic-mechanisms-contribute-to-the-lack-of-lps-induced-tolerance-in-rheumatoid-arthritis-synovial-fibroblasts/