Background/Purpose:

In macrophages, repeated stimulation of Toll-like receptor (TLR) 4 leads to adaptation of signaling pathways and epigenetic modifications resulting in a tolerant state of the cell and protecting inflamed tissues from inflammation-induced damage. We hypothesized that a lack of tolerization in rheumatoid arthritis synovial fibroblasts (RASF) significantly contributes to sustained inflammation and inflammation-induced damage seen in RA.

 The objective was to investigate tolerizable and non-tolerizable effects in RASF compared to macrophages.

Methods:

RASF and in vitro differentiated peripheral blood derived macrophages from healthy donors and RA patients were treated with LPS (100 ng/ml). 24h after the initial stimulation, cells were re-stimulated with LPS (10 ng/ml) for another 24h. Supernatants were collected from the second treatment period for ELISA and cells were harvested for isolation of RNA or protein extracts after a total treatment period of 48h. The expression of different genes, including cytokines and chemokines (IL6, CCL5, IL33, CXCL10), matrix metalloproteinases (MMP1, MMP3, MMP13), receptors (TLR2, TLR3, TLR4, MDA5, RIG1) as well as signaling molecules, activators and inhibitors of the TLR pathway (SHIP1, SOCS1, TNFAIP3, OAS1) was analyzed by quantitative Real-time PCR, Western blotting and ELISA. Nuclear factor-қB (NF-қB) and activator protein-1 (AP-1) promoter activities in RASF were evaluated by Dual-Luciferase reporter assays after repeated stimulation with LPS (100 ng/ml, 10 ng/ml).

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) maintained their production of IL6 after repeated TLR4 stimulation and secreted 13237 ± 5764 pg/ml IL6 after a single LPS stimulation and 12421 ± 7178 pg/ml IL6 after double stimulation. A lack of tolerizable effects after LPS stimulation of RASF was also found for MMP1, MMP3, MMP13, CCL5, IL33, TLR3 and TNFAIP3. Interestingly, the known interferon-responsive genes OAS1, RIG1, MDA5 and CXCL10 were tolerizable not only in macrophages but also in RASF. 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). TLR4 mRNA was not changed in macrophages or in RASF by LPS double stimulation suggesting that a change of TLR4 expression itself was not responsible for tolerization. Reporter gene activities for NF-қB and AP-1 were similar in single and double stimulated RASF, excluding changes in signaling molecules as the underlying mechanism for tolerizable/non-tolerizable effects.

Conclusion:

Based on the fact that neither TLR4 expression nor signalling pathways are altered by repeated LPS stimulation, 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 them aggressive in persistent inflammation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rheumatoid-arthritis-synovial-fibroblasts-lack-tolerization-of-inflammatory-cytokines-and-matrix-degrading-enzymes-after-repeated-lps-stimulation/