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 which protects inflamed tissues from damage. We have recently shown that in contrast to macrophages, rheumatoid arthritis synovial fibroblasts (RASF) lack these protective mechanisms and keep on secreting inflammatory cytokines and matrix degrading metalloproteinases also after repeated stimulation with LPS. The objective was to investigate mechanisms behind tolerizable and non-tolerizable effects in RASF.

Methods

RASF were treated with LPS (100 ng/ml). 24h after the initial stimulation, cells were re-stimulated with LPS (10 ng/ml) for another 24h. The expression levels of IL6, IL8, CXCL10, matrix metalloproteinases (MMP) 1 and MMP3, as well as RIG1 and 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. RASF (n=8) were transfected with siRNAs targeting cAMP responsive element binding protein 1 (CREB1) or scrambled siRNAs as control prior to stimulation with LPS (100 ng/ml, 24h). Activation of CREB1 after repeated LPS stimulation was analyzed in nuclear extracts (n=2) using p-CREB1 antibodies.

Results

RASF (n=10) maintained their production of IL6 after repeated TLR4 stimulation (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, MDA5 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. Silencing of CREB1 reduced the LPS-induced expression levels of the tolerizable genes CXCL10 (x-fold: ctrl 54.8 ± 64.9; siCREB1 34.5 ± 42.9; p<0.05), OAS1 (x-fold: ctrl 24.0 ± 21.1; siCREB1 15.6 ± 15.2, p<0.05) and RIG1 (x-fold: ctrl 14.8 ± 12.1; siCREB1 10.1 ± 8.6, p<0.05), whereas LPS-induced expression levels of the non-tolerizable genes IL6, IL8, MMP1 and MMP3 were not affected. Similar effects of CREB1 silencing were obtained when secreted protein levels of LPS-induced CXCL10, IL6, IL8, MMP1 and MMP3 where measured.  The phosphorylation of CREB1 was not changed by LPS double compared to single stimulation indicating that CREB1 activation was not impaired by double stimulation.

Conclusion

The expression of tolerizable genes in RASF is dependent on the transcription factor CREB1. Based on the fact that CREB1 activation is not altered by repeated LPS stimulation, epigenetic modifications on target gene promoters that effect the recruitment of CREB1 to promoters are likely to contribute to tolerization effects seen in RASF.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-role-of-the-transcription-factor-camp-responsive-element-binding-protein-1-in-lipopolysaccharide-induced-tolerance/