IL-1β/HMGB1 signalling promotes the inflammatory cytokines release via TLR signalling in human intervertebral disc cells

Abstract

Inflammation and cytokines have been recognized to correlate with intervertebral disc (IVD) degeneration (IDD), via mediating the development of clinical signs and symptoms. However, the regulation mechanism remains unclear. We aimed at investigating the regulatory role of interleukin (IL)β and high mobility group box 1 (HMGB1) in the inflammatory response in human IVD cells, and then explored the signalling pathways mediating such regulatory effect. Firstly, the promotion to inflammatory cytokines in IVD cells was examined with ELISA method. And then western blot and real time quantitative PCR were performed to analyse the expression of toll-like receptors (TLRs), receptors for advanced glycation endproducts (RAGE) and NF-κB signalling markers in the IL-1β- or (and) HMGB1-treated IVD cells. Results demonstrated that either IL-1β or HMGB1 promoted the release of the inflammatory cytokines such as prostaglandin E2 (PGE2), TNF-α, IL-6 and IL-8 in human IVD cells. And the expression of matrix metalloproteinases (MMPs) such as MMP-1, -3 and -9 was also additively up-regulated by IL-1β and HMGB1. We also found such additive promotion to the expression of TLR-2, TLR-4 and RAGE, and the NF-κB signalling in intervertebral disc cells. In summary, our study demonstrated that IL-1β and HMGB1 additively promotes the release of inflammatory cytokines and the expression of MMPs in human IVD cells. The TLRs and RAGE and the NF-κB signalling were also additively promoted by IL-1β and HMGB1. Our study implied that the additive promotion by IL-1β and HMGB1 to inflammatory cytokines and MMPs might aggravate the progression of IDD.

Keywords: high mobility group box 1 (HMGB1); interleukin (IL)β; intervertebral disc degeneration (IDD).

Figure 1. Supernatant levels of PGE2, TNF-α, IL-6 and IL-8 in the disc annulus fibrosus cells which were treated with IL-1β or HMGB1

Disc annulus fibrosus cells were treated with 0, 0.5, 1, 2 or 5 ng/ml IL-1β or with 0, 20, 40 or 80 ng/ml HMGB1 for 24 h, then the supernatant levels of PGE2, TNF-α, IL-6 and IL-8 were examined with ELISA methods. (A and B) Supernatant levels of PGE2 and TNF-α (A) or IL-6 and IL-8 (B) in the IL-1β-treated disc annulus fibrosus cells. (C and D): Supernatant levels of PGE2 and TNF-α (C) or IL-6 and IL-8 (D) in the HMGB1-treated disc annulus fibrosus cells. Results are presented as mean ± S.E.M. for quartic independent experiments. P values were calculated by Student t test between two groups, post the ANOVA test for the homogeneity of variance. *P<0.05, **P<0.01, ***P<0.001 or ns: no significance.

Figure 2. Additive inductions by IL-1β and HMGB1 of PGE2, TNF-α, IL-6 and IL-8 in disc annulus fibrosus cells

Disc annulus fibrosus cells were treated with 2 ng/ml IL-1β or (and) with 40 ng/ml HMGB1 for 0, 6, 12, 24 or 48 h, then the supernatant levels of PGE2 (A), TNF-α (B), IL-6 (C) and IL-8 (D) were examined with ELISA methods. Results are presented as mean ± S.E.M. for quartic independent experiments. P values were calculated by parametric ANOVA (Tukey–Kramer post hoc) test. *P<0.05, **P< 0.01, ***P<0.001 or ns: no significance.

Figure 3. Expression of MMPs in the IL-1β- or/and HMGB1-treated disc annulus fibrosus cells

Disc annulus fibrosus cells were treated with 2 ng/ml IL-1β or (and) with 40 ng/ml HMGB1 for 12 h, then the relative mRNA levels of MMP-1 (A), -3 (A), -9 (B) or TIMP metallopeptidase inhibitor 1 (TIMP-1) (B) to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were examined with real-time quantitative PCR (RT-qPCR) method; (C) western blotting assay for MMP-1, -3, -9 and TIMP-1 in protein levels in the IL-1β- or (and) HMGB1-treated disc annulus fibrosus cells. Results are presented as mean ± S.E.M. for quartic independent experiments. Parametric ANOVA (Tukey–Kramer post hoc) test was performed for statistical significance analysis. *P<0.05, **P<0.01 or ns: no significance.

Figure 4. Expression of TLRs and RAGE in mRNA levels in the IL-1β- or/and HMGB1-treated disc annulus fibrosus cells

Disc annulus fibrosus cells were treated with 0, 0.5, 1, 2 or 5 ng/ml IL-1β for 12 h, then the relative mRNA levels of TLR-2 (A), -4 (B), -6 (C) or RAGE (D) to GAPDH were examined with RT-qPCR method; (E and F) relative mRNA levels of TLR-2 and -4 (E), TLR-6 and RAGE (F) to GAPDH in the IL-1β- or (and) HMGB1-treated (for 12 h) disc annulus fibrosus cells. Results are presented as mean ± S.E.M. for quartic independent experiments. P values in A–D were calculated by the parametric ANOVA (Tukey–Kramer post hoc) test; and P value in E–F was calculated by Student t test. *P<0.05, **P<0.01, ***P<0.001 or ns: no significance.

Figure 5. siRNA-mediated TLR-4 knockdown inhibited the IL-1β- and HMGB1-induced CK release

(A) TLR-4 mRNA level in the disc annulus fibrosus cells, which were transfected with 30 or 60 nM siRNA-Control or siRNA-TLR-4 for 24 h; (B and C) Western blot analysis of TLR-4 in protein level in the siRNA-Control- or siRNA-TLR-4-transfected disc annulus fibrosus cells for 24 h; (D–G) Supernatant levels of PGE2 (D), TNF-α (E), IL-6 (F) or IL-8 (G) in the IL-1β- (5 ng/ml for 24 h) or HMGB1- (80 ng/ml for 24 h) treated disc annulus fibrosus cells. Results are presented as mean ± S.E.M. for quartic independent experiments. P values were calculated by Student t test between two groups, post the ANOVA test for the homogeneity of variance. *P<0.05, **P<0.01 or ns: no significance.

Figure 6. Regulation on NF-κB/p65 and IκBα by IL-1β and HMGB1 in disc annulus fibrosus cells

(A) Disc annulus fibrosus cells were treated with 2 ng/ml IL-1β or (and) with 40 ng/ml HMGB1 for 12 h, then the relative mRNA levels of NF-κB/p65 and IκBα to GAPDH were examined with RT-qPCR method. (B and C) Western blotting assay for (B) and relative levels of (C) NF-κB/p65 and IκBα in the disc annulus fibrosus cells, which were treated with 2 ng/ml IL-1β or (and) with 40 ng/ml HMGB1 for 24 h, with GAPDH as control; Student t test was performed for statistical significance analysis. *P<0.05, **P<0.01 or ns: no significance.