High mobility group box-1 induces pro-inflammatory signaling in human nucleus pulposus cells via toll-like receptor 4-dependent pathway

Abstract

Intervertebral disc (IVD) degeneration (DD) is associated with low back pain, the leading cause of disability worldwide. Damage-associated molecular patterns (DAMPs) that contribute to inflammation and trigger DD have not been well characterized. Extracellular high mobility group box-1 (HMGB1) protein has been implicated as a potent DAMP and pro-inflammatory stimulus in the immune system. In this study, we show that HMGB1 and IL-6 levels increase in patients with advanced DD in comparison to early DD. This study further tested the hypothesis that HMGB1 promotes inflammatory signaling driving DD in human nucleus pulposus (NP) cells and tissue. Immunofluorescence and western blot analysis confirmed the expression of HMGB1 and its extracellular release by NP cells under cell stress. Gene expression and protein quantification indicate that HMGB1 stimulates the expression IL-6 and MMP-1 in a dose-dependent manner. The contributions of toll-like receptor (TLR) -2, -4 and receptor for advanced glycation end products (RAGE) as receptors mediating HMGB1 signaling was examined using small molecule inhibitors. Inhibition of TLR-4 signaling, with TAK-242, completely abrogated HMGB1 induced IL-6 and MMP-1 expression, whereas inhibition of TLR-2, with O-vanillin, or RAGE, with FPS-ZM1, had mild inhibitory effects. HMGB1 stimulation activated NF-ĸB signaling while TAK-242 co-treatment abrogated it. Lastly, effects of HMGB1 on matrix deposition was evaluated in a 3D culture system of human NP cells. These results implicate HMGB1 as a potent DAMP that promotes inflammation in NP cells and degradation of NP tissues. TLR4-HMGB1 axis is a potential major pathway to alleviate disc inflammation and mitigate DD. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Keywords: high mobility group box-1 protein; intervertebral disc degeneration; nucleus pulposus; pro-inflammatory cytokines; toll-like receptors.

Figure 1.

Analyses of human IVD tissues from patients with increasing disc degeneration grades. Relative gene expression levels for HMGB1 (A) and IL-6 (B) from patient tissues of various degeneration severities. Results are presented as mean ± SD (N = 17) * versus “Early” group; p < 0.05.

Figure 2.

LPS induced HMGB1 nuclear translocation and extracellular release in human NP cells. (A) Representative images of human NP cells cultured in control (untreated) conditions or in stress conditions (LPS) for 16 h. The localization of cellular HMGB1 (green) was evaluated relative to the nucleus (DAPI, blue) using immunofluorescence staining. Scale bar: 25 μm. (B) HMGB1 nuclear-to-cytosolic ratio for cells stimulated with and without LPS for 16 h, n > 10 cells per group. (C) Representative western blot analysis of secreted HMGB1 in cell medium supernatant from two independent experiments, and (D) densitometric analysis of western blot results.

Figure 3.

HMGB1-induced pro-inflammatory signaling molecules in human NP cells. Human NP cells were treated with 0, 0.5, 1, or 2 μg/ml of recombinant disulfide-HMGB1 for 24 h. Relative mRNA levels of IL-6 (A) and MMP-1 (D) normalized to GAPDH. Protein levels of IL-6 (B) and MMP-1 (E) in cell medium supernatant was determined using ELISA. Strong correlation as indicated by Spearman’s rank correlation coefficient was observed between HMGB1 treatment and IL-6 secretion levels (ρ = 0.91122, p < 0.05) (C) or MMP-1 levels (ρ = 0.877737, p < 0.05) (F). Results are presented as mean ± SD (n = 3 donors) and *p < 0.05.

Figure 4.

Inhibition of TLR-4, TLR-2 and RAGE signaling has a differential effect on IL-6 expression in human NP cells stimulated by HMGB1. Human NP cells were co-treated with 2 μg/ml of recombinant HMGB1 and varying doses of TLR-4 inhibitor, TLR4i (A, B) or TLR-2 inhibitor, TLR2i (C, D) or RAGE inhibitor, RAGEi (E, F) for 24 h. Relative mRNA levels of IL-6 were normalized to GAPDH (A, C, E) and protein levels of IL-6 in supernatant cell medium were assessed by ELISA (B, D, F). Results are presented as mean ± SD (n = 3 technical replicates) and *p < 0.05.

Figure 5.

Inhibition of TLR-4, TLR-2 and RAGE signaling has a differential effect on MMP-1 expression in human NP cells stimulated by HMGB1. Human NP cells were co-treated with 2 μg/ml of recombinant HMGB1 and varying doses of TLR-4 inhibitor, TLR4i (A, B) or TLR-2 inhibitor, TLR2i (D, E) or RAGE inhibitor, RAGEi (G, H) for 24 h. Relative mRNA levels of MMP-1 were normalized to GAPDH (A, D, G) and protein levels of MMP-1 in cell medium supernatant were assessed by ELISA (B, E, H). No significant cytotoxicity was observed as measured by LDH release from human NP cells supernatant cell medium treated with 2 μg/ml of recombinant HMGB1 and varying doses of TLR-4 inhibitor, TLR4i (C) or TLR-2 inhibitor, TLR2i (F) or RAGE inhibitor, RAGEi (I) for 24 h. Results are presented as mean ± SD (n = 3 technical replicates) and *p < 0.05.

Figure 6.

Comparison of blocking TLR-4, TLR-2 and RAGE in downregulating IL-6 and MMP-1 expression in human NP cells. Human NP cells were co-treated with 2 μg/ml of recombinant HMGB1 and 1 μM of TLR-4 inhibitor (TLR4i) or 100 μM of TLR-2 inhibitor (TLR2i) or 10 μM of RAGE inhibitor (RAGEi) for 24 h. Treatment with recombinant HMGB1 alone served as no inhibition control group (No Inh). Relative mRNA levels of IL-6 and MMP-1 were normalized to GAPDH (A, C) and protein levels of IL-6 and MMP-1 in supernatant cell medium were assessed by ELISA (B, D). Results are presented as mean ± SD (n = 3 donors) and *p < 0.05.

Figure 7.

Inhibition of IL-6 and MMP-1 signaling using a combination of receptor inhibitors in human NP cells stimulated with HMGB1. Human NP cells were co-treated with 2 μg/ml of recombinant HMGB1 and different combination of TLR-4 inhibitor, TLR4i or TLR-2 inhibitor, TLR2i or RAGE inhibitor, RAGEi for 24 h. Relative mRNA level of IL-6 (A) and MMP-1 (B) were normalized to GAPDH. Results are presented as mean ± SD (n = 3). * versus HMGB1; # versus TLR4i, & versus TLR2i, ^ versus RAGEi; p < 0.05.

Figure 8.

Inhibition with TLR4i decreased p-NFκB levels Human NP cells were either untreated or co-treated with 2 μg/ml of recombinant HMGB1 ± 1 μM TLR4i. p-NFκB levels were analyzed in the nuclear fractions of cells after treatments for 10, 30, or 60 min. Lamin was used as a loading control for cell nuclear fractions.

Figure 9.

HMGB1 promoted GAG loss in 3D cultured human NP cells, while co-treatment with TLR4i inhibited GAG loss. Human NP cells were cultured in 3D for 5 weeks for mature ECM deposition to develop. Beads were then cultured for an additional 2 weeks either untreated or cotreated with 2 μg/ml HMGB1 ± 1 μM TLR4i. Staining included alcian blue (GAG staining), picrosirius red (collagen staining), and H&E (cell morphology).

Figure 10.

Schematic of pro-inflammatory signaling molecules release upon HMGB1 stimulation and its receptor blocking. Following cell stress with LPS, stores of the nuclear protein HMGB1 gets translocated from nucleus to cytosol and gets released into the extracellular milieu in human NP cells. This secreted HMGB1 further promotes expression of inflammatory cytokine, IL-6 and catabolic molecule, MMP-1 that are known to further promote and drive DD. Blockade of HMGB1 signaling receptors using various small molecule inhibitors showed a differential inhibition of the pro-inflammatory cytokines in human NP cells. Various sites of targeted pharmacological intervention are indicated by color-coded inhibitory lines and their effects on transcription and release of pro-inflammatory cytokine and catabolic molecule are indicated by respective color-coded arrow lines.