Effects of interleukin‐17A in nucleus pulposus cells and its small‐molecule inhibitors for intervertebral disc disease

Abstract Intervertebral discs (IVD) degeneration, which is caused by ageing or mechanical stress, leads to IVD disease, including back pain and sciatica. The cytokine interleukin (IL)‐17A is elevated in NP cells during IVD disease. Here we explored the pharmacotherapeutic potential of IL‐17A for the treatment of IVD disease using small‐molecule inhibitors that block binding of IL‐17A to the IL‐17A receptor (IL‐17RA). Treatment of NP cells with IL‐17A increased expression of cyclooxygenase‐2 (COX‐2), IL‐6, matrix metalloproteinase (MMP)‐3 and MMP‐13. These increases were suppressed by an IL‐17A‐neutralizing antibody, and small molecules that were identified as inhibitors by binding to the IL‐17A‐binding region of IL‐17RA. IL‐17A signalling also altered sulphated glycosaminoglycan deposition and spheroid colony formation, while treatment with small‐molecule inhibitors of IL‐17A attenuated this response. Furthermore, mitogen‐activated protein kinase pathways were activated by IL‐17A stimulation and induced IL‐6 and COX‐2 expression, while small‐molecule inhibitors of IL‐17A suppressed their expression. Taken together, these results show that IL‐17A is a valid target for IVD disease therapy and that small‐molecule inhibitors that inhibit the IL‐17A–IL‐17RA interaction may be useful for pharmacotherapy of IVD disease.

through, for example, the degradation of collagen fibres and hydrophilic proteoglycans within the extracellular matrix(ECM). 13 PGE2 is synthesized by two cyclooxygenase (COX) isoforms, COX-1 and COX-2. 14 COX-2 expression is induced in response to inflammatory stimuli 15 and mechanical stress, and its expression initiates the degenerative cascade. 16 COX-2 and PGE-2 play key roles in lower back pain and sciatica. 7,8,17 As such, COX-2 is considered an important mediator of the IVD degenerative process 8 and is a therapeutic target for IVD disease. 8 Recently, IL-17 was reported to be increased in NP cells of degenerated or herniated IVDs, along with IL-4, IL-6, IL-12 and interferon-γ.
IL-17 is a cytokine produced by the T helper 17 subset of CD4 + T cells and plays critical roles in various inflammatory disorders. 18,19 Its family is comprised of six members: IL-17A to IL-17F. 20 IL-17A binds to its cognate receptor (IL-17RA) and activates many intracellular signaling factors, including those of the nuclear factor kappa-light-chain-enhancer of activated B cells or mitogen-activated protein kinase (MAPK) pathways. 21 IL-17A also synergistically increases the release of inflammatory mediators in disc cells. 22,23 With respect to the relationship between IL-17A and other cytokines, IL-6 is one of a range of factors triggered when CD4 + T cells undergo differentiation into Th17 cells, which produce IL-17 in response to IL-6 and transforming growth factor β via Retinoic acid receptor-related Orphan Receptor (ROR)-γ. 24,25 IL-17A also induces expression of IL-6 in fibroblasts 26 and triggers a positive feedback loop of IL-6 signalling. 27 In IVD disease and degeneration, IL-6 can potentiate the catabolic actions of other cytokines, including IL-1 and TNF-α, 28 induce apoptosis 29 and cause loss of IVD matrix proteins including MMP family members, such as MMP-3 or MMP-13. 28,30 In addition, IL-6 and IL-6 receptor expression levels are responsive to IVD injury. 31 Thus, IL-6 plays an important role in the degenerative progression of NP cells and the various symptoms associated with degenerative disorders. Additionally, IL-17A may lead to an increase in COX-2 expression via the activation of MAPK pathways in disc degeneration. 32 Therefore, IL-17A may offer a potential target for therapeutic intervention for disc degeneration. However, there are few studies examining the role of IL-17 in disc cells. To address this, we focused on the crosstalk between IL-17A, IL-6 and COX-2 and investigated the effect of IL-17A on NP cells under hypoxic conditions (1% O 2 ), reflecting the in vivo context. The main objectives of this study were to examine the pharmacotherapeutic potential of IL-17A for the treatment of degenerative discs. We analysed the structure of IL-17A and the IL-17A receptor (IL-17RA) and identified small-molecule inhibitors that bind to the IL-17A-IL-17RA docking region and inhibit IL-17A signalling in NP cells. We evaluated the effects of these inhibitors as a potential new therapeutic strategy for disc diseases.

| Human samples and histology
Informed consent was obtained from each patient for the use of their IVD tissue according to the Declaration of Helsinki. Ethical approval was obtained from the Institutional Ethics Review Board of Tokai University School of Medicine. A total of 11 IVD tissues were dissected from six patients with lumbar herniation and five patients under 16 years of age with idiopathic scoliosis. The samples were evaluated according to Pfirrmann's magnetic resonance classification, 33 and grades III, IV and V were considered to be degenerated IVD and grades I and II considered to be non-generated IVD. Using this classification, IVDs from all six lumbar herniated disc patients were considered degenerated while those from all five idiopathic scoliosis patients were non-degenerated. The samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and embedded in paraffin. The sections were deparaffinized in xylene, rehydrated through graded ethanol and incubated with IL-17A antibody (#bs-2140R; Bios) diluted in 1% bovine serum albumin in PBS overnight at 4°C. Samples were then stained with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA). Staining was visualized using diaminobenzidine (Nakarai Tesque, Kyoto, Japan). Nuclei were stained with haematoxylin. All specimens were viewed under a microscope (IX70; Olympus, Tokyo, Japan). Positively immunolabelled cells were calculated as a percentage of the total number of cells per high-power field in each section.

| Isolation of NP cells, cell treatments and hypoxic culture conditions
Rat NP cells were isolated from male Sprague-Dawley rats (11 weeks old) using a modified method previously reported by Risbud et al. 5 Briefly, IVDs from the lumbar and coccygeal regions were dissected from rats under deep anaesthesia using aseptic conditions. The gellike NP was separated from the AF, and the NP tissue was minced by pipetting. The isolated cells were maintained in Dulbecco's modi-

| Real-time RT-PCR analysis
Total RNA was extracted from NP cells using RNAeasy mini columns (Qiagen, Hilden, Germany). Before elution from the column, RNA was treated with RNase-free DNase I (Qiagen). The purified, DNAfree RNA was converted to cDNA using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA).
Template cDNA and gene-specific primers were added to Power SYBR Green Master Mix (Applied Biosystems), and mRNA expression was quantified using the Step One Plus Real-time PCR System (Applied Biosystems). β-actin was used to normalize gene expression.
Melting curves were analysed to verify the specificity of the RT-PCR and the absence of primer dimer formation.

| Plasmids
The COX-2 promoter luciferase constructs phPES2-1432/+59 were kindly provided by Dr. Akihiko Hiyama (Tokai University, Kanagawa, Japan). 37 We used the vector pGL4.74 (Promega, Madison, WI, USA) containing the Renilla reniformis luciferase gene as an internal transfection control. Western blot data are presented as band intensities normalized to that of the loading control (β-actin).

| Colony-forming assay
To assess spheroid colony formation, single-cell suspensions of | 5541 dishes and cultured in 1 mL of MethoCult H4230 methylcellulose medium (Stem Cell Technologies) and were treated with 10-100 ng/ mL of IL-17A and 50-200 μg/mL of STK for 10 days. Colonies (>10 cells) were counted using an inverted microscope.

| Statistical analysis
All measurements were performed at least three times, and the data are presented as the mean ± standard deviations (SD). Differences between groups were analysed using Student's t test or one-way analyses of variance. Dunnett's test was used as post hoc test. Significance was set at P < 0.05.

| Induction of IL-17A expression in NP cells of human herniated discs
We first classified the level of degeneration in IVD samples according to Pfirrmann's magnetic resonance classification 33  Alcian blue staining was used to detect the sulphated glycosaminoglycans of NP cells on day 8, and we observed that IL-17A treatment lead to a significant decrease in sulphated glycosaminoglycans ( Figure 2E).

| IL-6 increases COX-2 expression in primary NP Cells
Next, because IL-6 mRNA was prominently increased by IL-17A stimulation, we evaluated the effects of IL-6 in rat NP cells. Treatment with IL-6 led to a significantly increased expression of COX-2, MMP-3 and MMP-13 mRNA, like IL-17A treatment, but had no effect on IL-17A mRNA expression compared with untreated control cells ( Figure 2F). In addition, we confirmed that COX-2 protein expression ( Figure 2G,H) and COX-2 promoter transcriptional activity were significantly increased by IL-6 treatment compared with untreated control cells ( Figure 2I). These results suggest that IL-6 induces expression of COX-2 and MMPs, but not IL-17A.  Results shown as mean ± SD; n = 4, *P < 0.05. B, C, Western blot analysis of NP cells treated with 50 ng/mL IL-17A for 24 hours under hypoxic conditions. IL-17A treatment led to a significant increase in expression of IL-6 and COX-2 proteins. Results shown as mean ± SD; n = 3, *P < 0.05. D, Effects of IL-17A on COX-2 promoter activity in NP cells. Luciferase activity was assessed to show COX-2 transcription in NP cells after treatment with 50 ng/mL of IL-17A. IL-17A treatment significantly increased COX-2 promoter transcriptional activity. Results shown as mean ± SD; n = 3, *P < 0.05. E, Alcian blue staining quantifying of NP cells treated with 50 ng/mL IL-17A. Optical density of the extracted dye was measured at 670 nm. Results shown as mean ± SD; n = 3, *P < 0.05. F, Real-time PCR analysis of NP cells treated with 50-100 ng/mL IL-6 for 24 hours. The control cells were not treated with IL-6. Treatment with IL-6 led to significantly increased expression of COX-2, MMP-3 and MMP-13 mRNA but had no effect on IL-17A mRNA expression. Results shown as mean ± SD; n = 3, *P < 0.05. G, H, Western blot analysis of NP cells treated with 50 ng/mL IL-6 for 24 hours under hypoxic conditions. IL-6 treatment led to a significant increase in expression of COX-2 proteins. Results shown as mean ± SD; n = 3, *P < 0.05. I, Effects of IL-6 on COX-2 promoter activity in NP cells. Luciferase activity was used to show COX-2 promoter transcription in NP cells after treatment with 50 ng/mL of IL-6, which significantly increased COX-2 promoter transcription activity. Results shown as mean ± SD; n = 3, *P < 0.05 MAPK inhibitors for 24 hours under hypoxia. We found that all inhibitors significantly suppressed IL-17A-mediated induction of COX-2 mRNA expression compared with cells treated with IL-17A alone (Figure 6A). IL-6 mRNA expression was significantly suppressed by SB and PD treatments, but SP treatment did not significantly suppress IL-6 mRNA compared with cells treated with IL-17A alone ( Figure 6B).

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These results suggest that some expression of COX-2 and IL-6 may be regulated through at least one of the MAPKs.
Considering these results, we analysed the phosphorylation sta- With respect to the relationship between IL-6 and IL-17A, some reports have indicated that IL-6 is elevated in response to IL-17Aactivated STAT3 in some tumour cells. 39,40 In our study, IL-6 was increased by IL-17A stimulation and down-regulated when IL-17A signalling was suppressed using an anti-IL-17A antibody or an IL-17A inhibitor. These results suggest that IL-17A may be one of the elements that controls expression of IL-6 in NP cells. Previous studies have reported that IL-6 is secreted by IVD cells in the absence of macrophages 41 and that its expression is elevated in injured IVD cells or herniated discs. 31,42 Others have shown that IL-6 down-regulates disc matrix formation and promotes disc degeneration 7,9,28,30 and contributes to expression of inflammatory mediators such as TNF-α and PGE2, 28,30 which lead to neuropathic pain. 29,43 We found that IL-17A treatment increases IL-6, COX-2,  We evaluated the effects of the STK small-molecule inhibitor on the intracellular IL-17A signalling pathway. A previous study reported that IL-17A increased COX-2 expression via the activation of MAPK pathways in NP cells under normoxic conditions. 32 In our study, treatment with MAPK inhibitors showed that p38 and ERK inhibitors suppressed not only COX-2 but also IL-6 mRNA expression; however, the JNK inhibitor suppressed only COX-2 expression.
Phosphorylation of p38 and ERK increased significantly, and JNK phosphorylation tended to increase with IL-17A treatment after 15-30 minutes under hypoxic conditions. This indicates that p38 and ERK are important for regulation of IL-6 and COX-2 expression. The STK small-molecule inhibitor was effective for suppressing p38 phosphorylation, suggesting that p38 coordinates both IL-6 and COX-2 regulation, and that STK particularly affects expression of IL-6 or COX-2 by suppressing p38 phosphorylation. Inhibition of p38 was reported to down-regulate the induction of IL-6 in NP cells 47  These results also demonstrate that STK small-molecule inhibitor is useful to suppress degeneration of IVDs.
In addition, we verified the effects of the STK small-molecule inhibitor in human NP cells. Although there are some differences in the conditions used for analysis of rat NP cells and human cells (eg, concentration of inhibitor and treatment period), the STK smallmolecule inhibitor also down-regulated IL-6 and COX-2 expression in human NP cells. This emphasizes the utility of small-molecule inhibitors for IL-17A-IL-17RA as a pharmacological therapy for IVD disease. Additionally, we analysed the effects of STK on human NP cell spheroid colony-forming capability, which reflects their ability to self-renew, as well as the ability to produce ECM. 45,49 The number of human NP cell spheroid colonies was decreased by treatment with IL-17A and was further improved by STK small-molecule inhibitor treatment. These findings demonstrate that the STK small-molecule inhibitor can facilitate self-renewal and ECM production, in response to the catabolic effects initiated by IL-17A signalling and provide insights for new therapeutic option for IVD disease.
The major limitations of this study are described below. We used both rat NP cells and human NP cells, and the notochondal phenotypes are not identical, with the presence of a portion of notochondal cells in adult discs differing in rats and humans. Thus, there is the possibility that the IVD degeneration process in rats is not analogous to that in humans. Our observations using human NP cells included sufficient samples to be statistically analysed; however, it is difficult to collect IVDs from patients of all the same ages in order to have a more controlled patient group. Therefore, concern over age variation of our patients and the effect it has on the degeneration process of IVDs still remains.
F I G U R E 7 Effects of IL-17A and STK treatments on human NP cells under hypoxia. A, B, IL-6 mRNA expression in human NP cells treated with or without 50 ng/mL of IL-17A and 50-100 μg/mL STK for 24 hours (A) and 48 hours (B). Treatment with 100 μg/mL STK significantly suppressed expression of IL-6 mRNA at both 24 and 48 hours. Results shown as mean ± SD; n = 4, *P < 0.05. C, D, COX-2 mRNA expression in human NP cells treated with or without 50 ng/mL of IL-17A and 50-100 μg/mL STK for 24 hours (C) and 48 hours (D). Treatment with 50 μg/mL STK significantly suppressed expression of COX-2 mRNA at 48 hours (D). Results shown as mean ± SD; n = 4, *P < 0.05. E, Quantification of colonies formed in NP cells treated with 0-100 ng/mL IL-17A after 10 days of culture in methylcellulosebased medium. F, Quantification of colonies formed in NP cells treated with 50 ng/mL IL-17Aand 0-200 μg STK after 10 days of culture in methylcellulose-based medium. Results shown as mean ± SD; n = 3, *P < 0.05 In summary, the present study shows that IL-17A increases expression of IL-6, COX-2, MMP-3 and MMP-13. Further, inhibition of IL-17A binding to IL-17RA suppressed IL-17A signalling and reduced expression of these factors in NP cells under hypoxic conditions, and the small-molecule inhibiters that bound to the IL-17A-IL-17RA-binding region were effective in NP cells. Although further studies on the detailed mechanisms of action are required, our study offers insight into the potential utility of small-molecule inhibiters of IL-17A-IL-17RA as a pharmacological therapy in IVD disease.