Inhibition of IL‐17 prevents the progression of traumatic heterotopic ossification

Abstract Traumatic heterotopic ossification (HO) is the abnormal formation of bone in soft tissues as a consequence of injury. However, the pathological mechanisms leading to traumatic HO remain unknown. Here, we report that aberrant expression of IL‐17 promotes traumatic HO formation by activating β‐catenin signalling in mouse model. We found that elevated IL‐17 and β‐catenin levels are correlated with a high degree of HO formation in specimens from patients and HO animals. We also show that IL‐17 initiates and promotes HO progression in mice. Local injection of an IL‐17 neutralizing antibody attenuates ectopic bone formation in a traumatic mouse model. IL‐17 enhances the osteoblastic differentiation of mesenchymal stem cells (MSCs) by activating β‐catenin signalling. Moreover, inhibition of IL‐17R or β‐catenin signalling by neutralizing antibodies or drugs prevents the osteogenic differentiation of isolated MSCs and decreases HO formation in mouse models. Together, our study identifies a novel role for active IL‐17 as the inducer and promoter of ectopic bone formation and suggests that IL‐17 inhibition might be a potential therapeutic target in traumatic HO.

destruction and requires inflammation. This inflammatory microenvironment activates a resident pool of interstitial progenitors that aberrantly undergo chondrogenesis and further ectopic bone formation. 3 Following trauma and inflammation, ectopic bones are formed in soft tissue through endochondral ossification. 4 However, the pathological mechanism of trauma-induced HO is not clear.
Clinical therapy is now limited to anti-inflammatory drugs, radiation or surgical excision of the already formed bone, which is associated with a high recurrence rate. 5,6 Previous studies have shown that inflammation plays an important role in trauma-induced HO and FOP. 7 Many cytokines in the inflammatory microenvironment can activate the progenitors, induce chondrogenesis/osteogenesis and lead to bone formation. 8,9 IL-17 can be produced by many types of cells, including T helper 17 (Th17) cells, CD8+ T cells, innate lymphoid (ILC3s) cells and natural killer T cells. 10 The effects of IL-17 on inflammation and bone are largely unknown. Previous studies have provided evidence of a catabolic function for IL-17 in bone homeostasis in that IL-17 induces the differentiation of osteoclasts, thereby explaining the development of bone resorption in patients with rheumatoid arthritis (RA). 11,12 In support of this evidence, inhibition of IL-17 with neutralizing antibodies reduces its bone erosion effects. 13 In addition to bone destruction, recent studies have provided evidence that IL-17 promotes osteoblast differentiation and subsequent bone formation. Some studies suggest that IL-17 induces the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. 14,15 Clinical trials performed with IL-17 blocking monoclonal antibodies have clearly shown that IL-17 inhibition is an effective treatment for ankylosing spondylitis (AS), a disorder characterized by new bone formation. 16,17 Notably, recent studies have shown that IL-17 promoted the osteoblast differentiation of isolated MSCs, whereas an inhibitory effect of IL-17 on whole bone was observed. 18 Taken together, these evidence indicate that the effect of IL-17 is complex in that it can promote and inhibit bone formation. The effect of IL-17 on bone may depend on different diseases and the interactions with other cells.
The Wnt/β-catenin signalling pathway has been proven to play a critical role in promoting osteogenic differentiation of MSCs. In addition, β-catenin is a central molecule that is necessary to maintain bone homeostasis and mechanotransduction through the maintenance of osteocyte viability. 19 The Wnt/β-catenin signalling pathway is essential for bone mass maintenance by regulating the activity of osteoblasts directly. β-Catenin conditional activation mice also showed OA-like changes in the knee joint. 20 Based on these observations, we hypothesize that IL-17 released during the inflammation phase of traumatic HO activates β-catenin signalling, leading to overgrowth of bony tissue during disease progression. The key questions that need to be addressed are the molecular mechanisms involved in the inflammation-regulated HO process.
We have previously demonstrated that activated β-catenin is associated with traumatic HO formation. 21 In this study, we show that IL-17 is highly induced in traumatic HO and promotes bone formation via β-catenin signalling. Furthermore, we reveal that IL-17 enhanced the osteoblast differentiation of MSCs isolated from the mice, which function as the crucial promoter in traumatic HO. Inhibition of IL-17 activity effectively attenuated traumatic HO progression in the mouse model.

| Patients and specimens
The study was approved by the ethics committee of Shanghai Jiao Tong University Affiliated Sixth People's Hospital, and written informed consent was obtained from the patients or their legal guardians. Traumatic HO was identified by X-ray and CT radiography from 30 patients (18 males and 12 females, previously healthy, age ranging from 20 to 57 years) who had previously suffered an elbow fracture that was treated by external or internal fixation (within 1 week from the initial injury). These patients returned for surgical resection of HO. Osteogenesis (15 patients, 2-3 months after injury) or maturation stage HO (15 patients, 8 months after injury) was defined based on the time since their injury occurred. Healthy muscles were collected from 8 patients who underwent traumatic forearm amputation. The muscles were used as baseline controls. Blood samples (5 mL per person) were collected 1 day before the clinical surgery.
Blood samples from 10 healthy individuals were used as baseline controls. All the samples were processed immediately to collect serum, which was then stored in a −80°C freezer. The serum specimens were processed for ELISA.

| Mice
Male 6-week-old BALB/c mice were anaesthetized with an intraperitoneal injection of pentobarbital sodium. A 1-cm longitudinal skin incision was made on the lateral aspect of the Achilles tendon to expose its full length. The Achilles tendon was then divided sharply at its midpoint with a surgical knife. For the sham operation, the incision was made through the skin without touching the Achilles tendon. The incised skin was closed with absorbable sutures. The mice were injected with IL-17 antibody (5 mg/kg) twice per week.

| Histology
Mice were killed by carbon dioxide (CO 2 ) inhalation and perfusion fixed with 10% buffered formalin via the left ventricle for 5 minutes.
Then, the ankles with Achilles tendons were dissected and fixed in 4% paraformaldehyde for 24 hours. All of these specimens were decalcified in a 10% EDTA solution for 1 month, embedded in paraffin and cut into 5μm sections for staining.
Sections were stained with 0.1% Safranin O and 0.02% Fast Green (Sigma-Aldrich) according to the manufacturer's instructions.
Immunohistochemical staining was carried out with primary antibodies against IL-17, IL-17R (Abcam, Cat No. ab11370) and βcatenin (Cell Signaling Technology, Cat No. 4370) with a 1:1000 dilution of an appropriate secondary antibody. Protein expression was visualized with a DakoCytomation EnVision staining kit.
The mean density of the positive area was measured by Image-Pro Plus 6.0 (IPP) image analysis software. Three random slides were selected, and five random fields of images per sample were taken.

| μ-CT
Achilles tendons and total hindlimbs from mice were fixed overnight in 4% paraformaldehyde. μ-CT was performed using a SkyScan with a voltage of 60 kV and a resolution of 18 μm, according to standard nomenclature. The region of interest (ROI) was set as the entire tibia to ensure that all the heterotopic bone was included within the ROI. Three-dimensional (3D) images were reconstructed using NRecon, and HO bone volumes were analysed by CTAn software.

| Serum IL-17 analysis
The concentration of IL-17 in the serum was determined by the IL-17 Quantikine ELISA Kit (human: D1700; mouse: M1700. R&D Systems) following the manufacturer's instructions.

| Isolation of cells from mice
Bone marrow cells were harvested from the femur by flushing with PBS and seeded at a density of 1 × 10 6 into 10-cm culture dishes (Corning, NY, USA) at 37°C and 5% CO 2 . Non-adherent cells were discarded after 24 hours, and attached cells were cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone) with 10% foetal bovine serum (FBS).

| ALP and Alizarin red S staining
For ALP staining, cells were stained with 5-bromo-4-chloro-3-ind olyl-phosphate/nitro-blue tetrazolium solution (Sigma-Aldrich) for 45 minutes at 37°C to visualize ALP activity. For Alizarin red S staining, cells were fixed in 4% paraformaldehyde for 10 minutes and rinsed 3 times with deionized water. The cells were then stained with 40 mmol/L Alizarin red S (Sigma), pH 4.0, for 10 minutes. Finally, the cells were rinsed 3 times with deionized water with gentle agitation.

| Quantitative analysis of ALP activity
Cells were washed twice with PBS and solubilized with lysis buffer (10 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, complete protease inhibitor, and 1% NP-40). ALP activity was assayed using p-nitrophenylphosphate (Sigma-Aldrich) as a substrate. The protein content was measured using the BCA Protein Assay kit (Thermo Scientific) according to the manufacturer's instructions.
The ALP activity was expressed as Sigma unit/min/mg of protein.

| Quantitative analysis of mineralization
The calcium deposits from osteoblast cells were washed 3 times with PBS and incubated for 24 hours at 4°C in 0.5 M HCl. Then, the calcium content in the HCl supernatants was measured using the Calcium Colorimetric Assay Kit (BioVision).

| RNA isolation and real-time PCR
Total RNA from the cells was prepared with TRIzol Reagent (Invitrogen).

| Western blot
The cells were washed in ice-cold PBS before lysis with a cell lysis buffer (Cell Signaling Technology). All samples were clarified by centrifugation at 12 000 rpm for 10 minutes at 4°C. Then, the protein concentrations were determined using the BCA Protein Assay kit (Thermo Scientific). Equal amounts of total protein lysates were separated by SDS-PAGE, and bands were transferred to a nitrocellulose membrane. Membranes were probed with specific antibodies to β-catenin and β-actin (Cell Signaling Technology) and then reprobed with appropriate secondary antibodies labelled with IR dyes.
Bound antibodies were detected with an Odyssey Infrared Imaging System (LI-COR Biosciences). Densitometric analysis of the protein bands was performed with Image-Pro Plus 4.5 software (Media Cybernetics).

| Statistical analyses
The data are represented as the means ± standard deviation (SD).
Comparisons between groups were performed using Student's t test, and one-way ANOVA was used for multiple comparisons. All of the experiments were repeated at least 3 times, and representative experiments are shown. Differences were considered significant at P < .05.

| IL-17 is overexpressed in patients with HO
Patients with HO after elbow fracture were identified by X-ray imaging. The surgical HO specimens were collected at the immature stage (2-3 months after initial injury) and maturation stage (>8 months after initial injury). 22

H&E and Safranin O and Fast
Green (SOFG) staining showed a thick layer of cartilage adjacent to the bone at the immature stage. However, we observed a larger cancellous bone and marrow and a thinner cartilage layer at the maturation stage ( Figure 1A,B). We next tested osteoclast activity.
The TRAP staining results showed that the number of TRAP + cells was significantly increased at the immature stage and decreased at the maturation stage ( Figure 1C,G). The expression of IL-17 and IL-17R was significantly elevated at the immature stage and decreased at the maturation stage ( Figure 1D,H,E,I). Furthermore, immunohistochemistry staining showed that the expression of β-catenin was significantly increased at the immature stage and decreased at the maturation stage ( Figure 1F,J). IL-17 concentrations in the serum of patients with HO were examined by ELISA, and significantly elevated IL-17 levels were observed in patients with HO compared with healthy controls. The IL-17 level was decreased at the maturation stage compared with the immature stage ( Figure 1K).

| IL-17/β-catenin signalling is activated in a traumatic HO mouse model
To explore the role of IL-17 in HO progression, we used a traumatic HO mouse model of the Achilles tendon. Heterotopic bone was formed at the injury site at 4 weeks and grew up to 8 weeks after initial Achilles tendon resection (Figure 2A,F). Immature bone was observed at 6 weeks and developed cancellous bone with marrow at 8 weeks ( Figure 2B). Similar to human HO, TRAP staining showed that the number of TRAP + cells in the ectopic bone increased at the initial stage (week 4) after tenotomy and decreased later at week 8.
Continuous osteoclast bone resorption produced a large bone marrow cavity ( Figure 2B,G). The immunohistochemical staining showed that the expression of IL-17 ( Figure 2C,H), IL-17R ( Figure 2D,I) and β-catenin ( Figure 2E,J) increased at week 4, peaked at week 6 and decreased at week 8. Taken together, the Achilles tendon HO mouse model shows a similar mechanism as that observed in human HO specimens, suggesting that a high level of IL-17 may contribute to the pathogenesis of HO.

| IL-17 antibody treatment decreases the formation of traumatic HO
We

| Inhibition of β-catenin suppresses the formation of traumatic HO
To evaluate the role of the β-catenin signalling pathway in traumatic HO progression, the mice were injected with IL-17R anti-  Figure 4A,D). SOFG staining showed that new bone formation was significantly inhibited when the mice were treated with IL-17R antibody or XAV-939 ( Figure 4B). In addition, the expression of β-catenin was decreased in HO tissue after local injection with IL-17R antibody or XAV-939 ( Figure 4C,E). Collectively, these results indicate that the IL-17R/β-catenin pathway is an important pathomechanism in traumatic HO.

| IL-17 enhances osteogenesis through the βcatenin pathway
To examine the effects of IL-17 on osteogenesis, MSCs were har-

| D ISCUSS I ON
Heterotopic ossification is a pathological process that can occur as a result of trauma or as a consequence of genetic mutations. However, we still have limited knowledge about the exact pathogenesis of HO.
Recent studies have shown that the immune system plays a pivotal role in the development of HO. 23 In this study, we found that IL- Bone loss induced by ovariectomy was increased in mice deficient in IL-17R. 40 We demonstrated that the expression of IL-17R was in- The HO formation process has been anecdotally associated with enhanced osteoblast activity, and β-catenin is one of the most important anabolic signalling pathways for osteoblast differentiation. 41 However, there is no effective therapy for traumatic HO. 47 In the present study, we demonstrated that injuries to the Achilles tendon reliably induced HO and increased active IL-17 levels throughout HO progression. We found that the inhibition of IL-17 activity Therefore, our findings suggest that inhibition of IL-17 could be a new paradigm for the treatment of traumatic heterotopic ossifications.

ACK N OWLED G EM ENTS
This study is financially supported by the National Natural Science

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.