IFN‐γ+IL‐17+Th17 cells regulate fibrosis through secreting IL‐21 in systemic scleroderma

Abstract This study aimed to explore the function of IFN‐γ+IL‐17+Th17 cells on fibrosis in systemic scleroderma (SSc). Blood and skin samples were collected from 20 SSc cases and 10 healthy individuals. The percentage of IFN‐γ+IL‐17+Th17 cells was detected using flow cytometry. The in vitro induction of IFN‐γ+IL‐17+Th17 cells was performed adopting PHA and rIL‐12. Gene expression was detected via quantitative real‐time polymerase chain reaction (qRT‐PCR), whereas western blot analysis was adopted for protein analysis. The distribution of IFN‐γ+IL‐17+Th17 cells was significantly increased in SSc cases and positively correlated with SSc stages (P = .031), disease duration (P = .016), activity (P = .025) and skin scores (P < .001). In vitro, IFN‐γ+IL‐17+Th17 cells could promote the expressions of α‐SMA and COL1A1, revealing increased fibroblasts’ proliferation and enhanced collagen‐secreting capacity. In addition, IL‐21 expression was significantly increased in co‐culture medium of IFN‐γ+IL‐17+Th17 cells and fibroblasts (P < .001). IL‐21 neutralizer treatment resulted in the down‐regulation of α‐SMA and COL1A1. IL‐21 was confirmed as an effector of IFN‐γ+IL‐17+Th17 cells in fibrosis process. The distribution of IFN‐γ+IL‐17+Th17 cells was significantly increased in SSc cases and positively correlated with disease activity. IFN‐γ+IL‐17+Th17 cells could promote fibroblast proliferation and enhance collagen‐secreting ability via producing IL‐21, thus contributing to fibrosis in SSc.

cells, a subgroup of T helper cells, belong to CD4 + T lymphocytes.
The main function of Th17 cells is to recruit and activate neutrophils and induce pro-inflammatory signal pathways. 8 Th17 cells hold high plasticity. In addition to IL-17, Th17 cells also secrete other cytokines under specific inflammation conditions, including TNF-α, IFN-γ, IL-21 and IL- 22. 9 Th17 cells are linked to multiple autoimmune diseases, such as psoriasis, 10 inflammatory bowel diseases, 11 rheumatoid arthritis 12 and SSc. 13 In mouse bleomycin (BLM) model of SSc, the percentages of Th17 cells were positively correlated with skin and lung inflammation scores and fibrosis severity. 14 Th17 cells might promote the proliferation of fibroblasts and enhance cytokine production, thus contributing to fibrosis in SSc model.
For example, Ramstein et al 16 reported that the frequency of IFNγ + IL-17 + Th17 cells was high in sarcoidosis, and their percentage was higher than Th1-cell percentages. In SSc, the levels of serum IL-17 and IFN-γ were significantly increased and showed close association with disease activity. 17 However, whether IFN-γ + IL-17 + Th17 cells were involved in the pathogenesis of SSc remained unclear.
In this study, we aimed to explore the distribution of IFN-γ + IL-17 + Th17 cells in SSc cases and their association with disease severity.
In addition, possible mechanisms of IFN-γ + IL-17 + Th17 cells affecting SSc progression were also investigated.

| Study subjects
Our investigation protocols were approved by the Ethics Committee of First Medical Center of Chinese PLA General Hospital. All eligible participants signed written informed consents before sample collection.
Twenty eligible SSc patients were consecutive dermatological outpatients or inpatients of our hospital. SSc patients were diagnosed based on the classification criteria for SSc proposed by the American College of Rheumatology in 2013. 18 Based on lesion and clinical symptoms, patients with other skin diseases, such as contact dermatitis and psoriasis, were excluded from our study. In addition, 10 healthy individuals were recruited from the Medical Center of our hospital and employed as healthy controls. The healthy individuals had no history of skin diseases. The cases and controls were matched in age and gender. According to disease history, clinical symptoms and pathological changes in skin, the patients were dived into early and non-early stages. 18 Patients in early stage group experienced no treatment of cyclophosphamide shock or methotrexate.
If patients had been treated by prednisone, the maximum dose was less than 30 mg/day. Moreover, their disease duration was less than 5 years. For patients in non-early stage group, the above-mentioned limitations were not applied. The basic features of the patients were collected from their medical records. The disease activity of the patients was estimated according to European Scleroderma Study Group disease activity score for SSc (Valentini disease activity index). 19 Skin involvement among the patients was estimated using the modified Rodnan skin score. 20

| Specimens
Full-thickness skin biopsy specimens (3 cm) were obtained from diseased lesions for the patients. In addition, site-matched normal skin tissues were also obtained from the healthy controls. Then, the obtained skin samples were embedded in paraffin for immunohistochemical analysis.
Peripheral blood samples (5 mL) were collected from both of SSc cases and healthy individuals. Then, the blood samples were diluted using phosphate buffer saline (PBS) and subjected to peripheral blood mononuclear cell (PBMC) isolation via the method of ficoll density-gradient centrifugation. PBMCs were cultured using RPMI media (Sigma Chem. Co.) at 37°C with the presence of 5% CO 2 .

| Dermal fibroblast isolation and culture
Dermal fibroblasts were isolated from skin biopsy according to the descriptions by Philippeos et al 21 Isolated dermal fibroblasts were cultured using DMEM with 10% FBS. Cell medium was incubated in a humid atmosphere at 37°C with the presence of 5% CO 2 . The cell medium was changed every 3 or 5 days; when cell confluent reached 80%, cell passage was performed.

| Co-culture of IFN-γ + IL-17 + Th17 cells and dermal fibroblasts
Dermal fibroblasts were cultured in 48-well plate using complete cell medium, and kept serum starvation for 12 hours prior to coculture. Then, dermal fibroblasts were co-cultured with induced IFN-γ + IL-17 + Th17 cells (1:2 dilution) for 3 days. The cells were harvested for subsequent detection. Dermal fibroblasts cultured with CD4 + T cells without stimulation were adopted as controls. In addition, for IL-21 inhibition experiments, the cultured dermal fibroblasts were pre-incubated using neutralizing antibody (0.1 µg/mL, PeproTech) for 1 hour, and then, co-culture experiments were performed.

| Flow cytometric analyses
To determinate the frequency of IFN-γ + IL-17 + Th17 cells in peripheral blood, flow cytometric analyses were performed. At first, the cells were prepared for cellular cytokine staining. 1 × 10 6

| Immunohistochemistry assay
Formalin-fixed skin tissues were cut into 4 μm-thick sections for histological examinations. At first, the tissue sections were incubated with 3% H 2 O 2 in absolute methanol to quench endogenous peroxidase. After washed three times adopting PBS, the sections were blocked using 2% goat serum. Then, the sections were incubated with specific primary antibodies overnight at 4°C, and adopted antibodies were as follows: anti-IFN-γ antibody (1:100, Abcam) and anti-IL-17 antibody (1:50, Abcam). Subsequently, the sections were washed three times employing PBS and incubated with rabbit secondary antibodies for 2 hours at room temperature. Later, the sections were stained through avidin-biotin method using diaminobenzidine. Five high power microscopes were adopted for each tissue specimen, and the percentages of positive cells were automatically analysed by Image-Pro Plus 6.0 software (Media cybernetics). The positivity was defined as the percentages of positively stained cells in all cells for each high power microscope, and average value for five randomly selected views was calculated for final positivity.

| Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells using TRIzol reagent (Invitrogen), and its quality was estimated utilizing a NanoDrop 2000 ultraviolet spectrophotometer (Thermo Scientific). 5 μg RNA was synthesized into the first chain of cDNA, which was performed using PrimerScript RT reagent kit (Takara). Then, qRT-PCR was car-

| Western blot
Protein samples were isolated from harvested cells using 500 μL radio-immunoprecipitation assay (RIPA) buffer (Thermo Scientific) with 1 mmol/L phenylmethane sulfonyl fluoride. Then, protein samples were obtained through centrifugation. Protein samples of same volume were subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane (PVDF) (0.45 µm pore size; EMD Millipore). Later, the membranes were blocked by 5% skimmed milk and incubated with primary antibodies at 4°C overnight. GAPDH acted as loading control. Then, the membranes were incubated employing secondary antibody at room temperature for additional 2 hours. Blotting results were analysed applying an ECL blotting analysis system (GE Healthcare Life Sciences). Each trial was repeated at least three times.

| Statistical analysis
All data analyses were performed using SPSS 18.0 software (SPSS, Inc), and GraphPad Prism version 5.0 (GraphPad) was adopted for figure plotting. The distribution of continuous variables was estimated using the method of Kolmogorov-Smirnov test. If data were in normal distribution, their comparison between two groups was performed via Student's t test; otherwise, Mann-Whitney U test was adopted. Continuous data were shown by mean ± standard deviation (SD), median and range. Differences in classified variables between groups were analysed using chi-square test. In addition, correlation analysis was performed through Pearson correlation method. P < .05 indicated statistical significance of the results.

| Baseline characteristics of the study participants
There were 20 SSc patients and 10 healthy individuals in our study.
The SSc group included 11 males and 9 females, whose average age was 56.15 ± 12.02 years. There were 5 men and 5 women in healthy control group, with an average age of 53.60 ± 12.04 years. The case and control groups were matched in age (P = .308) and gender (P = .883). In SSc group, 9 (45%) cases were confirmed with limited SSc, and the rest 11 (55%) ones with diffuse SSc. Their average disease duration was 3.40 ± 1.60 years, and their mean Valentini score  Table 1.

| The frequency of IFN-γ + IL-17 + Th17 cells was increased in SSc patients
The frequency of IFN-γ + IL-17 + Th17 cells in peripheral blood was detected for SSc cases and healthy individuals using flow cytometric assay ( Figure 1A). Analysis results demonstrated that compared to healthy individuals, SSc patients showed significantly heightened frequency of IFN-γ + IL-17 + Th17 cells (P < .001) ( Figure 1B). In addition, immunohistochemistry assay was adopted to analyse the number of cells which were positive to IFN-γ and IL-17 staining in collected skin tissues ( Figure 1C).
The percentages of positively stained cells of IFN-γ and IL-17 were significantly higher in SSc specimens than in healthy ones (P < .01) ( Figure 1D).

| Association of IFN-γ + IL-17 + Th17 cell distributions with clinical characteristics of SSc
The association of IFN-γ + IL-17 + Th17 cells' distributions with SSc severity was analysed in our study. We found that patients in non-early stage group exhibited higher frequency of IFN-γ + IL-17 + Th17 cells than those in early stages (P = .031). SSc patients in both groups showed increased frequency of IFN-γ + IL-17 + Th17 cells compared to healthy individuals (P < .001 for both) ( Figure 3A). In addition, correlation analyses were performed to investigate the association of

| IFN-γ + IL-17 + Th17 cells promoted dermal fibroblasts' proliferation and their collagensecreting capacity
Dermal fibroblasts were isolated from SSc skin tissues and cultured in vitro in our study. We co-cultured dermal fibroblasts with IFN-γ + IL-17 + Th17 cells for 3 days and performed immunohistochemistry to investigate the number of dermal fibroblasts which were immunolabelled by α-SMA. Fibroblasts cultured alone were employed as controls. Compared to control group, the number of positively stained cells of α-SMA was significantly increased in co-culture groups (P < .05 for all) ( Figure 5A). Collagen-secreting capacity of fibroblasts was estimated through COL1A1 protein. Western blot analysis was adopted to investigate the expressions of α-SMA and COL1A1. Three days after co-culture, the cells and culture supernatants were harvested for qRT-PCR and Western blot analyses. As displayed in Figure 5B,C, the expressions of α-SMA and COL1A1 were obviously up-regulated at both mRNA and protein levels, revealing increased fibroblast number and enhanced collagen-secreting capacity.

| IFN-γ + IL-17 + Th17 cells contributed to fibrosis through producing IL-21 in SSc
To explore mechanisms underlying the function of IFN-γ + IL-17 + Th17 cells on fibrosis, we investigated cytokine concentration in co-culture medium. In our preliminary experiments, we found that the level of IL-21 was highest in co-culture medium. Moreover, published articles had demonstrated that IL-21 might induce CD4 + T cell differentiation towards Th17 cells 22 and that it also wielded pro-fibrogenic function. 23 Therefore, IL-21 was considered as an effector in our current study. QRT-PCR analysis demonstrated that the mRNA level of IL-21 was significantly increased in co-culture medium of IFNγ + IL-17 + Th17 cells and fibroblasts (P < .001) ( Figure 6A). Moreover, compared to healthy ones, SSc skin specimens showed remarkable up-regulation of IL-21 mRNA (P < .01) ( Figure 6B).
In addition, to further confirm the function of IL-21 on fibrosis progression mediated by IFN-γ + IL-17 + Th17 cells, IFN-γ + IL-17 + Th17 cells were pre-treated with neutralizing anti-IL-21 antibody before co-culture. Then, 3 days after co-culture, we found that the mRNA level of IL-21 was decreased, in comparison with cells without F I G U R E 1 Representative flow cytometric assay for IFN-γ + IL-17 + Th17 cell distributions in peripheral blood for SSc cases and healthy individuals (A). The frequency of IFN-γ + IL-17 + Th17 cells was significantly higher in SSc cases than in healthy individuals (B). Representative immunohistochemistry staining images for IL-17-positive and IFN-γ-positive cells in SSc skin specimens and healthy controls × 250 (C). The percentages of positively stained cells of IFN-γ and IL-17 were significantly higher in SSc specimens than in healthy ones (D). **P < .01, ***P < .001 pre-treatment (P < .001) ( Figure 6C). Moreover, the levels of α-SMA and COL1A1 mRNAs also exhibited a decreasing tendency (P < .01) ( Figure 6D). All data suggested that IL-21 secreted by IFN-γ + IL-17 + Th17 cells played an important role in fibrosis in SSc.

| D ISCUSS I ON
SSc is a fatal disease and exhibits the accumulation of collagen in skin and internal organs, thus resulting in multiple organ fibrosis and high death rate. 24 According to the involvement of organs, multiple antifibrosis treatments have been developed for SSc patients, but their efficacy is unsatisfactory. 25 Because of the unclear aetiology of this disease, it remains a great challenge to identify key triggering events which drive fibrosis progression in SSc. It is generally accepted that the dysregulation of immune system is closely correlated with the progression of SSc. 26 Th17 cells could produce pro-inflammatory cytokines and play important roles in autoimmune diseases, like SSc. 27 In our current study, we explored the function of IFN-γ + IL-17 + Th17 conditions, thus producing IFN-γ. 16 IFN-γ + IL-17 + Th17 cells have been reported to be implicated in several autoimmune disorders, such as sarcoidosis 16 and coronary artery atherosclerosis. 28 However, the distribution of IFN-γ + IL-17 + Th17 cells has been rarely reported. In this study, we found that the percentage of IFN-γ + IL-17 + Th17 cells was significantly higher in SSc blood and skin specimens than in healthy controls. Moreover, the levels of IFN-γ and IL-17 were obviously increased in SSc, compared to healthy controls. In addition, we also found that SSc patients with high percentage of IFN-γ + IL- Th17 cells hold high plasticity, and they could produce diverse cytokines under different inflammation conditions. 33 Lei et al reported that with the presence of IL-21 in SSc mouse model, CD4 + cells could differentiate into Th17 cells. 22 Moreover, increased levels of IL-21 produced by Th17 cells contribute to fibrosis process. 23 Thus, we have been suggested that IL-21 might act as an effector of IFN-γ + IL-17 + Th17 cells in fibrosis process. In our study, we found that the levels of IL-21 were obviously increased in co-culture cell medium blasts, produce multiple cytokines and modify inflammation microenvironment, thus contributing to fibrosis process. 14 In addition to IL-21, IFN-γ + IL-17 + Th17 cells might produce other cytokines to promote fibrosis, but relevant factors were not explored in our study.
Additionally, in vivo experiments are required to verify our results.
In conclusion, the distribution of IFN-γ + IL-17 + Th17 cells is significantly increased in SSc cases and shows positive association with high disease activity score. In vitro, IFN-γ + IL-17 + Th17 cells could promote the proliferation of fibroblasts and enhance their collagen-secreting ability. IFN-γ + IL-17 + Th17 cells may contribute to fibrosis through IL-21 pathway in SSC.

ACK N OWLED G EM ENTS
None.

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
XX conceived and designed the experiments; AL conceived and performed the experiments; YZ prepared figures. HZ wrote the main manuscript text. All authors reviewed the manuscript. With the F I G U R E 6 IL-21 mRNA level was significantly increased in the co-culture medium of fibroblasts and IFN-γ + IL-17 + Th17 cells (A). Moreover, compared to healthy skin, SSc skin also showed upregulated IL-21 mRNA (B). The treatment with IL-21 neutralizer could reduce the level of IL-21 mRNA in co-culture medium (C). In addition, IL-21 neutralizer treatment resulted in the down-regulation of α-SMA and COL1A1 mRNAs (D). **P < .01, ***P < .001 approval of First Medical Center of Chinese PLA General Hospital Ethics Committee, written informed consent was obtained from every patient.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.