Effect of Crosstalk Between Th17 and Th9 Cells on the Activation of Dermal Vascular Smooth Muscle Cells in Systemic Sclerosis and Regulation of Tanshinone IIA

Background: To evaluate the effect of T helper 17 (Th17) cells and Th9 cells on the activation of dermal vascular smooth muscle cells (DVSMCs) in systemic sclerosis (SSc) and regulation of tanshinone IIA. Methods: The expression of interleukin 17 receptor (IL-17R) and interleukin 9 receptor (IL-9R) in the skin of SSc patients was assessed by immunouorescence. The expression of IL-9 and IL-9R mRNA in peripheral blood mononuclear cells (PBMCs) of SSc patients were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The proportion of Th9 cells in PBMCs of SSc patients was sorted by ow cytometry. The effect of IL-9 on the differentiation of Th17 and IL-17 on that of Th9 was detected by ow cytometry. The proportion of Th9 and Th17 cells in SSc patients was detected by ow cytometry. The level of collagen I, III, α-SMA, IL-9R, IL-17R, JNK, P38, and ERK were analyzed using Western blot (WB). Results: Th9 cells were highly expressed in SSc. IL-9 stimulated the differentiation of immature T cells into Th17 cells. IL-17 induced the differentiation of immature T cells into Th9 cells. Tanshinone IIA inhibited the differentiation of immature T lymphocytes into Th17 and Th9. WB showed that the combined action of IL-17 and IL-9 upregulated the inammation and proliferation of DVSMCs. Anti-IL17, anti-IL9, and tanshinone IIA inhibited the functional activation of DVSMCs. Conclusions: The current results suggested that Th17 and Th9 cells induced the activation of DVSMCs in SSc through crosstalk in vitro, and tanshinone IIA inhibited the process. the synergistic effect of IL-9 and IL-17 on functional activation of DVSMCs DVSMCs IL-17, IL-9, IL-17 and IL-9, IL-17 neutralizing antibody and IL-9 neutralizing antibody, IL-17 and IL-9 and tanshinone IIA, SSc serum, SSc serum/IL-17 neutralizing antibody/IL-9 neutralizing antibody, and SSc serum and tanshinone IIA for 3 days. The proteins were collected for WB detection of collagen I, collagen III, α-SMA, P-P38, P38, P-ERK, and ERK. The results showed that IL-17, IL-9, and SSc serum signicantly promote the expression of collagen I, collagen III, α-SMA, P-P38, and P-ERK. In addition, IL-9 and IL-17 neutralizing antibodies and tanshinone IIA inhibited the synergistic effect of IL-9 and IL-17 10).


Background
Systemic sclerosis (SSc) is an autoimmune disease [1,2]. Autoimmune abnormality, vasculopathy, and broproliferative alterations are three hallmark pathological processes in SSc that are responsible for the most severe clinical manifestations of the disease. These modi cations also determine the clinical outcome of the disease and mortality [3]. Vasculopathy and brosis occur in the early stages and exist throughout the course of the disease and are regulated by autoimmunity. Vasculopathy is characterized by vascular endothelial cell in ammation and smooth muscle cell proliferation, which eventually leads to vascular brosis and stenosis [4][5][6]. However, the pathogenesis of SSc-related vasculopathy is not yet understood.
Recent studies have shown an increase in Th17 cells and IL-17 in the skin and peripheral blood of SSc patients [7]. IL-17A induces in ammation of vascular endothelial cells [8]. We also observed that the proliferation and migration of dermal vascular smooth muscle cells (DVSMCs) from media to intima induced by Th17 cells as well as the phenotype transformation from contraction type to synthetic type, which results in over-production of collagen, may play a key role in the pathogenesis of SSc [9][10][11].

However, whether Th17 cells and other lymphocytes act synergistically in inducing the function of
DVSMCs is yet to be elucidated. Recently, IL-9 and IL-9-producing CD4 + T cells (Th9 cells) have been found in SSc, suggesting that IL-9 axis might be involved in SSc pathogenesis [12,13]. Other studies have shown that IL-9 induces the differentiation of Th17 cells [14,15]. Nevertheless, none of the studies have speci cally addressed whether Th17 and Th9 cells interact among each other to promote the functional activation of DVSMCs in the pathogenesis of SSc.
Thus, the present study elaborated on the effect of Th17 and Th9 cells on SSc patient-derived DVSMCs. We also studied the regulatory effect of tanshinone IIA on this process. Interestingly, we observed that Th17 and Th9 cells might induce the activation of DVSMCs in vitro in SSc through "dialogue," and tanshinone IIA alleviates this effect.

SSc patients and healthy controls
A total of 10 patients with SSc (4 men and 6 women) were included in this study after they provided informed consent. All patients were diagnosed according to the 2013 classi cation criteria for SSc and presented different degrees of clinical manifestations of microangiopathy (for example, Raynaud phenomenon, digital ulcers, telangiectasia, pulmonary hypertension, and renal crisis). In addition, 10 ageand sex-matched healthy individuals (5 men and 5 women), who underwent surgery for non-cutaneous disease evaluation, were enrolled in this study. Blood samples were withdrawn from SSc patients and healthy individuals. The skin tissues of SSc patients were obtained to isolate DVSMCs.

Isolationof SSc patient-derived DVSMCs
The skin tissues of the lesions were disinfected using 75% alcohol and excised surgically under sterile conditions, followed by 6-8 washes in sterile phosphate-buffered saline (PBS) and then soaked in 75% alcohol for 5-10 min. Subsequently, the tissues were washed with PBS containing two types of antibiotics (penicillin and streptomycin). The dermal vascular tissue was isolated under a dissecting microscope, minced, and trypsinized. The undigested tissue was removed using a 200-mesh lter and centrifugation for 10 min at 1500 rpm. Finally, the cells obtained were resuspended and added to the smooth muscle cell basal medium.

Tissue immuno uorescence
The skin tissue of SSc patients was trimmed, dehydrated, embedded, sliced, baked, dewaxed and rehydrated. Skin tissue slices were blocked with 1% bovine serum albumin (BSA) for 30 min and incubated with primary antibody overnight at 4 °C. After incubation with the secondary antibody at room temperature for 1 h, the cell nuclei were stained with 49,6-diamidino-2-phenylindole (DAPI) was used to stain the cell nuclei. The images were scanned using a uorescence microscope (Olympus, Tokyo, Japan).

Cell immuno uorescence
DVSMCs were plated in 24-well plates with coverslips. At 90-100% con uency, the cells were stimulated for 24 h, followed by xation with 4% paraformaldehyde for 15 min and permeabilization with 0.5% Triton X-100 for 15 min. Then, the cells were blocked with 1% BSA for 30 min and incubated with primary antibody overnight at 4 ℃. After incubation with a secondary antibody at room temperature for 1 h, DAPI was used to stain the cell nuclei. The images were scanned using a uorescence microscope.
Enzyme-linked immunosorbent assay (ELISA) The concentration of IL-9 was detected using the ELISA kit (MultiSciences, Hangzhou, China) according to the manufacturer's instructions. An equivalent of 100 μL standard protein samples and cell culture supernatants were added and incubated for 2 h at room temperature. After washing 4-6 times, each well was incubated for 1 h with 50 μL primary IL-9 antibody and then with 100 μL of enzyme-labeled antibody for 1 h at room temperature. After the termination of the reaction, the OD was measured at 450 nm maximum absorption and 630 nm wavelength. The calibrated OD value was obtained by subtracting the 630-nm value from that of 450 nm. The IL-9 concentration data were presented as mean ± SD values. Peripheral blood mononuclear cells (PBMCs) and CD4 + T cells sorting and ow cytometry detection PBMCs were obtained by density gradient centrifugation using Ficoll-Hypaque were cultured in RPMI 1640 medium supplemented with 10% FCS and antibiotics from SSc patients and healthy controls. CD4 + T cell subset was sorted using magnetic-activated cell sorting (MACS). The induced cells were incubated with FITC-labeled anti-human CD4 antibody at 4 °C for 30 min. In order to further analyze the production of IL-9 or IL-17, the cells were permeated with permeable buffer and stained with PE-conjugated antihuman IL-9 or anti-human IL-17 at room temperature for 30 min. At the end of the staining, all stained cells were analyzed by ow cytometry.

Western blot
The proteins of DVSMCs were extracted using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with the protease inhibitor phenylmethanesulfonyl uoride uoride (PMSF). The proteins were separated by 8% SDS-PAGE and transferred onto PVDF membranes. Subsequently, the membranes were blocked with 5% milk for 2 h at room temperature, probed with primary antibodies, including collagen I (

Statistical analysis
The data were statistically analyzed using the SPSS version 20.0 software (SPSS Inc., Chicago, IL, USA).
Quantitative data are expressed as the means ± standard deviation (SD). Data between the patients and healthy individual groups were analyzed using Student's t-test. Data among multiple groups were analyzed using one-way analysis of variance (ANOVA) combined with Bonferroni correction. P-value <0.05 was statistically signi cant.

Correlation between Th9 and SSc
Compared to normal subjects, IL-9R and IL-17R were highly expressed in the skin lesions of SSc patients (Fig. 1). IL-9 and IL-9R mRNA levels of PBMCs and IL-9 levels in SSc serum were increased (Fig. 2). Flow cytometry showed that the proportion of CD4 + IL-9 + T cells in PBMCs of SSc patients was signi cantly higher (Fig. 3). The proportion of Th9 was associated with SSc.
2. Crosstalk between Th17 and Th9 cells in vitro and regulation of tanshinone IIA 2.1 IL-9 and SSc serum promote Th17 differentiation and the reversal effect of tanshinone IIA The results showed that the proportion of Th17 cells in IL-9 and SSc serum groups was signi cantly higher than that in the control group; however, the proportion of Th17 cells in the IL-9 neutralizing antibody group was signi cantly lower than that in the control group, and IL-9 neutralizing antibody decreased the promoting effect of serum on Th17 cells (Fig. 4A). These ndings indicated that IL-9 stimulates the differentiation of immature T lymphocytes into Th17 cells in vitro, as well as the differentiation of immature T lymphocytes into Th17 cells. The data showed that tanshinone IIA decreases the proportion of Th17 and inhibits the effect of SSc serum on Th17 cells as compared to the control (Fig. 4B). Moreover, the content of IL-17 in the culture supernatant of each group was detected by ELISA. These results showed that IL-9 and SSc serum signi cantly promotes the secretion of IL-17 by immature T lymphocytes, while IL-9 neutralizing antibody and tanshinone IIA signi cantly inhibits the secretion of IL-17 by immature T lymphocytes and reverses the triggering effect of IL-9 and SSc serum (Fig. 4C).

IL-17 and SSc serum promote Th9 differentiation and the reversal effect of tanshinone IIA
The results showed that the proportion of Th9 cells in the IL-17 and SSc serum groups was signi cantly higher than that in the control group; however, the proportion of Th9 cells in IL-17 neutralizing antibody group was signi cantly lower than that in the control group and IL-17 neutralizing antibody decreased the promoting effect of serum on Th9 cells (Fig. 5A). These ndings indicated that IL-17 stimulates the differentiation of immature T lymphocytes into Th9 cells in vitro, and IL-17 in the serum of SSc patients stimulates the differentiation of immature T lymphocytes into Th9 cells. In addition, tanshinone IIA decreases the proportion of Th9 and inhibits the promoting effect of SSc serum on Th9 cells as compared to the control (Fig. 5B). Moreover, the content of IL-9 in the culture supernatant of each group was detected by ELISA. The results showed that IL-17 and SSc serum signi cantly promoted the secretion of IL-9 by immature T lymphocytes, while IL-17 neutralizing antibody and tanshinone IIA signi cantly inhibited the secretion of IL-9 by immature T lymphocytes and reversed the promoting effect of IL-17 and SSc serum (Fig. 5C).
3. Effects of IL-17 and IL-9 on the functionalactivation of DVSMCs and regulation of tanshinone IIA 3.1 IL-9 and SSc serum promote the expression of IL-17R in DVSMCs, and tanshinone IIA reverses this effect The DVSMCs of SSc patients were isolated and incubated with IL-9, IL-9 neutralizing antibody, SSc serum, SSc serum and IL-9 neutralizing antibody, tanshinone IIA, IL-9 and tanshinone IIA, and SSc serum and tanshinone IIA for 3 days. The expression of IL-17R was detected by immuno uorescence. The results showed that IL-9 and SSc serum signi cantly promote the expression of IL-17R, and IL-9 neutralizing antibody and tanshinone IIA reversed the promoting effect of IL-9 and SSc serum (Fig. 6).

IL-17 and SSc serum promote the expression of IL-9R in DVSMCs, and tanshinone IIA reverses this effect
The DVSMCs were incubated with IL-17, IL-17 neutralizing antibody, SSc serum, SSc serum and IL-17 neutralizing antibody, tanshinone IIA, IL-17 and tanshinone IIA, and SSc serum and tanshinone IIA for 3 days. The expression of IL-9R was detected by immuno uorescence. The results showed that IL-17 and SSc serum signi cantly promote the expression of IL-9R, and IL-17 neutralizing antibody and tanshinone IIA reversed the promoting effect of IL-17 and SSc serum (Fig. 7).

IL-9 and SSc serum promote the functional activation of DVSMCs, and tanshinone IIA reverses this effect
The DVSMCs were incubated with IL-9, IL-9 and IL-9 neutralizing antibodies, IL-9 and tanshinone IIA, SSc serum, SSc serum and IL-9 neutralizing antibodies, and SSc serum and tanshinone IIA for 3 days. Collagen I, collagen III, α-SMA, P-P38, P38, P-ERK, and ERK were detected by WB. The results showed that IL-9 and SSc serum signi cantly promote the expression of collagen I, collagen III, α-SMA, P-P38, and P-ERK. Moreover, IL-9 neutralizing antibody and tanshinone IIA reversed the effects of IL-9 and SSc serum (Fig. 8).

IL-17 and SSc serum promote the functional activation of DVSMCs, and tanshinone IIA reverse this effect
The DVSMCs were incubated with IL-17, IL-17 and IL-17 neutralizing antibodies, IL-17 and tanshinone IIA, SSc serum, SSc serum and IL-17 neutralizing antibodies, and SSc serum and tanshinone IIA for 3 days. Collagen I, collagen III, α-SMA, P-P38, P38, P-ERK, and ERK were detected by WB. The results showed that IL-17 and SSc serum signi cantly promote the expression of collagen I, collagen III, α-SMA, P-P38, and P-ERK. IL-17 neutralizing antibody and tanshinone IIA reversed the effects of IL-17 and SSc serum (Fig. 9).

Discussion
Th9, a de ned subset of T helper cells, has been identi ed by the potent production of IL-9 [16], which is a protein of 144 amino acids with a secretory signal sequence of 18 amino acids. Interestingly, IL-9 binds to IL-9R on target cells [17]. Although the role of Th9 cell subset has been described recently in many types of in ammatory diseases (namely, atopic diseases, parasitic in ammation, experimental autoimmune encephalomyelitis, and in ammatory bowel diseases) [18][19][20][21][22][23], the role in the pathogenesis of rheumatic diseases, such as SSc, is yet unknown.
The presence of high IL-9 levels and the strong expression of Th9 in SSc raise the concern of whether Th9 cells might participate in SSc pathogenesis. In this study, the strong expression of IL-9R has been demonstrated recently in the skin tissue of patients with SSc. Also, IL-9 expression was observed in the PBMCs from SSc patients. The majority of IL-9-producing cells in the skin were identi ed as Th9 cells. Among peripheral blood mononuclear cells, Th9 cells are the major source of IL-9, which is signi cantly expanded in SSc patients as compared to the controls. The participation of IL-9 and Th9 in the process of in ammation and brosis in SSc is yet elucidated.
Our previous research has con rmed that IL-17 and Th17 enhance the proliferation, collagen synthesis and secretion, and migration of SSc patient-derived DVSMCs via mitogen-activated protein kinase (MAPKs) signaling pathway [9]. In addition, the previous study also demonstrated that tanshinone IIA (C19H18O3), a fat-soluble pharmacologically active component of the Chinese herb Sativa miltiorrhiza that is a well-known traditional Chinese medicine used for the treatment of cardiovascular and connective tissue diseases, exerts an inhibitory effect on the functional activation of SSc patient-derived DVSMCs [10].
The present study focuses on the co-effects of Th17 and Th9 cells on SSc patient-derived DVSMCs. We also studied the regulatory effect of tanshinone IIA on this process. Th17 and Th9 promote each other's differentiation from immature T lymphocytes via characteristic cytokines IL-17 and IL-9. IL-17, IL-9, and SSc serum promoted the expression of collagen I, collagen III, α-SMA, P-P38, and P-ERK, inducing the functional activation of DVSMCs. Increased collagen synthesis and secretion of DVSMCs indicates the aggravation of angiopathy and brosis in SSc. Nevertheless, IL-17 and IL-9 neutralizing antibodies and tanshinone IIA reverse the effects.

Conclusion
The results of this study revealed the effect of T cell interaction on the functional activation of SSc VSMCs. Thus, the correlation between cellular immune abnormality and angiogenesis in the pathogenesis of SSc is detected, which provides a theoretical and experimental basis for SSc to nd new therapeutic targets and drugs.

Consent for publication
All the authors provided consent for the publication of this article.

Availability of data and materials
Data and materials were all available.

Competing interests
We con rm that all the listed authors have participated actively in the study, and have seen and approved the submitted manuscript. The authors do not have any possible con icts of interest.    The proportion of CD4+IL-9+ T cells in PBMCs of SSc patients was signi cantly higher than that of healthy controls.  IL-9 neutralizing antibody and Tanshinone IIA inhibit the differentiation of immature T lymphocytes into Th17. A and B. The proportion of immature T lymphocytes differentiated into Th17 in each group was detected using ow cytometry. C. The content of IL-17 secreted by immature T lymphocytes in each group was detected by ELISA. IL-9 concentration 20 ng/mL, anti-IL-9 concentration 5 μg/mL, Tanshinone IIA concentration 50 μM. * vs. control group, P<0.05, *** vs. control group, P<0.001, ## vs. IL-9 group, P<0.01, ### vs. IL-9 group, P<0.001. Figure 5 IL-17 neutralizing antibody and Tanshinone IIA inhibit the differentiation of immature T lymphocytes into Th9. A and B. The proportion of immature T lymphocytes differentiated into Th9 in each group was detected using ow cytometry. C. The content of IL-9 secreted by immature T lymphocytes in each group Page 17/21 was detected by ELISA. IL-17 concentration 100 ng/mL, anti-IL-17 concentration 8 μg/mL, Tanshinone IIA concentration 50 μM. * vs. control group, P<0.05, *** vs. control group, P<0.001, ### vs. IL-9 group, P<0.001.