IL-17 Upregulates MCP-1 Expression via Act1 / TRAF6 / TAK1 in Experimental Autoimmune Myocarditis

Myocarditis is a myocardial inammatory inltration heterogeneous disease. At present, various interventions are not effective in the treatment of myocarditis. IL-17, an important pro ‐ inammatory factor secreted mainly by Th17 cells, can promote the expression of multiple cytokines. MCP-1 is an important cytokine that mediates mononuclear cell inltration. Studies have found that IL-17 could stimulate the expression of MCP-1 to mediate inammatory inltration. But the mechanism by which IL-17 induces MCP-1 expression in experimental autoimmune myocarditis (EAM) remains unclear. The purpose of this study is to establish an EAM model to explore the role of Act1/TRAF6/TAK1 cascade in the induction of MCP-1 by IL-17. In the present study, we found that in EAM, IL-17 could stimulate the expression of MCP-1 by activating Act1/TRAF6/TAK1 cascade. After interfering TAK1 with si-TAK1, myocardial tissue inammation was greatly alleviated, and both MCP-1 mRNA and protein expression were downregulated. In conclusion, IL-17 can activate AP-1, NF-κB via Act1/TRAF6/TAK1 upregulation of MCP-1 expression in EAM. Studies have shown that TAK1 could activate NF-κB and AP-1, and then mediate the expression of the target gene. [21] . To assess the role of Act1/TRAF6/TAK1 pathway on IL-17-mediated inammatory inltration, we treated EAM mice with si-TAK1 and chose 21 day as the analysis time point. We found that the inammatory inltration was reduced in the si-TAK1 group, but it was still higher than that in the control group (P < 0.05). We next tested the mRNA expression and protein content of MCP-1. The results showed that the mRNA and protein levels of MCP-1 was down-regulated after blocking the signaling pathway (P < 0.05). Later, we tested the mRNA and protein levels of NF-κB and AP-1, and found that the levels of NF-κB and AP-1 was signicantly inhibited in EAM mice treated with TAK1 siRNA (P < 0.05).


Introduction
Myocarditis is a myocardial disease resulting from many causes, including microbial infection, autoantigens, drugs. The incidence of myocarditis is increasing year by year [1] . EAM is a myocardium in ammation mediated by CD4 + T cells. The pathological features of EAM were in ammatory cell in ltration, myocardial cell necrosis and collagen deposition in cardiac broblasts and myo broblasts [2] .
Studies have shown that persistent in ammation of the myocardium can lead to myocardial remodeling, which could evolve into dilated cardiomyopathy (DCM) with heart failure [1][2][3] .
Interleukin-17(IL -17), because of its HVS13 herpes virus gene showed high homology, originally called CTLA-8. It was renamed IL-17, when it was rst cloned in 1993 from the mouse activated T cell hybridoma [4] . Th17 cells, as a newly discovered subset of CD4 + T cells, are involved in many in ammatory diseases and are the main source of IL-17 [5] . IL-17 could bind to IL-17R to promote the expression of several chemokines and participate in several physiopathological processes, including immunologic defense, in ammatory in ltration and the progression of cancer [6] . Studies have shown that treatment of mice with viral myocarditis using IL-17-neutralizing antibody signi cantly reduced myocardial in ammatory in ltration [7] . Monocyte chemokine protein 1 (MCP-1) is a major chemokine of mononuclear cells in vivo, which can drive monocytes or macrophages to gather in the direction of in ammation [8] . Our previous study found that MCP-1 was upregulated by IL-17 in cardiomyocytes, however, how IL-17 regulated MCP-1 expression remains unclear. [9] .
It is found that after IL-17 is combined with IL-17R, IL-17R could recruit Act1 with the same domain through its SEFIR domain [10][11] . In addition, Act1 has a TRAF6 binding domain, which can bind downstream TRAF6 molecules and cause their ubiquitin activation [11][12] . Tang et al. found that the activation of TRAF6 then activated the P38 MAPK signaling pathway of human periodontal membrane cells [13] . TGF-β activated kinase 1 (TAK1) is a member of the MAP3K family, is considered to be an important regulatory downstream regulator of TRAF6 and can mediate the activation of downstream IKK kinases and MAPK signaling pathways. IKK can regulate the activation of NF-κB through IκB phosphorylation, and MAPK signal transduction pathway is the key pathway to activate transcription factor AP-1. [14][15] . Huang et al. demonstrated that IL-17A could activate Act1/TRAF6/TAK1 axis to mediate NF-κB activation, which regulates CXCL2 expression in airway epithelial cells. [16] . Meanwhile, studies showed that IL-17 could activate AP-1 in chondrocytes to stimulate collagenase 3 expression [17] .
Furthermore, activated AP-1 can upregulate the expression of MCP-1 in carcinoma cells [18] . Therefore, we speculate that IL-17 can mediate the expression of MCP-1 in EAM by activating Act1/TRAF6/TAK1 signal pathway. Based on the above ndings, we intend to establish an EAM mouse model to explore the role of IL-17 in regulating MCP-1 expression through Act1/TRAF6/TAK1 in the pathogenesis of EAM.

Establishment of the EAM model
Male BALB/c mice (18-22g) were purchased from Henan Experimental Animal Research Center. The animal experiments were approved by the Animal Experiment Committee of Zhengzhou University. For induction of EAM, the mice were subcutaneously injected with 200µg of the mixture of cardiac myosin polypeptide (MyHC-α614−629 :SLKLMATLFSTYAS)(GL Biochem, China) with CFA (Sigma-Aldrich, USA) on day 0 and day 7 [19] . The control group was given an equal volume of saline and CFA mixture.

Histology and Immunohistochemistry
The hearts were immersed in 4% paraformaldehyde, covered in para n and cut into slices. Then hematoxylin-eosin staining was used to observe and take pictures under the microscope. Double-blind scoring was performed according to the area percentage of in ammation. The score of myocarditis was divided into 4 grades. 0 grade was no in ammatory cell in ltration; in grade 1, the in ammatory area was less than 25%; in grade 2, the in ammatory area was 25-50%; in grade 3, the in ammatory area was 50% − 75%; in grade 4, the in ammatory in ltration was more than 75%. For immunohistochemistry (IHC), para n sections were dewaxed, hydrated and blocked. Then MCP-1 and IL-17 antibodies (A nity, China) were incubated at 4 ℃. On the next day, the primary antibodies were washed off with PBS, then incubated with secondary antibody for 1 hour. After washing, DAB was used as dyeing substrate, and sections were re-stained with hematoxylin. The IHC sections were observed with a microscope and analyzed with Image J software.

Flow cytometry
The spleens cells of mice were analyzed by ow cytometry on day 14, day 21, day 28 post the rst immunization. Mice spleen cells were prepared according to the previous procedure [20] . To nd Treg cells, we rst stained the surface with FITC-CD4 antibody and Percy-CY5.5-CD25 antibody (Ebioscience, USA). For Th17 cells, we stimulated the cells with 50ng/mL phorbol ester (Solarbio, China) and ionomycin (Solarbio, China) of 1mg/mL, and then added the Golgi blocker monensin (Solarbio, China) 0.7ug / mL, incubated at 5% CO2 at 37 ℃ for 4 hours, and stained the surface with FITC-CD4 antibody (Ebioscience, USA). Then, the cells were xed and broken according to the instructions of the kit (Multi science, China).
Next, Treg cells were labeled with PE-Foxp3 antibody and Th17 cells were labeled with APC-IL-17 antibody (Ebioscience, USA). After incubation, the samples were measured by Navios (Beckman, USA).

Enzyme-linked immunosorbent assay
The expression of IL-17 and MCP-1 in mice serum was tested by enzyme-linked immunosorbent assay (ELISA). After taking blood from mice eyeballs, the serum was extracted and detected by IL-17 and MCP-1 ELISA kits (Elabscience, China).
Quantitative RT-PCR Total RNA was isolated according to the procedure of the RNA extraction kit (Takara, Japan) and transformed into cDNA by the reverse transcriptase kit. (TOYOBO, Japan). The PCR primer sequences are shown as below: Reverse 5′-GTCTGGGATGGAAATTGTGA-3′  The relative expression of genes was expressed as a ratio of gene expression and GAPDH expression.
Each assay was performed three times.

Western Blotting
Total protein was extracted with RIPA lysate (Solarbio, China) containing phenyl methane sulfonyl uoride (PMSF) (Solarbio, China) and quanti ed. Then, the protein was separated by 10% SDS-PAGE gel electrophoresis and transferred to membranes (Epizyme, China). The membrane was incubated with speci c antibodies at 4°C. On the next day, the membrane was washed off by TBST, then incubated with secondary antibodies, washed and colored. The optical density of the strips was analyzed by Image J software and calculated the average gray value of each strip.

Statistics
The results were analyzed by SPSS 22.0 software. Data were presented as the mean±SD. Comparisons among multiple groups were performed by one-way ANOVA, and comparisons among groups were performed by LSD-t test, with a test level of α = 0.05.

Establishment of EAM model
In order to explore the progression of EAM, we set three different time points. We found that the heart weight of EAM mice was greatly increased on days 14, 21 and 28 post induction (P < 0.05); the heart weight/body weight ratio of the EAM was also higher than the control (P < 0.05). Then we analyzed the results of HE staining in heart and found that the in ammatory cells began to in ltrate on 14 day, reached the peak on 21 day, and began to decline on 28 day; no in ammatory cell was observed in the control group. At the same time, we made a double-blind score on the degree of in ammation. We found that the pathological score of the EAM group was greatly higher than that of the control group (P < 0.05), and reached the highest value on day 21.
Expression of IL-17 and MCP-1 in EAM Subsequently, we explore the expression of IL-17 and MCP-1 in EAM. First, we performed IHC of the heart. As shown in Fig. 3, positive cells began to appear in the myocardium of EAM mice on 14 day. A high number of positive cells appeared in myocardial in ammatory lesions and stroma on 21 day, and decreased on 28 day. No positive cells were found in the control group at all times. Next, we analyzed the optical density and found that the EAM group had higher optical density of IL-17 and MCP-1 compared with the control group (P < 0.05), and the optical density of IL-17 and MCP-1 expressed on 21 day was higher than those on 14 day and 28 day(P < 0.05). Then, we extracted myocardial mRNA for qRT-PCR and found that the expression of IL-17 and MCP-1 mRNA in EAM mice increased on 14 day, reached the peak on 21 day, and decreased on 28 day(P < 0.05). Consistent with the IHC results, the expression levels of IL-17 and MCP-1mRNA in group EAM were greatly higher than those in the control group (P < 0.05). Meanwhile, we used ELISA kit to detect the levels of IL-17 and MCP-1 in serum. We found that the levels of IL-17 and MCP-1 were signi cantly increased in the EAM group (P < 0.05).

The proportions of Th17 cells and Treg cells in EAM
In order to further investigate the relationship between IL-17 and EAM, we tested the proportion of Th17 cells in the spleen of mice. In line with IL-17 expression, the proportion of Th17 cells greatly increased in the EAM group (P < 0.05), and reached the peak on 21 day. In parallel, we also analyzed the proportion of Treg cells. As the antagonistic cells of TH17 cells, the proportion of Treg cells in EAM group decreased signi cantly (P < 0.05). Then, we analyzed the correlation between Th17 cells, Treg cells and the in ammatory in ltration of EAM. The results further con rmed that Th17 cells were positively correlated with the in ammatory in ltration of EAM, and Treg cells were negatively correlated with the in ammatory in ltration of EAM(P < 0.05).

The signaling pathway activated by IL-17 in EAM
In order to study the signal transduction mechanism and transcription factors of MCP-1 secreted by myocardial tissue induced by IL -17, mRNA was extracted from mouse myocardial tissue to detect the expression of Act1, TRAF6, TAK1, p65 and c-jun. As shown in Fig. 7, the mRNA levels of Act1, TRAF6, TAK1, p65 and c-JUN were upregulated in the EAM group (P < 0.05). Interestingly, we found that the change trend at the gene expression was consistent with the change trend in in ammation. At the same time, we tested all the molecules from the protein level, and found that compared with the control group, the protein expressions of Act1, TRAF6, TAK1, p-p65 and p-c-Jun were greatly increased (P < 0.05). Those suggested that IL-17 could activate Act1/TRAF6/TAK1 axis to up-regulate the expression of MCP-1 in EAM.

Blocking TAK1 diminishes immune cell in ltration during EAM
Studies have shown that TAK1 could activate NF-κB and AP-1, and then mediate the expression of the target gene. [21] . To assess the role of Act1/TRAF6/TAK1 pathway on IL-17-mediated in ammatory in ltration, we treated EAM mice with si-TAK1 and chose 21 day as the analysis time point. We found that the in ammatory in ltration was reduced in the si-TAK1 group, but it was still higher than that in the control group (P < 0.05). We next tested the mRNA expression and protein content of MCP-1. The results showed that the mRNA and protein levels of MCP-1 was down-regulated after blocking the signaling pathway (P < 0.05). Later, we tested the mRNA and protein levels of NF-κB and AP-1, and found that the levels of NF-κB and AP-1 was signi cantly inhibited in EAM mice treated with TAK1 siRNA (P < 0.05).

Discussion
Myocarditis is an in ammatory disease of the cardiovascular system with a predilection for adolescents and children that may eventually lead to DCM and heart failure. Chronic in ammation caused by virus infection or autoimmune reaction is the main cause of DCM [1,22] . MCP-1 is an important in ammatory factor in vivo, which could mediate the in ltration of mononuclear cells. [23] . For example, in patients with bronchial asthma, MCP-1 can chemotaxis monocytes and macrophages to the alveoli, leading to airway in ammation and remodeling [24][25] . Our previous study also found that MCP-1 can lead to a large number of in ammatory cell aggregation through its receptor CCL2 in viral myocarditis [26] . Therefore, it is speculated that reducing the expression of MCP-1 is of great signi cance for the occurrence and development of myocarditis.
In previous studies, we found that IL-17 could up-regulate MCP-1 and aggravate cardiomyocyte injury [9] . In this study, we explored the relationship between IL-17, MCP-1, and myocarditis. We performed IHC of mouse hearts by constructing an EAM mouse model. The results showed that with the development of EAM, both IL-17 and MCP-1 levels increased and peaked on 21 day, and decreased on 28 day. Meanwhile, we also measured its mRNA expression and its content in serum, and consistent results were obtained. It is suggested that IL-17 and MCP-1 are involved in the in ammatory in ltration process of EAM.
Th17 cells are helper T cells that differentiate in response to stimulation by IL-6 and IL-23 [27] . Evidence has proved that Th17 cells are associated with many autoimmune diseases, including Sjogren's syndrome, rheumatoid arthritis, Scleroderma, and so on [28][29] . Conversely, Treg cells, as indispensable cells for immune homeostasis, can play suppressive roles in a variety of autoimmune diseases [29][30] .
From our analysis, we found that the proportion of Th17 cells in EAM group increased greatly, while the opposite result was found for Treg cells. We further performed a correlation between the proportion of Th17 cells, Treg cells and the degree of in ammatory in ltration. The analysis showed that the severity of EAM was closely related to the proportion of Th17 cells and Treg cells. Those results suggested that the Th17 cells/Treg cells ratio may be used as a new therapeutic target for EAM to ameliorate myocardial in ammatory in ltration, and also further proved that IL-17 plays a key role in the in ammatory in ltration of EAM. But in EAM, by which signal IL -17 up-regulates MCP -1 expression in myocardial tissue, it remains unclear.
It has been documented that the intracellular region of IL-17R contains a SEFIR domain. Interestingly, Act1 contains both TRAF6 domains and a SEFIR domain. And Act1 is also the only one to contain a SEFIR domain in addition to the IL-17R family. Act1 can bind to a variety of TRAFs, and at the same time, it acts on cell receptors containing the SEFIR domain, such as IL-17R, through the interaction of the SEFIR-SEFIR domain [11,31−32] . Liao et al reported that TRAF6 could aggravate cardiac hypertrophy through TAK1 dependent signaling [33] . Tadashi et al found that IL-17A could activate the Act1/TRAF6/TAK1 signaling pathway to regulate cardiomyocyte contractility and death [34] . Therefore, we speculated that IL-17 regulates MCP-1 expression by activating Act1/TRAF6/TAK1 axis in EAM. In the present study, our data also support the hypothesis that the up-regulation of MCP-1 by IL-17 is dependent on the Act1/TRAF6/TAK1 signaling pathway. Promoters are DNA sequences that play an initiating role during transcription. Our previous studies revealed that MCP-1 promoter sequence contains NF-κB, AP-1 binding site [16,35−36] . Therefore, we further explored whether TAK1 activates NF-κB and AP-1 to upregulate MCP-1 expression. We found that the levels of NF-κB and AP-1 increased signi cantly in EAM group, and the elevation was correlated with the degree of in ammatory in ltration. This suggests that in EAM, IL-17 may regulate MCP-1 expression through the Act1/TRAF6/TAK1 cascade to mediate in ammatory cell in ltration.
In conclusion, this study shows that IL-17 regulates the in ammatory in ltration of EAM by regulating the expression of MCP-1 through Act1 / TRAF6 / TAK1, which will help to clarify the pathological mechanism of myocarditis and provide a new direction for clinical practice.

Con icts of interest
The authors declare no con icts of interest. Availability of data and material All data generated or analysed during this study are included in this published article.

Code availability
Not applicable for this section.

Authors' contributions
Xiao Huang designed the study, analyzed the data and revised the manuscript. Zhuolun Li analyzed the results and revised the manuscript. Xinhe Shen revised the manuscript. Na Nie performed the ELISA. Yan Shen designed the study, wrote the manuscript, and revised it.

Ethics approval
The animal experiments were approved by the Animal Experiment Committee of Zhengzhou University.

Consent to participate
Not applicable for this section.

Consent for publication
Not applicable for this section.      The correlation between Th17 cells, Treg cells and myocarditis score. a) correlation between Th17 cells and myocarditis score; b) correlation between Treg cells and myocarditis score.