Role of Interleukin 17A in Aortic Valve Inflammation in Apolipoprotein E-deficient Mice

Interleukin 17A (IL17A) is reported to be involved in many inflammatory processes, but its role in aortic valve diseases remains unknown. We examined the role of IL17A based on an ApoE−/− mouse model with strategies as fed with high-fat diet or treated with IL17A monoclonal antibody (mAb). 12 weeks of high-fat diet feeding can elevate cytokines secretion, inflammatory cells infiltration and myofibroblastic transition of valvular interstitial cells (VICs) in aortic valve. Moreover, diet-induction accelerated interleukin 17 receptor A (IL17RA) activation in VICs. In an IL17A inhibition model, the treatment group was intra-peritoneally injected with anti-IL17A mAb while controls received irrelevant antibody. Functional blockade of IL17A markedly reduced cellular infiltration and transition in aortic valve. To investigate potential mechanisms, NF-κB was co-stained in IL17RA+ VICs and IL17RA+ macrophages, and further confirmed by Western blotting in VICs. High-fat diet could activate NF-κB nuclear translocation in IL17RA+ VICs and IL17RA+ macrophages and this process was depressed after IL17A mAb-treatment. In conclusion, high-fat diet can lead to IL17A upregulation, VICs myofibroblastic transition and inflammatory cells infiltration in the aortic value of ApoE−/− mice. Blocking IL17A with IL17A mAb can alleviate aortic valve inflammatory states.

Aortic valve stenosis remains as the most prevalent valvular disease in Western countries [1] , affecting over 25% of all patients over the age of 65 [2] . Currently, there is no effective pharmacological therapy for aortic valve stenosis and surgical or interventional valve replacement serves as the only curative treatments. However, in patients who underwent transcatheter aortic valve replacement, up to 30% had oxygen dependence [3] and 60% had significant lung disease, which has been associated with increased morbidity and mortality [4] . Others reported unexpected side effects including chronic kidney disease and liver disease, all of which are considered to contribute to deaths after aortic valve surgery [5] .
Inflammation plays a key role in aortic valve stenosis, ultimately leading to aortic valve calcification. Low density lipoprotein (LDL) cholesterol has also been suggested to underlie the progression of inflammatory aortic valve stenosis [6] . High-fat diet (HFD) can induce significant aortic valve calcification in ApoE -/mice [7] , significantly increasing transvalvular peak jet velocity and markedly decreasing aortic valve area (AVA) and AVA index. Similarly, significant calcium deposits in aortic valve of HFD-treated ApoE -/mice by alizarin red staining [7] . Currently, ApoE -/mice fed with HFD are the common animal models for aortic valve inflammation study [7][8][9] .
In recent years, interleukin 17A (IL17A) was reported to coordinate local tissue inflammation via inducing the release of proinflammatory cytokines and neutrophil-mobilizing chemokines in several cell families [10,11] . And recent studies implicate that IL17A can also accelerate atherosclerotic plaque development via its receptor IL17RA [12,13] , and vascular inflammation decreased in the IL17A-deficient mice [14] . Besides, IL17A is primarily produced by Th17 cells, a subset of CD4 + T cells, and Th17 cells have also been observed in atherosclerotic plaques both in human and animals [15] .
These findings have stroked our view for uncovering the character of IL17A/IL17RA performance in aortic valve inflammation.
We therefore sought to accurately examine the role of IL17A in aortic valve inflammation using HFD mouse models, and blocking IL17A with an anti-IL17A antibody (mAb) in ApoE -/mice.

Affymetrix Microarray Data and Data Analysis
Microarray data of GSE77287 were obtained from the Gene Expression Omnibus (GEO; http://www. ncbi.nlm.nih.gov/geo/) database. Calcific aortic valve tissues were extracted from patients with calcific aortic valve disease (CAVD) who underwent aortic valve replacement, and normal controls were obtained from non-CAVD cardiac transplant recipient hearts [16] . Gene expression profiles of three samples of calcific aortic valves and three age-matched normal controls were performed using Affymetrix Gene Chip microarrays [16] . The raw data and annotation files were downloaded for subsequent analysis, based on Affymetrix Human Gene 2.0 ST Array. The original data were preprocessed with background correction, normalization and calculating expression using the 'affy' package in R (version 3.4.2). Heatmap and PPI network were applied as indicated [17] .
All the mice were sacrificed at 20th week, and aortic valves were collected and stored at -80°C until cutting. All the animal procedures were performed according to the Helsinki Declaration and institutional guidelines at the laboratory animal center of Huazhong University of Science and Technology.

Quantitative Assessment of Colocalization
Intensity correlation analysis (ICA) [18] was performed on immunofluorescent images to assess the percentage of labelled IL17A + CD4 + T cells (Th17 cells), IL17RA + CD4 + T cells, IL17RA + VICs and IL17RA + macrophages in aortic valves. Briefly, WCIF Image J software was used to determine colocalized red and green pixel areas. We obtained intensity correlation quotient (ICQ), ∑N(A i -a)(B i -b) value, with dependent staining 0<ICQ≤+0.5. The percentage of colocalization was calculated relative to total aortic valve area for that field. Online manual for correlation analysis is referred to http://wwwfacilities. uhnresearch.ca/wcif/imagej/colour_analysis.htm.

Plasma Lipid and ELISA Analysis
Total plasma cholesterol and triglycerides were enzymatically measured using the Cholesterol/ Triglyceride Assay Kit (Roche-Hitachi, Switzerland) according to the manufacturer's instructions. Serum IL17A and other cytokines levels were measured with IL17A, IL6 and TNF-α ELISA (Elabscience, China) according to the manufacturer's protocols.

Cell Harvest and Western Blotting Analysis
VICs were isolated from murine aortic valve leaflets and cultured as previously described with some modifications [16] . Briefly, aortic valve leaflets were digested in collagenase (2.5 mg/mL in M199 medium) for 30 min at 37°C. After removing immune and endothelial cells by vortex, a milder solution of collagenase medium was used (0.8 mg/mL) for 3 h at 37°C. Cells were collected by centrifugation at 1200 × g for 5 min, and then resuspended and cultured in DMEM containing 4.5 g/L glucose, penicillin G, streptomycin, and 10% fetal bovine serum. Cells were harvested and homogenized in RIPA Lysis and Extraction Buffer with protease and phosphatase inhibitor cocktails (Pierce, USA). Nuclear proteins were prepared using NE-PER Nuclear Extraction Reagents (Pierce, USA). All these procedures were performed according to the manufacturer's protocols. Equal amounts of protein were resolved by SDS-PAGE (10% resolving gel with 4% stacking) and transferred to PVDF membranes. Membranes were blocked with buffer containing 10% non-fat milk and 5% BSA in TBS-T (50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, and 0.1 % Tween- 20), and then incubated with NF-κB (p65) (1:500 dilution, Bioworld Technology, T429, USA) and β-actin (1:1000 dilution, Santa Cruz, sc-8432, USA). Blots were washed in TBS-T and incubated with appropriate HRP-conjugated secondary antibodies. The immune complexes were then visualized using ECL reagent (Beyotime, China). Image J software was used to analyze the area and density of protein band.

Quantitative Real-time RT-PCR Validation
The target genes IL17A and IL17RA were validated by quantitative real-time RT-PCR. Total RNA was isolated and cDNA was transcribed using PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Japan) according to the manufacturer's instructions. Primers were searched from Primerbank (https://pga.mgh. harvard.edu/primerbank/) and listed in table 1. All data were normalized to GAPDH. 2 −ΔΔCt methods were recruited to calculate the relative expression level.

Statistics
Statistical calculations were performed using GraphPad Prism 7 (GraphPad Software Inc., USA). With unpaired Student's t-test or sign test of the normal approximation means, data were expressed as mean±SEM. The P value <0.05 was considered significant.

Up-regulation of IL17A-IL17RA Axis in Aortic Valve of HFD ApoE -/-Mice
Firstly, IL17A-IL17RA-related genes from the analysis of GSE77287 were selected based on PPI network. Relative expression of these genes was shown in fig. 1A-1C.
Furtherly, as shown in fig Table 1 The primers of IL17A-IL17RA-related genes  (20 weeks), serum levels of cytokines (pg/mL) as well as lipid profile in the serum were determined by ELISA. Data are expressed as mean±standard error of the mean. n.s., not significant   1K). However, IL17RA + CD4 + T cells and macrophages showed no significant difference ( fig. 1J and 1L).
The results were further confirmed in VICs by Western blotting as shown in fig. 3E and 3F. As VICs are the main component of aortic valve and contribute to normal function, HFD ApoE -/mice led to significant NF-κB intranuclear translocation in VICs (fig. 3E). After IL17A mAb treatment, the NF-κB translocation was depressed ( fig. 3F).

Validation of IL17A and IL17RA Expression Data by Real-time RT-PCR
The expression levels of IL17A-IL17RA genes were detected using qRT-PCR. The result showed that Immunofluorescence analysis of NF-κB nuclear translocation in IL17RA + VICs and IL17RA + macrophages was performed. Normal diet (ND) ApoE -/mice were compared with their high-fat diet (HFD) counterparts, whereas IL17A mAb-treated group was compared with irrelevant IgG-treated group. Translocation of NF-κB nuclear was assessed by NF-κB staining (arrows). IL17RA + VICs and IL17RA + macrophages were respectively imaged by merged colors of αSMA + IL17RA + DAPI (A, C), CD68 + IL17RA + DAPI (B, D). Scale bar=40 μm. Portion of NF-κB nuclei-positive area or cell particle in total L17RA + VICs or IL17RA + macrophages was set as criteria to estimate NF-κB nuclear translocation. Separately, immunofluorescent staining images suggested obvious intranuclear translocation of NF-κB in IL17RA + VICs (A) (P<0.05) and IL17RA + macrophages (B) (P<0.01) in HFD group as compared with ND group. Meanwhile, IL17A mAb treatment illustrated blocking of NF-κB intranuclear translocation in IL17RA + VICs (C) (area%, P<0.01; particle%, P=0.064) and IL17RA + macrophages (D) (area%, P<0.01; particle%, P<0.05). E: HFD induced the increase of NF-κB, as shown by the increase in NF-κB levels in nuclear extract Compared to ND. F: IL17A mAb treatment inhibited intranuclear translocation of NF-κB, as shown by the decrease in NF-κB levels in nuclear extract compared to control group. Data were analyzed using the Student's t test.

DISCUSSION
Aortic valve disease is a significant culprit in cardiovascular disease and results from a complex interaction between hypercholesterolemia state and chronic inflammation. The development of aortic valve disease is characterized by three primary processes: lipid accumulation, inflammation, and ultimate calcification [23,24] . Inflammation is the core point to all stages of aortic valve pathology. Previous pathological studies suggested that stenotic aortic valve is rich in inflammatory cells and exhibits myofibroblastic differentiation in VICs-the most prevalent cells in aortic valve and responsible for maintaining normal valve structure and function. In inflammatory conditions, quiescent VICs will be activated into myofibroblasts [25,26] . αSMA, a cytoskeletal isoform of actin, is the phenotypic marker for VICs. The transition from VICs to myofibroblasts is also a critical change in valvular calcified pathology, markedly increasing the αSMA positive staining area. Overexpression of αSMA increases the VICs contractility and calcific nodule formation, whereas knockdown of αSMA with siRNAs reverses these changes [27] . Myofibroblastic transition is thought to contribute directly to the thickening and stiffening of valve and the subsequent calcification [28] . CD4 + T cells and macrophages are the principal infiltrated inflammatory cell types in various valvular heart diseases [29] . Inflammatory macrophages, as indicated by CD68, promote valvular cells calcification in a cathepsin S-dependent manner in aortic valve inflammatory disease [30] . Our current study employed HFD ApoE -/mice model as a wellestablished model for aortic valve inflammation, and displayed the increase of cholesterol and IL17A during aortic valve inflammation. At the same time, we observed significantly increased infiltration of inflammatory cells such as macrophages and CD4 + T cells, and myofibroblastic transition of VICs.
Suppressing IL17A with IL17A mAb weakened levels of these inflammatory responses.
IL17A, which is primarily secreted by Th17 cells, participates in local tissue inflammation via inducing release of proinflammatory cytokines and neutrophil-mobilizing chemokines in various cell types [10] . IL17RA is expressed in smooth muscle cells under basal conditions [31] . In atherosclerotic disease, IL17A/IL17RA are involved in systemic and vascular inflammation in response to HFD and are implicated in the progression of the atherosclerotic plaque [12] . However, the role of IL17A in different stages of aortic valve inflammation remains unknown. In the current study, our data revealed that IL17A + CD4 + T cells were increased in HFD aortic valve. Immunofluorescent staining also stated that IL17RA was raised significantly in VICs. Blockade of IL17A reversed the changes. Taken together, these findings indicated that IL17A and IL17RA were involved in VICs myofibroblastic transition, and CD4 + T cells and macrophages infiltration in aortic valve inflammatory state.
Colocalization analysis is a vital section of coexpression study. Unlike the usual dye-overlap method easily misled by subjectivity, it identifies protein pairs and calculates colocalization area. The same results may also be acquired by using flow cytometry, but it will run out of almost all the treasured tissues. Additionally, we can also inquire a statistically testable, single-value assessment ICQ between the stained protein pairs. We used this co-expression method to assess IL17RA colocalized in the specific cells, not just IL17RA positive area in the whole aortic valve. At last, we obtained the results that IL17RA was significantly activated in myofibroblastic VICs in the HFD aortic valves.
In our study, IL17A/IL17RA contribute to inflammation by mean of cellular infiltration and activation, e.g., T cells and macrophages infiltration and VICs myofibroblastic transition. Furtherly, it can be speculated that IL17A activates VICs via IL17RA and subsequently triggers activation of NF-κB, thereby inducing NF-κB-dependent gene transcription, as also reported in some publications for several other cell types [32,33] . On the other hand, as the number of VICs is various in different experiment groups, in order to avoid the bias, we used (NF-κB + IL17RA + VICs)/ (IL17RA + VICs)% value, instead of NF-κB positive area or cells in the whole aortic valve, to assess NF-κB translocation. At last we illustrated that the NF-κB was responsible for IL17A/IL17RA-related inflammation in VICs on HFD or IL17A mAb treatment mice.
In conclusion, we demonstrated for the first time that IL17A/IL17RA were involved in VICs myofibroblastic transition and inflammatory cells infiltration in the HFD ApoE -/mice, which is the downstream regulation mediated by NF-κB. These conditions were reversible by inhibition of IL17A. Therefore, blocking IL17A could probably provide potential prophylactic and therapeutic potent for different stages of aortic valve diseases in the future.

Open Access
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