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Epithelial-Mesenchymal Transition in Asthma Airway Remodeling Is Regulated by the IL-33/CD146 Axis

Epithelial-mesenchymal transition (EMT) is essential in asthma airway remodeling. IL-33 from epithelial cells is involved in pulmonary fibrosis. CD146 has been extensively explored in cancer-associated EMT. Whether IL-33 regulates CD146 in the EMT process associated with asthma airway remodeling is still largely unknown. We hypothesized that EMT in airway remodeling was regulated by the IL-33/CD146 axis. House dust mite (HDM) extract increased the expression of IL-33 and CD146 in epithelial cells. Increased expression of CD146 in HDM-treated epithelial cells could be blocked with an ST2-neutralizing antibody. Moreover, HDM-induced EMT was dependent on the CD146 and TGF-β/SMAD-3 signaling pathways. IL-33 deficiency decreased CD146 expression and alleviated asthma severity. Similarly, CD146 deficiency mitigated EMT and airway remodeling in a murine model of chronic allergic airway inflammation. Furthermore, CD146 expression was significantly elevated in asthma patients. We concluded that IL-33 from HDM extract-treated alveolar epithelial cells stimulated CD146 expression, promoting EMT in airway remodeling in chronic allergic inflammation.


INTRODUCTION
Asthma is a disease that is characterized by airway inflammation, airway remodeling, and airway hyperresponsiveness (1). Airway remodeling is described as a change in the composition, thickness or volume of airway walls, including subepithelial fibrosis, and increased smooth muscle composition, in asthmatic patients compared to normal individuals (2). Epithelial-mesenchymal transition (EMT) is a pathophysiological process induced by multiple signaling pathways centered on TGF-β and refers to the loss of function of epithelial cells and their transformation to mesenchymal cells, including a decrease in E-cadherin, and an increase in N-cadherin expression (3)(4)(5)). An increasing number of studies have demonstrated that increased EMT plays an important role in airway remodeling in asthma (5,6).
CD146 was originally acknowledged as a tumor marker for melanoma (MCAM). As a multifunctional molecule (7), CD146 plays diverse biological roles in tumors, atherosclerosis, systemic sclerosis, and other diseases (8)(9)(10). CD146 in macrophages promotes cell adhesion and foam cell formation (8). CD146 in CD4 + T cells is associated with Th17 differentiation in systemic sclerosis (9). CD146 is also associated with pulmonary infections, in which it promotes the adherence of bacteria or viruses to airway epithelial cells (11)(12)(13)(14). Increased expression ofCD146 in gastric cancer leads to decreased expression of E-cadherin and increased expression of N-catenin and vimentin (15). CD146 also regulates the EMT process in hepatocellular carcinoma via the MAPK1 signaling pathway, which exacerbates the invasion and metastasis of hepatocellular carcinoma (16). These studies suggest that the EMT process is associated with cancer progression. Increased expression of CD146 in the airway epithelial cells of asthma patients was recently discovered, and IL-13 (a type 2 inflammatory cytokine) regulates the expression and function of CD146 in airway epithelial cells (11,12). Although the regulation of EMT by CD146 has been extensively reported in studies of tumor metastasis (17), the roles of CD146 in asthma EMT and airway remodeling have not been explored.
Interleukin-33 (IL-33) is a member of the IL-1 cytokine family and is expressed in fibroblasts, endothelial cells, epithelial cells, and other cell types (18,19). Once bound with the membrane receptor ST2, IL-33 activates the MyD88/NF-κB signaling pathway (20) and induces the type 2 response in CD4 + T cells, which release IL-4, IL-5, and IL-13 (19). Serum IL-33 and the soluble form of ST2 are closely associated with asthma disease progression (21) and exacerbation (22). Moreover, recent studies have shown that IL-33 is involved in asthma airway collagen deposition, suggesting that IL-33 may be involved in the EMT process in the lung (23)(24)(25). The regulation of IL-33 signaling related to CD146 expression and the EMT process in asthma, however, remains largely elusive. In the present study, we demonstrated that IL-33 increased the expression of CD146, which promoted the EMT process in asthma.

Animals and a Murine Model of Asthma
Specific pathogen-free (SPF) female C57BL/J mice aged 6-8 weeks were obtained from the Laboratory Animal Center, To establish a murine model of asthma, the mice were intranasally administered house dust mite (HDM, Greer Laboratories, Lenoir, NC, USA) extract (25 µg of HDM extract dissolved in 40 µL of phosphate-buffered saline) 5 days/week for 5 weeks. All mice were treated with HDM extract under isoflurane anesthesia and were ultimately sacrificed (26).

Cell Culture
The mouse pulmonary epithelial cell lines MLE-12 and A549 were obtained from ATCC (VA, USA) and cultured in DMEM containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 µg/ml streptomycin in a 5% CO 2 atmosphere at 37 • C. MLE-12 or A549 cells were seeded in 6-well plates or 24-well plates overnight and then treated with HDM extract or the cytokine IL-33 for the indicated durations. Primary alveolar epithelial cells from mice were purified using 0.1% collagenase, 0.25% trypsin, and DNase I and were selected with mouse IgG (36111ES60, Yeasen, China) as previously described (27). To exclude the potential effects of lipopolysaccharide (LPS) contamination, HDM extract was treated with the ToxinEraser TM endotoxin removal Kit (L00338, Genscript, China). The purified product was the major constituent of HDM.

Cell Transfection
MLE-12 cells were seeded and incubated overnight before transfection. The CD146 expression plasmid, an siRNA plasmid, or blank vehicles (Abmgood, China) were mixed with Lipofectamine 2000 (Invitrogen, USA) in DMEM without FBS, penicillin or streptomycin for 25 min and were then transfected into MLE-12 cells at 60-80% density in DMEM for 48 h. The cells were treated with HDM extract or PBS for 24 h before total protein extraction.

Western Blotting
Total protein from the cells or tissues was lysed with RIPA buffer (89900, Thermo, USA) containing protease and phosphatase inhibitors (78443, Thermo, USA) on ice for 20 min. Then, the samples were centrifuged for 10 min, and the supernatants were collected and transferred into new EP tubes. The protein concentrations were measured by a BCA assay (P0012S, Beyotime, China). The proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes at 300 mA. The PVDF membranes were blocked with 5% skim milk powder for 1 h at room temperature and were then incubated with primary antibodies ( Table 1) at 4 • C overnight. The PVDF membranes were washed with TBST 4 times for 5 min each and were then incubated with goat anti-rabbit HRP IgG (EarthOx Life Sciences) or goat anti-mouse HRP IgG (EarthOx Life Sciences) for 1 h at room temperature. The PVDF membranes were washed with TBST 4 times for 7 min each. The specific antibody-bound proteins were visualized with the Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA) and the G:Box gel doc system (Syngene, UK).

Quantitative Real-Time PCR
Total RNA was extracted from cells using the TaKaRa Universal Total RNA Extraction Kit (Dalian, China) and was then used to synthesize cDNA using PrimeScript RT master mix (TaKaRa). The expression of specific RNAs was quantified by using SYBR Green Universal PCR master mix (TaKaRa) in a StepOnePlus Real-Time PCR System (ABI, USA). The primer sequences used for real-time PCR were synthesized by Genescript. The primer sequences are as follows:

Immunofluorescence
After treatment with HDM extract or PBS for 24 h, the culture medium was removed, and the MLE-12 cells were washed in PBS 3 times. The cells were then fixed in 4% paraformaldehyde at 4 • C for 15 min, followed by 3 washes with PBS. Afterwards, the cells were blocked with 5% goat serum for 1 h at room temperature and were incubated with mouse anti-E-cadherin, rabbit anti-N-cadherin or rabbit anti-SPD primary antibody at 4 • C overnight. The cells were washed with PBS 3 times and incubated with Alexa Fluor 555 donkey anti-mouse IgG (H+ L) or Alexa Fluor 647 donkey anti-rabbit IgG (H+L) at 37 • C for 1 h in the dark. Next, the cells were washed with PBS and stained with DAPI (4 ′ ,6-diamidino-2-phenylindole; Yeasen, China) at 37 • C for 10 min in the dark. Images were visualized with a ZEISS LSM710 confocal fluorescence microscope or an Olympus IX73 fluorescence microscope.

Airway Responsiveness
The FinePointe RC System (Buxco Research Systems, Wilmington, NC) was used to measured airway responsiveness. Mice were challenged with aerosolized PBS and methacholine to measure lung resistance. The airway resistance values were recorded for 3 min after each challenge. Then, we calculated the average airway resistance (28).

Differential Counts of Inflammatory Cells in BALF
The bronchoalveolar lavage fluid (BALF) was collected from mice and centrifuged to separate the supernatant and sediment. The sediment was resuspended in PBS and measured with a blood cell analyzer (ADVIA 2120i).

Histological Staining
Lung tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue sections were stained with H&E, PAS, and Sirius red. Images were visualized with a Zeiss Axio Examiner microscope.

Immunohistochemistry
Lung tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue sections were blocked with 5% goat serum for 30 min at 37 • C and incubated with mouse anti-E-cadherin at 4 • C overnight. Tissue sections were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. 3,3-Diaminobenzidine (DAB) was used as a color developer, and hematoxylin was used for counterstaining. Images were visualized with a Zeiss Axio Examiner microscope. Collagen I in the lung homogenates was measured using an ELISA kit (E-EL-M0325c, Elabscience, China). Soluble CD146 in human plasma (E-EL-H2403c, Elabscience, China) was measured using commercial ELISA kits. All ELISA experiments were performed according to the instructions provided by the manufacturers.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 5 (La Jolla, CA), and the data are displayed as the means ± SEM. Images from the Western blotting or immunofluorescence results were analyzed with ImageJ. Student's t-test or one-way ANOVA was applied to assess the statistical significance. A value of P < 0.05 was considered statistically significant ( * P < 0.05; * * P < 0.01; * * * P < 0.001; and #P > 0.1).

HDM Promoted CD146 Expression in Alveolar Epithelial Cells via IL-33/ST2 Signaling
Once inhaled into the respiratory tract, HDMs may directly stimulate alveolar epithelial cells. As shown in Figure 1A, HDM extract challenge increased CD146 transcripts in the mouse alveolar epithelial cell line MLE-12, which was further validated in the immunoblotting assay ( Figure 1B). Primary alveolar epithelial cells purified from the lung were subjected to SPD staining ( Figure 1C). Similarly, HDM extract increased CD146 expression in primary alveolar epithelial cells (Figure 1D).
In agreement with a previous study that showed that IL-33 was increased in asthma (29), HDM extract increased IL-33 expression (Figure 2A) and secretion ( Figure 2B) in alveolar epithelial cells. To explore whether HDM-mediated IL-33 induction was associated with the major HDM component Derp or LPS contamination, we removed endotoxin from HDM extract and treated epithelial cells with treated HDM that lacked LPS.  Again, IL-33 was increased in the cell lysate ( Figure 2C) or culture supernatant (Figure 2D) was increased.
To explore whether IL-33 is involved in CD146 expression, we stimulated epithelial cells with IL-33 and found that IL-33 directly promoted CD146 expression in mouse alveolar epithelial MLE-12 cells ( Figure 2E) and human alveolar epithelial A549 cells (Figure 2F). The ST2-neutralizing antibody decreased CD146 expression (Figure 2G), suggesting that IL-33/ST2 was required for CD146 expression in HDM-treated epithelial cells. In summary, HDM extract increased the expression of CD146 in alveolar epithelial cells, which was mediated by IL-33 and its receptor ST2.

CD146 Expression in Alveolar Epithelial Cells Was Dependent on p65
IL-33 binding to ST2 on epithelial cells may activate a series of downstream signaling pathways, including the MyD88, NF-κB, and MAPK pathways (30). As shown in Figure 3A, HDM extract activated MyD88 in MLE-12 cells. Similarly, HDM extract increased the phosphorylation of NF-κB p65 (Figure 3B). In the MAPK signaling pathway, p38 but not JNK, and p42 was activated in MLE-12 cells treated with HDM extract (Figure 3C). More importantly, the p65 inhibitor antagonized the HDMinduced upregulation of CD146 (Figure 3D), highlighting the importance of NF-κB in CD146 expression. In contrast with the results observed with the p65 inhibitor, the p38 inhibitor showed insignificant effects on the expression of CD146 in MLE-12 cells treated with HDM extract (Figure 3E). Therefore, CD146 in HDM-treated alveolar epithelial cells was regulated by NF-κB p65.

HDM Promoted EMT in Alveolar Epithelial Cells via CD146
There is now evidence that asthma patients have more EMT than normal individuals (5,31). The cadherin switch, which is a fundamental event in EMT, was induced in MLE-12 cells treated with HDM extract. As shown in Figures 4A-C, HDM extract decreased the expression of E-cadherin and increased N-cadherin expression. In addition, HDM extract increased fibronectin and α-SMA levels in MLE-12 cells (Figure 4D), suggesting that HDM extract promoted EMT in alveolar epithelial cells. To explore the roles of CD146 in HMD-induced EMT, CD146 was either overexpressed via an expression plasmid or silenced with a siRNA plasmid in MLE-12 cells. Accompanied by CD146 elevation, E-cadherin was significantly decreased (Figure 5A). In contrast, CD146 silencing caused the increased expression of Ecadherin in epithelial cells (Figure 5B). E-cadherin expression was inversely correlated with CD146 expression, suggesting that CD146 may positively regulate EMT in alveolar epithelial cells. Moreover, the ST2-neutralizing antibody rescued Ecadherin expression in epithelial cells treated with HDM extract (Figure 5C). Considering that the ST2-neutralizing antibody decreased CD146 expression in epithelial cells treated with HDM extract (Figure 2G), we concluded that IL-33/ST2 contributed to CD146-mediated EMT in alveolar epithelial cells treated with HDM extract.

TGF-β and SMAD3 Played Dominant Roles in EMT in Alveolar Epithelial Cells Treated With HDM Extract
TGF-β has been shown to be the most common EMT inducer in asthma (5,32). Accordingly, HMD extract increased TGF-β levels in alveolar epithelial cells (Figure 6A). STAT3 and SMAD3 are downstream molecules of the TGF-β signaling pathway in the EMT process. Administration of HDM extract contributed minimally to STAT3 activation ( Figure 6B) but resulted in the phosphorylation of SMAD3 (Figure 6C) in alveolar epithelial cells. More importantly, a SMAD3 inhibitor (SIS3) partially but significantly increased E-cadherin expression in MLE-12 cells treated with HDM extract (Figure 6D), suggesting that TGF-β and SMAD3 regulate EMT in HDM-treated alveolar epithelial cells.

IL-33 Was Essential for CD146 Expression in a Mouse Model of Asthma
To demonstrate the significance of IL-33/ST2 in CD146 expression, we developed an asthma model in wild-type mice and IL-33 KO mice (Figure 7A). IL-33 deficiency reduced lung resistance in the murine model of asthma (Figure 7B). The number of total cells and eosinophils in BALF were decreased in the IL-33 KO mice treated with HDM extract (Figures 7C,D). Similarly, pulmonary tissue sections stained with H&E exhibited more inflammatory infiltration in WT mice than in IL-33 KO  Figure 7E). Total IgE in sera was significantly elevated in the HDM-treated mice; however, the IgE concentration was comparable in WT and IL-33 KO mice challenged with HDM extract (Figure 7F). The expression of type 2 cytokines, including IL-4, IL-5, and IL-13, was increased in the HDM extract-treated mice. IL-33 deficiency reduced IL-4, IL-5, IL-13, and IFN-γ levels in the lung tissue of the HMD-treated mice (Figure 7G). These results suggest that IL-33 deficiency may alleviate asthma disease severity.

mice (
To further explore EMT in asthma, collagen I in pulmonary tissue was quantified, and the results showed that the level of collagen I was decreased in IL-33 KO mice compared to WT mice treated with HDM extract (Figure 8A). As expected, pulmonary tissue sections stained with PAS ( Figure 8B) or Sirius red ( Figure 8C) revealed that collagen deposition and glycogen storage were more pronounced in WT mice than in IL-33 KO mice. Consistent with the previous in vitro observations, decreased CD146 ( Figure 8D) and elevated  (Figures 8D,E) levels were observed in IL-33 KO mice compared to WT type mice after HDM treatment. As observed in vitro, Myd88, NF-κB, and p38 may be involved in EMT in the mouse model of asthma (Supplementary Figure 1). In summary, IL-33 deficiency alleviated disease severity and decreased CD146 expression and EMT in asthma.

CD146 Deficiency Decreased EMT in a Mouse Model of Asthma
To further evaluate the roles of CD146 in the asthma-associated EMT process, we established an asthma model in WT mice and CD146 KO mice. As shown in Figure 9A, lung resistance was reduced in CD146 KO mice compared to WT mice treated with HDM extract. The IgE level in the asthmatic WT mice and CD146-deficient mice was comparable (Figure 9B). In pulmonary tissues stained with H&E, the inflammatory response was decreased in the CD146 KO murine model of asthma (Figure 9C). Pulmonary cytokines, including IL-4, IL-5, IL-13, and IFN-γ, were decreased in CD146 KO mice compared to WT mice after HDM treatment ( Figure 9D). Of note, IL-33 levels in asthmatic WT or CD146-deficient mice were comparable ( Figure 9E). Because CD146 regulated EMT in alveolar epithelial cells, the level of collagen I was significantly decreased in the mouse model of asthma with a CD146 KO background (Figure 10A). Similarly, collagen deposition and glycogen storage in asthmatic CD146 KO mice were decreased, as evidenced by PAS ( Figure 10B) and Sirius red staining (Figure 10C), respectively. Furthermore, CD146 deficiency caused an increase in E-cadherin in the asthma model (Figures 10D,E), suggesting that CD146 may orchestrate EMT in asthma.

Soluble CD146 Was Elevated in the Plasma of Asthma Patients
We demonstrated that CD146 contributed to asthma pathogenesis in a mouse model. CD146 is not only expressed on the cell membrane but could also be released into circulation (33). To demonstrate the clinical significance of the study, we measured soluble CD146 (sCD146) levels in the plasma of asthma patients. As shown in Figure 11, the level of sCD146 was significantly increased in asthma patients compared to healthy controls. Considering that CD146 was increased in the airway epithelial cells of asthma patients (11,12), we hypothesized that CD146 may be important in asthma.

DISCUSSION
In the present study, we first demonstrated that HDM extract promoted CD146 expression in alveolar epithelial cells via IL-33, and this effect was blocked with an antibody against the IL-33 receptor ST2. CD146, which was upregulated with an expression plasmid or downregulated with an siRNA plasmid, was found to play essential roles in E-cadherin expression in alveolar epithelial cells, suggesting that CD146 may mediate EMT in asthma. In a chronic asthma model in IL-33-deficient mice, CD146 expression was decreased in the pulmonary tissues, accompanied by increased E-cadherin expression, suggesting that IL-33 is essential in the CD146 expression and airway remodeling observed in asthma. Accordingly, CD146 deficiency in this chronic asthma model caused elevated E-cadherin expression, suggesting that CD146 deficiency reduced EMT in asthma. Moreover, we found that the level of soluble CD146 was increased in asthma patients. Therefore, we hypothesized that CD146 may mediate airway remodeling in chronic asthma in a manner that was dependent on the IL-33 signaling pathway.
In pulmonary epithelial cells, HDM extract stimulated CD146 expression and IL-33 production. As an alarmin molecule (34) and mucosal response amplifier (35), IL-33 binding with its receptor ST2 promoted CD146 expression. It has been demonstrated that IL-33 receptor knockout decreases the airway inflammatory response but induces the persistence of IL-5 + IL-13 + type 2 innate lymphocytes to maintain certain characteristics of asthma (36). Consistent with the above observation, we observed that IgE levels were comparable in the WT and IL-33 KO murine asthma models. In HDM-treated CD146 KO mice, the IL-33 concentration was similar to that in HDM-treated WT mice and was accompanied by comparable IgE levels in WT and CD146 KO mice treated with HDM extract. These results suggest that IL-33 was not indispensable for IgE induction in asthma.
EMT has been reported to be intricately involved in airway remodeling in asthma (37,38). In contrast, inhibition of the EMT process may slow airway remodeling in asthma (39). The increased expression of IL-33 in airway epithelial cells is closely related to the severity of asthma (40), and IL-33 has been shown to not only exacerbate airway inflammation (41) but also promote airway remodeling in asthma (42)(43)(44). Downstream signaling molecules of ST2, including MyD88, NF-κB p65, and MAPK, are then activated. However, only NF-κB p65 was indispensable for the CD146 expression observed in alveolar epithelial cells after stimulation with HDM extract. Because CD146 dimerization may activate NF-κB p65 (45), the reciprocal regulatory mechanisms between CD146 and NF-κB p65 warrant further study.
CD146 has been shown to be expressed by diverse cell types with multiple functions (7). In mouse tracheal epithelial cells, CD146 expression was accompanied by IL-13-mediated eotaxin-3 expression, suggesting that CD146 is an enhancer of the IL-13 response (46). In human primary nasal airway epithelial cells stimulated with TLR agonists, the absence of CD146 decreased expression of the inflammatory chemokine IL-8 (47), suggesting that CD146 may amplify inflammation. Consistent with the roles of CD146 in the inflammatory response, IL-4, IL-5, IL-1, and IFN-γ levels were significantly reduced in CD146-deficient mice with chronic asthma. Moreover, CD146 was directly linked to EMT in alveolar epithelial cells, and this relationship was dependent on the TGF-β/Smad-3 signaling pathway.
CD146 has been shown to be expressed on not only epithelial cells but also other cells, including endothelial cells (48), subpopulations of T cells (49), and mesenchymal stromal cells (MSCs) (50). All of these cell types may be involved in asthma pathogenesis and tissue remodeling (51,52). In addition to epithelial cells, the roles of other CD146 + cells in EMT and airway remodeling in asthma need to be elucidated in the future. Moreover, CD146 is shed from the cell membrane via MMP-3 activity (53). Elevated sCD146 levels in the plasma of asthma patients may enhance the production of vascular endothelial growth factor receptor (VEGFR) and VEGF2 (54). Therefore, we hypothesized that CD146 also regulated neovascularization, which is closely associated with EMT in asthma (55).
In summary, we expanded the role of CD146 in the EMT process from cancer metastasis to airway remodeling in asthma. We proposed that the binding of IL-33 to ST2 on HDMstimulated airway epithelial cells promoted CD146 expression, which further amplified the inflammatory response, EMT and airway remodeling.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by ethics committee of the First Affiliated Hospital of Nanjing Medical University (2017-SR-298). The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Animal Care and Use Committee of Nanjing Medical University (IRB: 1709011).