Brg1 Promotes Airway Mucus Hypersecretion via IL- 13 and the JAK1/2-STAT6 Signaling Pathway in Asthma

Wenjing Zou (  1016278469@qq.com ) Children's Hospital of Chongqing Medical University maozhu xu Children's Hospital of Chongqing Medical University Jie Hu Children's Hospital of Chongqing Medical University Lili Yang Children's Hospital of Chongqing Medical University Gang Gen Children's Hospital of Chongqing Medical University Zhou Fu Children's Hospital of Chongqing Medical University Zhengxiu Luo Children's Hospital of Chongqing Medical University


Background
Asthma is a common, serious, and chronic airway in ammatory disease in children. The clinical manifestations are mainly recurrent wheezing, shortness of breath, coughing, and chest tightness, and it is often accompanied by expiratory air ow limitation. According to current research, there are currently approximately 334 million asthma patients worldwide, and there are as many as 40 million asthma patients in China. In the past decade, the global asthma incidence has increased from 2.4% to 4.3% [1] . Due to the complexity of its etiology and pathogenesis, the clinical treatment of asthma is facing substantial challenges. Recent studies suggest that epigenetic regulation (heritable changes in gene expression that occur in the absence of alterations in DNA sequences) may in part mediate the complex gene environment interactions that can lead to asthma [2] . Therefore, it is interesting to study the pathogenesis of asthma from the perspective of epigenetics. Brahma-related gene 1 (Brg1), a chromatin remodeling protein encoded by the SMARCA4 gene, is the core component of the mammalian switch/sucrose non-fermentable (SWI/SNF) class of chromatin remodeling complex, with ATPase activity. We previously found that Brg1 plays an important role in the pathogenesis of asthma. High expression of Brg1 can be detected in the peripheral blood of children with asthma [3] . Previous studies have suggested that knocking down Brg1 in asthmatic mice can reduce chronic airway in ammation in asthma.Therefore, this study further explored the effect of Brg1 on asthmatic airway mucus hypersecretion. Mucus hypersecretion is an important clinical manifestation of asthma. Consequently, further exploration of whether the Brg1 gene can participate in the regulation of asthmatic airway mucus hypersecretion will provide a new direction for the treatment of asthma.
Mucus hypersecretion can be a critical contributor to asthma exacerbation and can be initiated by multiple intracellular signaling pathways [4] . MUC5AC is an important protein that encodes structurally related mucin glycoproteins, the principal macromolecules in airway mucus. MUC5AC overproduction is an additional disease feature of asthma. Signal transducer and activator of transcription 6 (STAT6) is necessary and su cient for inducing the MUC5AC gene [5] .

Animals
Female C57BL/6 mice (4-6 weeks old) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). AE2 and cubic cell-speci c Brg1-knockout (SFTPC-Cre/Brg / ) mice were generated by hybridizing SFTPC-rtTA/(TetO)7 transgenic mice (The Jackson Laboratory, CA, USA) with Brg1 loxP/loxP mice (kindly provided by Prof. Yujun Shi, West China hospital of Sichuan University). Brg1 deletion in SFTPC-Cre/Brg1 / mice was achieved by administering doxycycline (1 mg/mL) in drinking water containing 0.4% sucrose. The various control mice were Brg1 / mice (no Cre) that received doxycycline. Experimental mice were housed under speci c pathogen-free conditions and subjected to a 12 h/12 h dark/light cycle. The study was approved by the ethics committee of Chongqing Medical University.

Grouping and administration
Twenty female C57BL/6 mice were divided into control and asthma groups. Mice in the asthma group underwent house dust mite (HDM) challenge according to previous studies [6] . Brie y, mice received either 20 μg HDM (Greer, Los Angeles, CA, USA) dissolved in 30 μL normal saline (NS) (asthma group) or the same volume of NS (control group) by nasal inhalation on days 0, 14, 21, 23, 25, 27, and 29. A total of twenty SFTPC-Cre/Brg / mice were divided into control/Brg / and asthma/Brg / groups. The modeling method of the control and asthma groups of SFTPC-Cre/Brg / mice was the same as that of wild-type mice. All mice were sacri ced at 24 h after the last challenge and then euthanized by intraperitoneal injection of 4% pentobarbital (0.5 mg/kg).

Measurement of airway hyper-responsiveness (AHR)
AHR was determined using an invasive pulmonary function instrument (EMKA Technologies, Paris, France) within 24 h after the nal HDM challenge. Mice were anesthetized with 2% pentobarbital sodium, intubated, and placed in whole-body plethysmography chambers. Airway resistance and compliance were measured every second. Baseline and changes in airway resistance were determined using NS and methacholine (3.125, 6.25, 12.5, 25, or 50 mg/mL) respectively, which were nebulized at a volume of 20μL [6] . Lung resistance at each concentration was calculated.

Histological and immunohistochemical assessment of lung tissues
The left lung tissues were xed in 4% formalin buffer and embedded in para n. The para n blocks were then serially sectioned into 4-μm-thick slices and subjected to immunohistochemical (IHC) staining.
Antibodies against MUC5AC (1:300; Abcam, Massachusetts, USA) were used for IHC staining. Tissues or cells that were stained brown were recorded as being positive for MUC5AC. The intensity of IHC staining was quanti ed using Image-Pro Plus 6.0. Regarding the histochemical analysis, para n blocks were serially sectioned into 4-μm-thick slices and subjected to hematoxylin/eosin (H&E) or periodic acid-schiff (PAS) (Leagene Biotechnology Co. Ltd, Beijing, China) staining. Three individual areas per section and lung tissues per group were evaluated for in ammatory cell in ltration.

2.5.Broncho alveolar lavage uid (BALF) analysis and cell counting
The right lung of each animal was washed three times with 0.5 mL phosphate-buffered saline (PBS) in order to collect BALF. Approximately 1.3 mL of the instilled uid was consistently recovered. All the cells in BALF were collected by centrifugation and treated with red blood lysis buffer, after which they were counted by microscopy using a cell counter. The remaining in ammatory cells were smeared on slides, dried, and stained using Wright-Giemsa stain (Jiancheng Techno Co, Nanjing, China), according to the manufacturer's instructions, to determine differential cell counts in accordance with conventional morphological criteria. At least 200 cells per slide were evaluated to determine the differential cell counts.

Cell culture and Treatments
Human bronchial epithelial cells (16HBE) were obtained from the American Type Culture Collection (ATCC, USA) and cultured in Dulbecco's phosphate-buffered saline medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco, USA). The cells were cultured at 37°C in a 5% CO 2 atmosphere, and the growth status of the cells was evaluated under a microscope. Cells were grown to 85-90% con uence, passaged, and subjected to trypsin digestion. Short hairpin RNAs (shRNAs) against Brg1 (Brg1-shRNA), as well as a negative control shRNA (NC-shRNA),were designed and cloned into pGMLV-SC5 RNAi backbone adenoviral vectors (Genomedtech, Shanghai, China). 16HBE cells were then infected with the Brg1-shRNA adenovirus vectors and screened for the stable expression of green uorescent protein.

Immuno uorescence detection
The expression of MUC5AC in 16HBE cells was determined using immuno uorescence staining. Three groups of cells (16HBE, Brg1-sc, Brg1-sh) were made into cell slides, and the slides were removed 24 h later and xed with 4% paraformaldehyde. The slides were stained with primary antibodies, rabbit anti-Brg1 (1:200, Abcam, UK) for 12 h at 4°C. The sections were washed three times with PBS and stained for 30 min at room temperature with donkey anti-rabbit Cy5 secondary antibodies (1:500; Invitrogen, USA). Nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole) (Invitrogen, USA), and uorescence of the samples was captured using a confocal laser scanning microscope (A1þR, Nikon, Tokyo, Japan).
Harvested samples containing input (protein pretreated with nothing), IgG (protein pre-treated with A/G agarose and rabbit anti-IgG), anti-JAK1/2 (protein pre-treated with A/G agarose and rabbit anti-PI3K), and anti-Brg1 (protein pre-treated with A/G agarose and rabbit anti-Brg1) were analyzed by western blotting.

Chromatin Immunoprecipitation CHIP
16HBE and Brg1 knockdown 16HBE cells (Brg1-sh) were cultured for 48 h. One plate of cells (10 cm plate) was removed for formaldehyde cross-linking and ultrasonic disruption. We chose the CHIP kit from the American company Milipore, and all the experimental steps were performed in accordance with the kit's instructions. The concentration of Brg1 antibody used in CHIP is 1:20, and the antibody was purchased from Zen-bio Biotechnology Co., Ltd., Chengdu, China. The ultrasonic fragmentation product was removed, and the ultrasonic effect was tested by electrophoresis. The next day, the immune complex was precipitated and washed. After de-crosslinking, DNA fragments were recovered. The nal sample was dissolved in 30 uL Elution buffer. Finally, the recovered DNA was subjected to q-PCR analysis. The primer sequences of JAK1 were forward 5-GGCGTCGAAGCAAACTGTTT-3 and reverse 5-TACCGTTGTGGGCTACGTTC-3. The primer sequences of JAK2 were forward 5-GACAGACCTGAAGAGCAGCA-3 and reverse 5-CTGGCACCAGATCGTGTAGG-3.

Statistical analyses
Data are expressed as the mean ± standard error of the mean. GraphPad Prism software (version 5.0; GraphPad, San Diego, CA, USA) was used for the statistical analyses. Two-way analysis of variance was conducted to determine statistically signi cant differences in the tested variables among the different groups. If an overall test was signi cant, Tukey's test was used for speci c comparisons between groups. Statistical signi cance was set at p < 0.05. All experiments were repeated at least thrice with consistent outcomes.

Results
3.1. Knockout of Brg1 reduced airway in ammation and airway ventilation resistance in asthmatic mice Brg1 knockout mice and wild type mice asthma model was established using house dust mites (Fig.1A). The total number of airway in ammatory cells in the BALF of the Brg1 knockout mouse asthma group was signi cantly lower than that of the wild-type asthma group, and the difference was statistically signi cant (Fig.1B). The number of eosinophils in the BALF of the Brg / /asthma group was signi cantly lower than that in the WT/asthma group, and the difference was statistically signi cant (Fig.1C). There was no difference in the total in ammatory cells and eosinophils in BALF between the WT/Control group and Brg / /Control groups (Fig.1B-C). HE staining results of mouse lung tissue showed that the in ltration of in ammatory cells around the bronchus of the Brg / /asthma group was signi cantly less than that in the WT/asthma group (Fig.1D). The results of the invasive lung function test showed that the airway resistance of the Brg / /asthma group was lower than that of the WT/asthma group. There was no difference in airway ventilation resistance between the WT/Control group and Brg / /Control groups (Fig.1E).

Knockout of Brg1 alleviated airway mucus secretion and MUC5AC protein expression in asthmatic mice
PAS staining of mouse lung tissue showed that airway mucus secretion in the Brg / /asthma group was signi cantly lower than that in the WT/asthma group ( Fig.2A). Nucleic acid was extracted from lung tissue for qPCR detection. The results showed that the mRNA expression of MUC5AC in the lung tissue of the Brg / /asthma group was lower than that in the WT/asthma group (Fig.2B). Immunohistochemical analysis of lung tissue also showed that the expression of MUC5AC protein in the airway of the Brg / /asthma group was signi cantly reduced compared with that in the WT/asthma group (Fig.2C). We used ImageJ software to perform statistics on the immunohistochemical results of the MUC5AC protein.
The results also showed that the airway expression of MUC5AC protein in the Brg / /asthma group was lower than that in the WT/asthma group (Fig.2D). We collected mouse alveolar lavage uid for ELISA experiments, and the results showed that the MUC5AC protein content in the BALF of the Brg / /asthma group was lower than that of the WT/asthma group (Fig.2E). The above results suggest that knocking out the Brg1 gene can signi cantly improve airway mucus hypersecretion in asthma.

Knockdown of Brg1 reduced the expression of MUC5AC protein and inhibited the activation of the JAK1/2-STAT6 signaling pathway in asthma
Proteins were extracted from mouse lung tissues for western blot analysis. The western blot results showed that there was no difference in the expression of MUC5AC protein in the lung tissues of the WT/Control group and Brg / /Control group. The expression of MUC5AC protein in the lung tissue of the Brg / /asthma group was signi cantly lower than that of the WT/asthma group (Fig.3A). The expression of MUC5AC protein in lung tissue detected by Western blot was statistically analyzed (Fig.3B). Studies have shown that IL-13 is closely related to airway mucus hypersecretion during the asthma onset [8] . Therefore, we used ELISA experiments to detect the amount of IL-13 levels secretion in mouse BALF, and the results showed that IL-13 in the BALF of the Brg / /asthma group were signi cantly lower than those in the WT/asthma group (Fig.3C). It has been reported in the literature that excessive activation of the JAK1/2-STAT6 pathway an important reason for the high secretion of airway mucus in asthma [9] .
Western blotting revealed the activation of the JAK1/2-STAT6 signaling pathway in mouse lung tissue, and the results showed that the percentages of p-JAK1/2/JAK1/2 and p-STAT6/STAT6 in the Brg / /asthma group were signi cantly lower than those in the WT/asthma group (Fig.3D). The percentages of p-JAK1/2/JAK1/2 and p-STAT6/STAT6 in the western blot results were statistically analyzed ( Fig.3E-F). The above results suggested that knocking out of Brg1 could inhibit the activation of the JAK1/2-STAT6 pathway in asthma, thereby reducing airway mucus hypersecretion.

Knockdown of Brg1 reduced the expression of MUC5AC and inhibited the JAK1/2-STAT6 signaling pathway in 16HBE
We used adenovirus to knock down the Brg1 gene in bronchial epithelial cells (16HBE). To verify whether Brg1 knockdown was successful in 16HBE, we extracted the cell protein and performed western blot experiments. The results showed that the Brg1 protein expression of the cells in the Brg1 knockdown group (Brg1-sh) was signi cantly lower than that of the virus empty control group (Brg1-sc), while the Brg1-sc group had no difference compared with the blank control group (16HBE) (Fig.4A-B). At the same time, we extracted cell nucleic acid for qPCR and found that Brg1 mRNA levels in the Brg1-sh group were signi cantly lower than those in the Brg1-sc group, and the difference was statistically signi cant (Fig.4C). The results of the immuno uorescence staining of 16HBE showed that MUC5AC protein was expressed in the cells, and the uorescence intensity of the Brg1-sh group was lower than that of the Brg1-sc group (Fig.4D). The expression of MUC5AC protein in the Brg1-sh was lower than that in the Brg1sc group as determined by western blotting (Fig.4E). Concurrently, we also detected the changes of the JAK1/2-STAT6 pathway in 16HBE. The percentages of p-JAK1/2/JAK1/2 and p-STAT6/STAT6 in the Brg1-sh group were signi cantly lower than the Brg1-sc group, suggesting that Brg1 knockdown can signi cantly inhibit the activation of the JAK1/2-STAT6 signaling pathway (Fig.4F). We found that the results of 16HBE cell proteins p-JAK1/2/JAK1/2 and p-STAT6/STAT6 were signi cantly lower in the Brg1sh group than in the Brg1-sc group (Fig.4G-H). The above in vitro experimental results also suggest that knocking down Brg1 in bronchial epithelial cells can simultaneously reduce the expression of MUC5AC and IL-13 protein and inhibit the activation of the JAK1/2-STAT6 pathway.
3.5. Brg1 regulated the JAK1/2-STAT6 pathway partly by binding to the JAK1/2 gene promoter region Since Brg1 gene is an epigenetic regulatory factor, it can participate in the transcriptional regulation of a variety of genes. Therefore, we used house dust mite extract to treat bronchial epithelial cells, and then collected the cells to detect the combination of Brg1 and JAK1/2 using Co-IP. The results of Co-IP showed that HDM was used to stimulate 16HBE, and Brg1 interaction with JAK1/2 protein in 16HBE (Fig.5A). The abnormally increased Brg1 gene expression in asthma may be involved in the regulation of airway mucus hypersecretion in asthma. The results of the CHIP experiment showed that the Brg1 gene can be detected in the airway epithelial cells to bind to the promoter region of the JAK1/2 gene (Fig.5B-C). Knockdown of the Brg1 gene can inhibite asthmatic airway mucus hypersecretion and reduce MUC5AC and IL-13 secretion, possibly through inhibition of the activation of the JAK1/2-STAT6 pathway. The mechanism diagram of this study is shown in Fig.5D.

Discussion
In recent years, the incidence of asthma has gradually increased, with more than 300 million asthma patients worldwide. The pathological features of asthma mainly include airway epithelial injury, airway hyperresponsiveness, high mucus secretion and airway remodeling. Among them, the high secretion of airway mucus is an important factor that obstructs airway ventilation in asthma and affects the severity of the disease in children. Mucus hypersecretion is a pathophysiological feature of asthma and contributes to morbidity and mortality. Asthma is a disease closely related to genetic inheritance. In recent years, epigenetics has gained increasing attention in the pathogenesis of asthma. Studies have shown that there is epigenetic regulation of genes in many patients with asthma. Brg1 is a chromatin remodeling protein. Our previous studies have found that the expression of Brg1 gene is increased in asthmatic mice and children. Knockdown of the Brg1 gene in mouse models can signi cantly reduce airway epithelial damage and airway in ammation in asthma. We investigated the involvement of Brg1 in the regulation of Brg1 in airway mucus hypersecretion in asthma, focusing on the role of Brg1 in the JAK1/2-STAT6 pathway. In this study, Brg1 knockout reduced airway mucus secretion in asthmatic mice. One study showed that Brg1 regulated endothelial-derived IL-33 to promote ischemia-reperfusion -induced renal injury and brosis in mice [10] . This suggests that the Brg1 plays an important role in the regulation of epithelial cell function. Previous studies have shown that overlapping contributions of multiple SWI/SNF subunit (Brg1) mutations in the translocation of the endometrium to distal sites, with loss of cell integrity being a common feature in SWI/SNF mutant (Brg1) endometrial epithelia [11] . Another study found that the decreased expression of KDM2B and Brg1 produced similar effects to that of poly(I:C)treated cells, which could promote the in ammatory response of nasal mucosal epithelial cells. Brg1 may have an inhibitory effect on the development of nasal mucosal epithelial in ammation [12] . Brg1 promotes the transcription of endothelial Mrtfa and Mrtfb, which elevates the expression of SRF and SRF target genes that establish embryonic capillary integrity. These data highlight the new and temporally speci c role for Brg1 in embryonic vasculature and provide novel information about epigenetic regulation of Mrtf expression and SRF signaling in developing blood vessels [13] . Research on the proliferation and migration of lung cancer cells found that Brg1 regulates the transcription of CCNB1 and LTBP2 by changing histone modi cations on the target promoter, recruiting KDM3A (a histone H3K9 demethylase) to be removed from the target gene promoter Dimethyl H3K9, which activates transcription [14] . In a study on the of B cell activation process, it was found that activation of the JAK/STAT cytokine signaling pathway is particularly dependent on the regulation of the Brg1 gene [15] . The above research results and the Brg1 gene found in this study may be involved in the regulation of the JAK1/2-STAT6 signaling pathway. The abnormally high expression of Brg1 gene in asthmatic mice may promote the activation of JAK1/2-STAT6 signaling pathway, thereby increasing the expression of MUC5AC protein and increasing mucus secretion in asthmatic mice.
Studies have shown that the JAK1/2-STAT6 signaling pathway is an important pathway that mediates the hypersecretion of airway mucus in asthma. Activation of the JAK1/2-STAT6 signaling pathway can be observed in OVA-induced asthma [16] . Pseudomonas aeruginosa and its virulence factor pyocyanin inhibit FOXA2 (a key transcriptional regulator of mucus homeostasis) by activating the antagonistic signaling pathways EGFR-AKT/ERK1/2 and IL-4/IL-13-STAT6-SPDEF ), thereby exacerbating periodic proin ammatory reactions and excessive mucus secretion [17] . Srolo Bzhtang exhibited protective effects against cigarette smoke-induced airway in ammation and MUC5AC hypersecretion, which might be related to the downregulation of the IL-13/STAT6 signaling pathway [18] . In a study on gastrointestinal mucus, it was found that supplementation with gastric mucin (MUC5AC) signi cantly aggravated JAK/STAT signal transduction, indicating that the JAK/STAT signal pathway is involved in the regulation of MUC5AC protein [19] . Petra Dames et al. showed that IL-13 impairs ENaC-dependent sodium transport by activating the JAK1/2-STAT6 signaling pathway. These results improve our understanding of the mechanisms through which IL-13 functions as a key effector cytokine in ulcerative colitis, thereby contributing to the distinct pathology of this disease [20] . In addition to these classical pathways, IL-13 is also known to signal via MAP kinases. IL-13 actions via the heterodimeric type I receptor, as identi ed here in our present study using neutralizing antibodies, have been shown for different target genes and in different tissues to involve phosphorylation of JAK1/2 and subsequent phosphorylation and activation of the transcription factors STAT3 and STAT6 [20] . In this study, we also found that knocking out the Brg1 gene can reduce the secretion of IL-13 in the airways of asthma. Therefore, it may reduce the activation of the JAK1/2-STAT6 pathway regulated by IL-13, thereby reducing the expression of MUC5AC mucus protein.

Conclusions
In summary, on the basis of the abnormally high expression of Brg1 in asthma in a previous study, we further studied the effect of Brg1 on the hypersecretion of airway mucus in asthma, and found that knocking out the Brg1 gene in asthma models can reduced airway mucus secretion in mice and IL-13 and MUC5AC protein expression. We have veri ed from in vivo and in vitro experiments that knocking down the Brg1 gene may inhibit the activation of the JAK1/2-STAT6 pathway, while the reduction of IL-13 secretion can also reduce the activation of the JAK1/2-STAT6 pathway and reduce the secretion of MUC5AC protein. Our research provides new ideas for reducing the mucus hypersecretion of asthma in the future and for the treatment of asthma.