MiR-365-3p is Involved in IL17-Mediated Asthma in Mouse Model

BACKGROUND The IL-17 superfamily, which mediates cross-talk between the adaptive and innate immune systems, has been associated with severity of asthma. The role of miRNAs in the disease has been paid much attention. To explore the roles of IL-17 in asthma and the relationship between IL-17 and miRNAs, we used a model of severe asthma driven by chronic respiratory exposure to house dust mite (HDM) exposure in wild type and IL-17KO mice, followed with miRNA proling assays and analysis. METHODS Male and female C57BL/6 mice (6-8 weeks old) and IL-17KO mice (C57BL/6 background) were exposed to puried HDM extract intranasally for 5 days/week for 5 consecutive weeks. Sterile saline was used as the control. The parameters including airway responsiveness, inammatory cells in bronchoalveolar lavage uid (BALF), airway smooth muscle bundle, collagen deposition, and cytokine levels in BALF were examined. The miRNA prole of mouse lung tissue was analyzed by microarray assays. The dysregulation of miRNA related to IL-17 and asthma was validated by qRT-PCR. The in vitro cell culture experiment was performed to conrm the relationship between IL-17 and selected miRNA. The regulation of miRNA on predicted target gene was validated by administration of miRNA mimics. deciency reduce (AHR) dysregulated miRNAs, and its expression recovered in IL-17KO mice. Furthermore, miR-365-3p was decreased in mouse alveolar epithelial cells by IL-17 treatment. The transfection of miR-365-3p mimics decreased the expression of predicted target gene ARRB2. was decreased in HDM-exposed WT mice, and its expression was resumed in IL-17KO mice. The further in vitro studies validated the regulatory role of miR-365-3p on the target gene ARRB2. Our studies demonstrated for the rst time the IL-17 related miRNAs in asthmatic mice, which may provide experimental evidence for the IL-17 and miRNA targeting therapy of asthma. study, we established the severe asthma model by using the wild type and IL-17KO mice to investigate the role of IL-17 in the pathogenesis of severe asthma. The results showed that in the severe asthma models, IL-17 deciency could reduce the inltration of inammatory cells in the lung tissue, the level of Th2 cytokines, the airway smooth muscle mass, and collagen deposition, indicating that IL-17 plays an important role in multiple parts of severe asthma. These results support the previous in vitro ndings.


Introduction
Asthma is a chronic in ammatory airway disease characterized by reversible air ow limitation. The pathogenesis of asthma is extremely complex. A variety of in ammatory cells and cellular components are involved in the airway in ammation of asthma. In addition to airway in ammation, airway remodeling is considered as the main pathological basis for irreversible airway obstruction and airway hyperresponsiveness 1 .
Th17 cytokines can induce the production of multiple chemokines, thereby recruiting neutrophils and macrophages to eliminate pathogens. Th17 cells are the intermediate link between innate immunity and adaptive immune response and are considered as potential inducers of autoimmunity and tissue in ammation 2,3 . Th17 cells have been associated with autoimmune diseases such as experimental autoimmune encephalitis (EAE) and rheumatoid arthritis (RA). Recent studies showed that Th17 cells and cytokines are involved in the pathogenesis of asthma 4 . In previous studies, we found that Th17 cytokines including IL-17A / F and IL-22 can induce the migration and proliferation of airway smooth muscle cells (ASMCs) 5,6 . We also found that IL-17A / F can stimulate ASMCs to produce a variety of cytokines and chemokines, of which CXCL1,2 and 3 are the most secreted chemokines, and the supernatant of IL-17A / F stimulated cells can also promote the chemotaxis of ASMCs 7 . The above research results from in vitro experiments showed that Th17 cytokines may participate in the formation of airway remodeling by promoting the chemotaxis, proliferation and secretion of ASMC.
MiRNA is a type of endogenous non-coding RNA with regulatory functions found in eukaryotes, and its size is about 20-25 nucleotides in length. Recent studies have shown that miRNAs are involved in a variety of regulatory pathways and play a key role in a variety of diseases, including cancer, viral infections and in ammatory diseases of the lungs 8 . In recent years, the role of miRNAs in the development of asthma has received increasing attention. Recent publications con rmed the crucial regulatory role of miRNAs in the pathogenesis of asthma 9 . Interestingly, IL-17 can regulate the release of in ammatory factors and chemokines by astrocytes in experimental autoimmune encephalomyelitis through miRNA, which in turn aggravates the disease 10 . This provides a clue for elucidating the mechanism of IL-17 regulating severe asthma.
To explore the roles of IL-17 in asthma and the relationship between IL-17 and miRNAs, we used a model of severe asthma in wild type and IL-17 de cient mice, followed with miRNA pro ling assays and analysis. We demonstrated that IL-17 plays an important role in the pathogenesis of severe asthma by the recruitment of neutrophils, the induced expression of IL-4, IL-5, IL-13 and KC, and the promotion of airway remodeling. In the dysregulated miRNAs, the expression of miR-365-3p was decreased in HDMexposed WT mice, and its expression was resumed in IL-17KO mice. The further in vitro studies validated the regulatory role of miR-365-3p on the target gene ARRB2. Our studies demonstrated for the rst time the IL-17 related miRNAs in asthmatic mice, which may provide experimental evidence for the IL-17 and miRNA targeting therapy of asthma.

Methods
Animals C57BL/6 female mice (6-8 wk) were purchased from Charles River (Montreal, Canada). IL-17KO mice were prepared as previously described 11 . All animals were treated and maintained under a protocol approved by the Animal Care Committee of McGill University, following guidelines set by the Canadian Council on Animal Use and Care.

Antigen Administration
Wild type and IL-17KO mice were exposed to puri ed HDM extract (Greer Laboratories, Lenoir, NC) intranasally (25 mg of protein in 10 ml of saline) for 5 days/week for ve consecutive weeks. The equivalent volume of saline was used as the control. Each group contained 10 mice. The mice were killed at 24 h after the last exposure.

Preparation of bronchoalveolar lavage uid
The lungs were lavaged using a cannula inserted in the trachea and the lungs were instilled with 0.5 ml PBS. Cytospins were prepared at a density of 0.5 ´ 10 6 cells/ml. Differential cell counts were performed using standard morphological criteria on Hema-Gurr-stained cytospins (500 cells/sample) (Merck, Darmstadt, Germany).

Bronchoalveolar lavage cytokine analysis
Aliquots of cell-free bronchoalveolar lavage uid (BALF) were frozen in liquid N 2 and stored at -80°C. The levels of IL-4, IL-5, IL-13, IL-10, TNF-a, IFN-g and KC in BALF were analyzed by the Bio-plex system (Bio-Rad, Mississauga, Ontario, Canada) performed in the Goodman Cancer Centre Transgenic Core Facility, McGill University, Canada.

Immunohistochemistry staining
The immunohistochemistry staining for IL-17A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a-SMA (Santa Cruz Biotechnology) was performed on para n-embedded mouse lung tissue sections as previously described 12 .
miRNA expression array Equal amount of RNA sample from each mice of different groups was pooled respectively for miRNA pro ling assay using µPara o ® Micro uidic Biochip miRNA microarray (LC Sciences, Houston, USA).
Real-time quantitative RT-PCR Independent assays were performed using quantitative reverse transcription PCR (qRT-PCR) on all mouse samples for individual miRNA (miR-207, miR-5112, miR-2861, miR-340-5p, miR-6238, miR-181c-5p, miR-6239, miR-365-3p and miR-133b-3p) (Qiagen, Hilden, Germany) and predicted target genes (FASL, TRAF3, ARRB2, Sgk1, PIK3R3, ADAM10 and ADM) (Bio-rad, Foster City, USA). Data were presented relative to U6 for miRNA and β-actin for target genes based on calculations of 2 (−σσCt) . The primer sequences for target genes were listed in Table 1. Statistical signi cance was de ned as p < 0.05 as measured by the t-test using GraphPad Prism 5 software (GraphPad, San Diego, USA).  Luciferase reporter assay Sense and antisense sequences corresponding to a 406-bp fragment from the 3'UTR of ARRB2 with the predicted binding and mutated sites (position 236-243) were ampli ed from cDNAs of mouse lung tissue by using primers containing the SacI restriction site in the sense oligo and XbaI restriction site in the antisense oligo (Sense: 5'-ATCGAGCTCCTGTCCACCCGAGATACAC-3'; Antisense: 5'-AGCTCTAGAGGTACCCTGCAGATGTAGAA-3'; GenePharma, Shanghai, China). To construct luciferase reporter plasmids for ARRB2, the annealed synthetic oligos were cloned downstream to the re y luciferase into SacI-XbaI double digested pmirGLO Dual-Luciferase miRNA target expression vector (Promega, WI, USA). For the luciferase reporter assay, 293-T cells (ATCC) were co-transfected with 250 ng of luciferase reporter plasmid harboring the wild type/mutant binding sites of ARRB2 respectively along with 25nM mimic control/miR-365-3p mimic using X-tremeGENE siRNA Transfection Regent (Roche) in Opti-MEM (Gibco). After 48h of transfection, cells were washed in PBS and lysed in Reporter lysis buffer (Promega), and luciferase activity was measured in a FlexStation 3 microplate reader (Molecular Devices, Sunnyvale, CA, USA) using the Dual-Luciferase reporter assay kit (Promega) according to the manufacturer's instructions. Fire y luciferase activity was normalized to Renilla luciferase activity, and relative luciferase activity was calculated taking re y luciferase activity of empty pmirGLO transfected cells as 100 percent.

Western Blot
The protein samples of mouse lung tissue or MLE-12 cells were loaded (5 μg) on a 10% acrylamide SDS-PAGE gel (Bio-Rad, Hercules, USA) for protein separation, followed by transfer to PVDF membranes (Bio-Rad). The blots were then blocked with 1% BSA in 0.1% Tween 20/TBS for 1 h at room temperature and then incubated overnight at 4°C with antibodies speci c for ARRB2 (Novus Biologicals, Centennial, CO, USA). After washing with 0.1% Tween 20 in TBS, the membranes were incubated with a 1: 3000 dilution of goat anti-rabbit IgG HRP (EMD Millipore Corp, Burlington, Massachusetts, USA) in 1% solution of powdered milk in TBS/0.1% Tween 20. The membranes were exposed to ECL solution (Bio-Rad) and imaged by chemiluminescence (Clinx Science Instrument, Shanghai, China).

Statistical analysis
Statistical analysis for expression of miRNAs and mRNAs by qRT-PCR in mouse lung tissue was performed by unpaired test. The paired t-test was performed for cell culture experiments. Probability values of P < 0. 05 were considered signi cant. Data analysis was performed by using the GraphPad Prism 5 software (GraphPad, San Diego, CA, USA).

Results
The The results of the counts of various types of in ammatory cells in BALF showed that compared with the WT control mice, the number of various types of cells in the WT model group mice was signi cantly increased (P<0.05, P<0.01; Figure 2A). Compared with the WT model group, the total cell number, macrophages, neutrophils and lymphocytes in the IL-17KO model group were signi cantly reduced (P<0.05; Figure 2A). Observation of mouse lung tissue sections showed that compared with WT model mice, IL-17KO model mice had signi cantly thinner airway wall, airway smooth muscle and submucosal layer. The in ammatory cell in ltration was signi cantly reduced ( Figure 2B).
The cytokine levels in BALF were detected by ELISA. Compared with the WT control group, the Th2 cytokine levels including IL-4, IL-5 and IL-13 in the WT model group were signi cantly increased. In addition, the level of pro-in ammatory cytokine KC was also signi cantly increased in the WT model group. Compared with the WT model group, the levels of IL-4, IL-5, IL-13 and KC in the IL-17KO model group were signi cantly reduced ( Figure 3).
The immunohistochemical staining of airway smooth muscle cells on mouse lung tissues showed that the airway smooth muscle area in WT severe asthmatic mice was signi cantly increased, while that in IL-17KO model mice was signi cantly reduced ( Figure 4A). Similarly, the collagen deposition in the lung tissue of IL-17KO model mice was also signi cantly reduced ( Figure 4B).
The overall results suggested that IL-17 is involved in both in ammation and airway remodeling in severe asthma.
miRNA expression pro le in mouse lung tissue The miRNA expression pro les in the lung tissues of each group of mice were detected by using miRNA microarray assay. The results showed that the expression of 31 miRNAs in WT model mice changed by more than 1.5 times compared with WT control group, of which 26 miRNAs increased, 5 miRNAs decreased (Table 1). While the expression of miR-5112, miR-207, miR-2861, miR-340-5p, miR-6238, miR-181c-5p, miR-6239, miR-365-3p and miR-133b-3p was equivalent between IL-17KO model and IL-17KO control mice (Table 2). We further veri ed the expressions of the above-mentioned differentially expressed miRNAs in mouse lung tissue, the results showed that the expressions of miR-340-5p and miR-365-3p were signi cantly different between WT control and WT model group, while only miR-365-3p appeared equivalent expression between IL-17KO control and IL-17KO model group ( Figure 5).

Target gene prediction and veri cation of expression level
The target genes of miR-365-3p were predicted by the Targetscan database. Among these predicted target genes, TRAF3, ARRB2, Sgk1, PIK3R3, ADAM10 and ADM are the ones that have been reported to be related to asthma. The expression of these genes in mouse lung tissue was tested. The results showed that the expression of ARRB2 was signi cantly higher in WT control group compared with WT model group, and its expression was equivalent between IL-17KO control and IL-17KO model group ( Figure 6A). The expression of ARRB2 in the mouse lung tissue at protein level was detected by western blot ( Figure   6B).

Veri cation of the regulation of miR-365-3p on target gene
Since the reductive level of miR-365-3p was found in the WT model group and recovered in the IL-17KO mice, we investigated whether miR-365-3p expression could be affected by IL-17 stimulation in the mouse alveolar epithelial cells. The results showed that the expression of miR-365-3p was signi cantly reduced by the stimulation of different concentrations of IL-17 ( Figure 7A).
To validate the targeting role of miR-365-3p on ARRB2, the luciferase reporter assay was performed. As shown in Figure 7B, the transfection of miR-365-3p mimic could signi cantly reduce the luciferase activity, suggesting ARRB2 is the target gene of miR-365-3p. Furthermore, miR-365-3p mimic could reduce the expression of ARRB2 in MLE-12 cells, while the inhibitor signi cantly increased the expression level at both mRNA ( Figure 7C) and protein level ( Figure 7D).

Discussion
Our previous studies and other studies have demonstrated that IL-17 may play an important role in asthma. In the present study, we rst established the severe asthma model by using the wild type and IL-17KO mice to investigate the role of IL-17 in the pathogenesis of severe asthma. The results showed that in the severe asthma models, IL-17 de ciency could reduce the in ltration of in ammatory cells in the lung tissue, the level of Th2 cytokines, the airway smooth muscle mass, and collagen deposition, indicating that IL-17 plays an important role in multiple parts of severe asthma. These results support the previous in vitro ndings.
MicroRNAs play important regulatory roles in cell differentiation, cell cycle, and apoptosis 8 . Due to the role of multiple gene regulation, miRNAs have received much attention as biomarkers and target for novel therapeutics. Emerging studies explored the roles of miRNAs in severe asthma 14 . Different miRNAs play roles in biological mechanisms underlying Th2 and macrophage polarization, type 2 innate lymphoid cell biology regulation, steroid-resistant asthma phenotype, airway smooth muscle dysfunction and impaired anti-viral innate immune. We hypothesized that IL-17 may regulate asthma through miRNA. Thus, we further performed miRNA array assay in both WT and IL-17KO mice with or without HDM challenging.
MiR-365-3p was found up-regulated in multiple sclerosis and down-regulated in psoriasis 24 . Its level in serum was reported to be associated with the IL-17 25 . Depending on the different types of cancer, mir-365-3p could act as either an inhibitor or a promoter in tumorigenesis 26,27 . Besides the controversial role in tumorigenesis, miR-365 is considered to be a potential biomarker to identify the phenotypes of asthma, because the expression of miR-365 in severe asthma is lower than mild type 28 . Furthermore, miR-365-3p was up-regulated during the development of bronchopulmonary dysplasia of rat model 29 . To our knowledge, our study reported for the rst time the dysregulation of miR-365-3p in the mouse severe asthma model. We found miR-365-3p is highly expressed in IL-17KO mice and negatively related with IL-17 expression, therefore, miR-365-3p is likely to play an anti-in ammatory role in asthma.
We further predicted the target genes of miR-365-3p, the expression of target genes TRAF3, ARRB2, Sgk1, PIK3R3, ADAM10 and ADM was examined. The dysregulation of ARRB2 in WT model mice and the recovery in IL-17KO model mice were validated at both mRNA and protein levels. Furthermore, the targeting role of miR-365-3p on ARRB2 was con rmed by both luciferase reporter assay and transfection of mimic and inhibitor of miR-365-3p.
ARRB2 (beta-arrestin-2) is an intercellular molecular regulating G protein-coupled receptor function and associating with receptor internalization 30 , which is involved in CD4 + T cells migration, Th2 cell chemotaxis, and in ammatory cytokine production in asthma 31,32 . Increased ARRB2 expression in OVAchallenged mice and repressed in ammatory responses in ARRB2 de ciency mice 33 both demonstrated the key role of ARRB2 in the pathogenesis of asthma. Our study showed the association of IL-17 and ARRB2 via the regulation of miR-365-3p in the severe asthma mouse model.
Overall, through miRNAs expression pro ling in WT and IL-17KO severe asthma mouse models, we identi ed the association of miR-365-3p of IL-17 in severe asthma. The further analysis of target genes indicated ARRB2 is regulated by miR-365-3p. Thus IL-17 may play important roles through miR-365-3p/ARRB2 in severe asthma, which may provide the clue for the potential therapeutic targets.

Conclusions
Taken together, these ndings suggest that IL-17 plays an important role in the in ammatory response and airway remodeling, and miR-365-3p may be a key miRNA involved in this regulation.   House dust mite (HDM) extract induced the production of IL-17 in severe asthma mice. A. The level of IL-17A in mouse Bronchoalveolar lavage uid (BALF) was detected by ELISA, P<0.01. B.

Abbreviations
Immunohistochemically staining of mouse lung tissue sections, the brown area represents IL-17 positive.