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Aberrant cell migration contributes to defective airway epithelial repair in childhood wheeze

Abnormal wound repair has been observed in the airway epithelium of patients with chronic respiratory diseases including asthma. Therapies focusing on repairing vulnerable airways, particularly in early life, present an extremely novel treatment strategy. We report defective lower airway epithelial cell repair to strongly associate with common pre-school and school-aged wheezing phenotypes, characterised by aberrant migration patterns and reduced α5β1 integrin expression. Next generation sequencing identified the PI3K/Akt pathway as the top upstream transcriptional regulator of α5β1 integrin, where Akt activation enhanced repair and α5β1 integrin expression in primary cultures from children with wheeze. Conversely, inhibition of PI3K/Akt signaling in primary cultures from children without wheeze reduced α5β1 expression and attenuated repair. Importantly, the FDA-approved drug celecoxib, and its non-COX2-inhibiting analogue dimethyl-celecoxib, stimulated the PI3K/Akt-integrin α5β1 axis and restored airway epithelial repair in cells from children with wheeze. When compared with published clinical datasets the identified transcriptomic signature was also associated with viral-induced wheeze exacerbations highlighting the clinical potential of such therapy. Collectively, these results identify airway epithelial restitution via targeting the PI3K/Akt-integrin axis as a novel therapeutic avenue for childhood wheeze and asthma. We propose that the next step in the therapeutic development process should be a proof-of-concept clinical trial since relevant animal models to test the crucial underlying premise are unavailable. Research Cell biology Pulmonology


[Main Text: ] Introduction
Approximately 300 million individuals suffer from asthma globally (1) and it remains the commonest chronic respiratory disorder in children. The pathobiology of asthma is complex and poorly understood (2)(3)(4). Whilst current therapies ameliorate symptoms, none affect the longterm trajectory of lung function decline (5)(6)(7). Despite the significantly high global prevalence of asthma, and the magnitude of research investment, there are few new therapies that aim to target specific disease mechanisms. Thus, there is an unmet need for new therapeutic avenues to be explored.
In the airway, normal repair following injury is characterized by spreading and migration into a wound of leading edge cells that surround the wound site. This is followed by basal cell proliferation and differentiation into various epithelial cell subtypes. Migration alone can close small wounds whereas repair of large areas requires proliferation and migration. We previously 6 demonstrated that although primary airway epithelial cells (pAEC) from children with asthma are hyperproliferative, they are ineffective at repairing wounds in vitro (11), in part due to deficient production of the extracellular matrix (ECM) protein fibronectin (FN) (27). With the intent of intervening in early life to prevent irreversible airway damage and persistence of symptoms, we recruited children with recurrent wheeze, as well as healthy controls without wheeze, to test the hypothesis that dysregulated airway epithelial cell repair was also characteristic of this cohort. Furthermore, knowing that airway epithelial cell migration is facilitated via the binding of membrane-bound integrins on their cell surface to FN and other ECM proteins (28)(29)(30)(31), we also hypothesized that aberrant migration and dysregulation of integrin-related pathways contributes to deficient epithelial repair observed. Thus, we focused on the pattern and regulation of migration of leading edge cells following in vitro wounding.
In this study, we show that the defective airway epithelial cell repair phenotype associates with pre-school wheeze and physician diagnosis of asthma. Abnormal leading edge cell migration impairs repair following injury in pAEC cultures from children with wheeze associated with reduced expression of integrin α5β1. Transcriptomic analysis of the leading edge migrating cells identified a number of upstream regulators of integrin expression and migration, of which blunted PI3K/Akt signaling resulted in reduced expression of integrin α5β1 and, as a consequence, pAEC migration and repair following wounding. Importantly, we showed that the PI3K/Akt-integrin α5β1 axis is therapeutically targetable with the FDA-approved drug, celecoxib, and its experimental analogue, dimethyl-celecoxib. Furthermore, dysregulation of the PI3K/Akt-integrin α5β1 axis was also present in independent cohort datasets that associated with wheeze recurrence. Considering its safety profile is known and it is currently prescribed to young 7 children with juvenile arthritis, our findings warrant further preclinical assessment and initial trial of celecoxib in children with recurrent wheeze to improve symptom resolution and reduce length of stay in hospital via its enhancement of airway epithelial repair.

Defective airway epithelial cell repair phenotype associates with respiratory wheeze in children
Previous observations by our group identified a defective repair phenotype in pAEC to associate with physician diagnosis of asthma in school-aged children (11,27,32). This study assessed in vitro wound repair in pAEC cultures from 119 children aged from 1.5 to 17.7 years of age (Table   1). Defective pAEC repair was determined to associate strongly with respiratory wheeze in all children (Table 1). Specifically, defective pAEC repair associated with respiratory wheeze in pre-school aged children (≤5 years old) and school-aged children (>5 years old; Table S1).
Furthermore, the proportion of school-aged children (>5 years old) with physician diagnosis of asthma and complete pAEC repair was significantly lower than their age-matched non-asthmatic counterparts (Fisher exact test p = 1.223e-09; Table S2), corroborating our previously published observations (11,27,32). 9 Consistent with our previous findings (11,27,32), pAEC from children without any respiratory conditions demonstrated a rapid repair response that was completed by 72h post-wounding (p<0.050, Figure 1A, Movie S1). In contrast, pAEC from children with wheeze displayed significantly compromised wound repair capacity and failed to fully repair over the duration of the experiment (p<0.050, Figure 1B, Movie S2). As such, this study aimed to investigate the mechanisms regulating defective pAEC repair in children with respiratory wheeze. 10

Aberrant cell migration contributes to defective repair in airway epithelial cells from children with wheeze
When the migration component of repair was assessed, leading edge cells from children without wheeze migrated consistently towards the center of the wound ( Figure 1C, Movie S1). However, leading edge cells from children with wheeze had a highly variable trajectory distribution, lacking consistent directionality, with some cells migrating backwards into the leading edge ( Figure 1D, Movie S2). The significant reduction in distance migrated (p<0.050, Figure 1E), velocity (p<0.050, Figure 1F), directionality (p<0.050, Figure 1G) and overall forward motion (p<0.050, Figure 1H) contributed to the failure of pAEC from children with wheeze to fully repair.

Integrin α5β1 regulates leading edge cell migration in the airway epithelium
Previous investigations by our group identified that reduced FN synthesis and release contributed to the defective repair of pAEC from children with asthma (27). Furthermore, addition of exogenous FN did not completely restore wound repair (27) suggesting that additional mechanisms are contributing to the defective wound repair. We speculated that the expression of FN-binding integrins is also dysregulated from pre-asthmatics or recurrent wheezers and contributes to the defective repair that characterizes epithelial cells from this cohort.
In pAEC from children without wheeze, confocal imaging demonstrated strong staining intensity for integrin α5 along the first row of leading edge cells, following wounding, whereas staining intensity was progressively weaker in cells distal to the wound ( Figure 2C). Expression of integrin β1 was similar to 5 in cells along the leading edge but was almost undetectable in sites distal to the wound ( Figure 2D). In contrast, integrin α5 and β1 ( Figure 2F-H) protein staining of leading edge cells from children with wheeze was lower than that of their non-wheezing 12 counterparts ( Figure 2E-F) and integrin β1 staining was more diffuse in pAEC from children with wheeze ( Figure 2G) compared to that observed in pAEC from non-wheezing children.
To confirm the role of integrins during airway epithelial repair, functional blocking experiments were performed in pAEC from children without wheeze. Since integrin α5 only forms a functional heterodimer with integrin β1, and β1 can induce intracellular signaling to regulate cell migration, in these experiments, integrin β1 was blocked with anti-β1 integrin antibody. This resulted in the inhibition of wound closure of pAEC from children without wheeze in a concentration-dependent manner ( Figure 2I, Figure S3A) and dysregulated migration patterns ( Figure 2J-K, Figure S3B-F).

Altered transcriptional response to wounding and upstream regulators of integrin α5β1 expression in childhood wheeze
In order to determine major pathways with the capacity to influence migration and repair, we undertook whole genome transcriptional profiling of leading edge cells 24h following scratch wounding. Samples from children with wheeze displayed an altered transcriptional profile as they formed distinct clusters from their non-wheezing counterparts ( Figure 3A). A total of 1,154 genes were differentially expressed (fold change: |1.5|, Benjamini-Hochberg-adjusted p-value < 0.050; Figure 3B), with 664 upregulated (Table S3) and 490 downregulated genes (Table S4), between leading edge pAEC from children with and without wheeze. These differentially expressed genes corresponded to 24 canonical pathways (Table S5), where PI3K/Akt, cholecystikynin/gastrin, TLR, acute phase response and IL-6 signaling were the top five downregulated pathways ( Figure 3C). When the PI3K/Akt signaling pathway was interrogated further, a number of genes (17 up and 17 downregulated) were found to be significantly differentially expressed and highly interconnected suggesting this pathway's key biological role during wound repair ( Figure 3D).
We investigated global transcriptional expression patterns by leading edge cells from primary cultures of children with and without wheeze at 24h post wounding by employing prior knowledge built into the upstream regulator analysis package within Ingenuity ® Systems (IPA ® , QIAGEN, Redwood City, CA, USA, www.qiagen.com/ingenuity). Upstream regulator analysis identified 2 molecules to be activated and 33 to be inhibited that have previously been shown to alter gene expression of either integrin subunit individually or both subunits concurrently (Table   S6). Further analysis revealed upstream regulators of integrin α5 and/or β1 expression including 14 several growth factors and signaling molecules, such as PDGF BB, HRAS, PI3K (complex), ERBB2 and Akt ( Figure 3E, Table S6). We also conducted a comprehensive review of the literature to further refine the candidate regulator from this list known to be involved in airway epithelial cell migration and repair. The PI3K pathway, and its downstream signaling effector, Akt, were identified as the most extensively studied in the literature for their role in airway cell migration and repair regulation ( Table S7).

Inhibition of PI3K/Akt signaling abrogates pAEC migration and repair
Given our transcriptomic analysis and corroboration by prior knowledge (Table S7), we explored whether dysregulated signaling via the PI3K/Akt pathway occurs in pAEC from children with wheeze, thereby reducing the expression of integrin subunits α5 and β1 and impairing the capacity of cells to migrate and repair efficiently. Protein analysis of the Akt pathway determined that although total Akt expression was abundantly expressed, phosphorylated Akt at serine 473 was significantly reduced in pAEC from children with wheeze compared to nonwheezing controls (p<0.050; Figure 3F). To determine the effect of reduced PI3K/Akt signaling on wound repair, we initially treated pAEC cultures from non-wheezing children with Wortmannin, a broad-spectrum PI3K inhibitor (33,34). As expected, the levels of activated Akt were significantly reduced following treatment with Wortmannin (p<0.050, Figure S4A). This resulted in significant reduction in expression of both α5 (p<0.050, Figure S4B) and β1 (p<0.050, Figure S4C) integrin subunits. Finally, inhibition of PI3K signaling resulted in concentration-dependent attenuation of closure rates (p<0.050, Figure S4D) and migration of leading edge cells (p<0.050, Figure S4E-M).
We replicated key experiments using a specific inhibitor of Akt, MK2206 ( Figure 4). Treatment of pAEC from non-wheezing children with increasing concentrations of MK2206 (0.01-1 μM) also resulted in a concentration-dependent inhibition of Akt activation (p<0.050, Figure 4A), and reduced expression of integrin α5 on the cell membrane in as little as 12h (p<0.050, Figure 4B).
Conversely, Akt inhibition did not significantly affect integrin β1 expression following 12h or 48h treatment (p>0.050, Figure 4C). Treatment with MK2206 also reduced wound closure (p<0.050, Figure 4D) and leading edge cell migration, reflected by distance migrated, velocity, directionality and forward migration index compared to vehicle treated cultures from children without wheeze (all p<0.050 respectively, Figure 4E-M), recapitulating repair responses of cells from wheezers.

SC79 targets the Akt/α5β1 integrin pathway to enhance pAEC migration and repair in childhood wheeze
To determine whether activation of Akt could enhance α5β1 integrin expression, migration and wound repair, pAEC cultures of wheezing children were treated with increasing concentrations (0.5-20 μM) of the Akt activator, SC79 ( Figure 5). SC79 treatment augmented Akt signaling (2-20 μM, p<0.050; Figure 5A) and increased protein expression of both integrin subunits α5 (0.5-20 μM, p<0.050; Figure 5B) and β1 (0.5-5 μM, p<0.050; Figure 5C). Importantly, significantly enhanced wound closure of pAEC of children with wheeze in a concentration-dependent manner compared to vehicle and untreated controls (p<0.050, Figure 5D). Improved migration was reflected in greater distance migrated, increased velocity, enhanced directionality and towards the center of the wound (p<0.050, Figure 5E-M).

Celecoxib targets the PI3K/Akt-integrin α5β1 axis to enhance repair of epithelial cells from children with wheeze
Celecoxib, an FDA-approved selective COX2 inhibitor used in the treatment of rheumatoid arthritis, juvenile arthritis and pain management, has been reported to activate PI3K/Akt (35,36). Therefore, we tested whether celecoxib would enhance airway epithelial cell repair in wheezers through the activation of PI3K/Akt signaling and integrin α5 and β1 expression. Furthermore, we also tested dimethyl-celecoxib analogue which lacks inhibitory activity against COX2 to demonstrate that effects on epithelial repair were independent of COX2 inhibition.

Corroboration of a defective pAEC repair signature with independent chronic airway disease cohorts
In the absence of a suitable preclinical animal model of asthma or of intrinsic epithelial dysregulation of PI3K/Akt-integrin axis (37), we conducted meta-analysis of relevant published transcriptomic datasets. We interrogated datasets for evidence of these pathways being implicating in early life wheeze or asthma, as well as identified who would potentially benefit from celecoxib treatment. Specifically, we targeted our comparisons to datasets using ex vivo nasal samples from adults with physician-diagnosed stable, mild asthma (38), or viral-induced acute wheeze exacerbations in children (39). Comparison of our defective lower airway pAEC repair signature to the adult stable, mild asthma dataset (38) identified more than 80 common differentially expressed genes (Table S8), including genes associated with integrin and PI3K/Akt pathways like FN1, GRB2, SRC and SHC1 ( Figure S8). Furthermore, there was over 80% overlap of overrepresented pathways between the two datasets, such as integrin cell surface interactions and growth factor signaling (Table S9) (38). Significantly, evaluation of a published microarray dataset from a longitudinal viral-induced acute wheeze pediatric cohort (64 children) (39) identified five unique modules of co-expressed genes ( Figure 7A). The association of the identified modules to defective pAEC wound repair and recurrence of wheeze exacerbations was assessed as to identify if therapeutic targeting in this pediatric cohort would be efficacious at reducing the number of exacerbations. Indeed, one module of genes was found to associate significantly with recurrence of wheeze exacerbations and defective wound repair ( Figure 7B-C, Table S10). In fact, pathway analysis identified over 45% contingency between our defective repair dataset and the recurrent wheeze-associated gene module with overrepresented pathways also including integrin cell surface interactions and growth factor signaling (Table S11).

20
Network analysis of the identified gene module identified highly interconnected hubs like GRB2, PIK3AP1, ITGA5 and TLN1, which are known to be involved in PI3K/Akt signaling and integrin pathways ( Figure 7D). Our hypothesis that defective pAEC repair through dysregulated PI3K/Akt signaling is implicated in early life wheeze and asthma is identified via meta-analysis of relevant publicly available human gene expression data. Furthermore, these observations indicate that defective pAEC wound repair may persist in adult asthma. Thus, targeting of the PI3K/Akt-integrin axis, particularly in early life, could reduce wheeze frequency, impact on disease trajectory and persistence of early-onset asthma into adulthood.

Discussion
With a focus of identifying therapeutic targets prior to persistent asthma development, we assessed the reparative capacity of the airway epithelium of young children, including pre-school aged children with wheeze. As with our previous observations of defective airway epithelial cell repair in school-aged children with physician-diagnosed asthma (13,15,27), we now report defective airway epithelial cell repair to also associate with recurrent pre-school wheeze. We have demonstrated that aberrant cell migration underpins the defective repair process. Given our previous studies which indicate that exogenous addition of FN improves, but does not provide complete restitution of cultures from children with physician-diagnosed asthma (27), we turned our attention to FN-binding integrins. In the current study, we show reduced expression of integrin α5 and β1 in the airway epithelium of children with wheeze at baseline and following wounding compared to children without wheeze. Using whole cell transcriptomics, we identified several upstream transcriptional regulators of integrin α5 and β1 expression of which the PI3K/Akt signaling pathway was the top predicted regulator and was downregulated in children in cells from children with wheeze. Furthermore, we confirmed reduced Akt activation in pAEC from children with wheeze. Significantly, inhibition of this pathway in pAEC from nonwheezing controls resulted in decreased expression of integrin α5β1, reduced migration and defective repair, mimicking what was observed in pAEC from children with wheeze phenotype.
Conversely, activation of the PI3K/Akt pathway in pAEC from children with wheeze increased integrin α5β1 expression and enhanced epithelial cell migration and repair following wounding, recapitulating a normal response. We demonstrated that FDA-approved celecoxib and its experimental analogue, dimethyl-celecoxib, enhanced integrin expression and restored leading edge cell migration resulting in complete epithelial repair in cultures from children with wheeze.
In the absence of a suitable animal model of asthma or of epithelial dysfunction due to PI3K/Akt-integrin α5β1 downregulation (37), we interrogated published ex vivo pAEC transcriptomic datasets for evidence of these pathways being implicated in early life wheeze or asthma. We discovered that defective wound repair and dysregulated PI3K/Akt expression were associated with: i) adult asthma (38), ii) acute wheeze exacerbations and iii) wheeze recurrence in children presenting to hospital (39). Our observations of defective epithelial repair contributing to the development of respiratory symptoms are also supported by McErlean and colleagues (16) who identified inadequate nasal epithelial repair to differentiate adult asthmatics that experienced acute respiratory exacerbation. The presence of signatures of aberrant epithelial repair in both young children with recurrent wheeze and adults with more persistent symptoms, implies a trajectory that could be mitigated by early intervention to restore normal epithelial function. The in vitro normalization of epithelial cell migration and repair by celecoxib and its analogue without COX-2 inhibitory activity, suggests an easily testable hypothesis and indicate that a safe, early intervention is feasible.
Celecoxib is already approved for use in children with juvenile arthritis and could be repurposed for treatment of children with wheeze relatively expediently requiring only Phase II/III safety and efficacy clinical trials. Although celecoxib has been previously associated with an increased risk of myocardial infarction in adults, these adverse cardiovascular events appear less frequently than with other classical NSAIDs like Ibuprofen (40) and have not been reported in children.
Furthermore, adult asthma patients with aspirin-exacerbated respiratory disease can safely be treated with celecoxib (41). Importantly, no acute lung function decline has been observed following selective COX2 inhibition with celecoxib in asthmatics (42,43). Thus, this may highlight celecoxib as a new area for therapy and further investigation in adults with aspirinexacerbated respiratory disease to improve respiratory outcomes. Our study highlights that the capacity of celecoxib to aid repair in pAEC from children with wheeze is independent of COX2 inhibition. Unlike our findings, previous work in the oncology literature has suggested that celecoxib treatment of cancer cell lines may inhibit PI3K/Akt signaling at high micromolar concentrations (44,45). This discrepancy may highlight dose-dependent and cell-specific responses to celecoxib treatment in vitro. Our observations warrant future investigations on the modulation of the PI3K/Akt/integrin α5β1 axis in the airway epithelium to aid barrier restitution and limit inflammation following injury. In addition, further studies are needed for the therapeutic evaluation of celecoxib in the clinical setting, and consideration of the development of aerosolized formulations (46,47), or analogues, to improve bioavailability in vivo and enhance epithelial repair in the airways of patients with wheeze or asthma.
The extensive patient cohort used in this study reflects the general pediatric population and therefore most children with wheeze providing cells had mild, but persistent symptoms. Despite the relatively simple phenotyping of the cohort we have observed abnormal epithelial cell repair in cultures from children with recurrent wheeze and/or physician-diagnosed asthma used in this and previous studies (15,27). This raises the possibility that a heterogenous group of wheezing and respiratory disorders in young children might represent the clinical expression of an underlying vulnerable epithelial endotype. The reported observations are unlikely to be as a 24 result of therapy since none were receiving inhaled corticosteroids or bronchodilators for at least one month prior to recruitment into the study. In addition, we utilized standard culture and conditional reprogramming methods that conserve the wound repair phenotypes of pAEC (48).
Proteins of interest that were differentially expressed in cells traditionally cultured in monolayers were similarly expressed in the conditionally reprogrammed derived cells. These findings further support our hypothesis that intrinsic abnormalities exist in the airway epithelium of children that associate with established clinical phenotypes, such as recurrent wheeze and asthma.
In summary, we have identified a novel vulnerable epithelium endotype implicated in young children with wheeze that is characterized by abnormal capacity of airway epithelial cells to migrate during wound re-epithelization. Specifically, dampened signaling via the PI3K/Akt pathway leads to reduced protein expression levels of α5 and β1 integrin subunits and stimulation of the PI3K/Akt pathway restores α5 and β1 integrin protein expression and promotes normal migration and wound repair. Enhancing repair of damaged airways in individuals with wheeze and asthmatics could minimize the impact of virus infections, reduce chronic inflammation, minimize airway damage and wheeze recurrence. This study has provided further insight regarding important intrinsic epithelial vulnerability in childhood wheeze and asthma, identified FDA-approved celecoxib as a potential therapy and suggested a rationale for investigating epithelium-centered therapies in young children with wheeze. These observations warrant a clinical trial to assess efficacy of celecoxib to accelerate airway epithelial repair and reduce symptoms of wheeze in children.

Study participants and sample collection
Two cohorts were used in this study: 91 (59 male) children with recurrent wheeze, and 110 (67 male) children with no history of wheeze (Table S12) respiratory questionnaires (49,50). Although stable at the time of sampling, all children with recurrent wheeze had mild respiratory symptoms, such that none had received glucocorticosteroids (inhaled or oral) or -agonists at least one month prior to recruitment and airway sampling. Tracheobronchial AEC were obtained via trans-laryngeal, non-bronchoscopic brushings of children through an endotracheal tube as previously described (11,27,51,52).

Cell culture
This study utilized conditional reprogramming (48,53) and traditional culturing (11,52) methods to establish ex vivo human primary airway epithelial cells from children. The remainder of ex vivo cells were used for RNA extraction and gene expression profiling as previously described (11). All cell culture experiments were performed in primary submerged monolayers in BEGM™ (LONZA).

96-well Essen Woundmaker
To assess wound repair kinetics, linear scratch wounds were created using a commercial monolayer wounding kit (54). Wound closure was calculated through image analysis by the accompanying live-cell imaging system (Essen Bioscience, Ann Arbor, MI, USA).

Leading edge cell tracking and quantification of migration parameters
Manual Tracking, a publicly available ImageJ2 plugin was utilized for its capacity to manually

Data interpretation and quantification of migration parameters
Graphical representation and analysis of the cell migration parameters was performed using the

Gene expression analysis by qPCR
The mRNA expression of FN-binding integrin subunits and housekeeping gene, PPIA, was determined with gene-specific primers listed in the Supplementary (Table S13). Gene expression 29 was determined by using two-step reverse transcription and real time PCR assays, as previously described (11). Relative gene expression was calculated using the 2 -ΔΔCT method by normalization to PPIA housekeeping gene and an endogenous tissue control (57).

In-Cell™ and On Cell™ Western Assays
The protein expression of integrin subunits α5 and β1, phospho-Akt (serine residue 473) and total Akt was quantified via the In-Cell™ and On-Cell™ Western Assays, as described (14).
Briefly, cells were plated at 1.2 × 10 5 cells/cm 2 onto 96-well plates pre-coated with fibronectin, collagen and bovine serum albumin and maintained as described earlier (11,48,52). Briefly, following relevant treatments and controls, confluent cell monolayers were fixed with 4% (w/v) PFA for 20 minutes at room temperature and then either permeabilized with 0.1% (v/v) Triton X- Protein expression was normalized to cell number using the DRAQ5 and Sapphire700 stains.
The activated Akt expression levels were normalized to total Akt expression.

Immunocytochemistry and microscopy
For protein expression and localization analysis of integrin α5 and β1 in pAEC cultures, fluorescent immunocytochemistry and confocal microscopy was utilized. For imaging of cultured pAEC following wounding, pAEC of children with and without wheeze were cultured on 0.8 cm round glass coverslips (pre-coated with fibronectin, type I collagen and bovine serum albumin). Cultures were grown to confluence, scratch wounded with sterile p300 pipette tip and 24h post wounding fixed using 4 % (w/v) PFA for 20 minutes at room temperature and washed three times for 5 minutes per wash with 1x PBS. The cells were stored in 1x PBS at 4°C until required. For immunocytochemical staining for integrin α5 and β1 expression patterns, cultured pAEC were equilibrated to room temperature and incubated with Zuk's blocking buffer for 1h at room temperature, as previously described (11). Following blocking, primary antibodies against human integrin subunits α5 (1:100; clone EPR7854, Abcam) and β1 (1:1000; clone P5D2, Abcam) diluted in Zuk's blocking buffer were incubated for 1h at room temperature followed by Subsequently, the single-end sequence reads were mapped to the reference genome (Homo sapiens GRCh38 v91 -Ensembl) using STAR (version 2.6.0c). The count reads were then indexed to the metadata table using htseq-count (version 0.6.1p1). Alignment quality was assessed with MultiQC (version 1.5) (58). Sample variance was inspected with principal component analysis (PCA) and heatmap. To establish a signature gene expression profile from pAEC from children with or without wheeze, DESeq2 (version 1.20.0) was used to identify genes that had statistically significant differential expression (Benjamini-Hochberg-adjusted p-value<0.050, fold changes |1.5|) between "wheeze leading edge 24h" and "non-wheeze leading edge 24h".

Data interpretation
NetworkAnalyst (59,60) and Ingenuity ® Pathway Analysis (IPA ® , QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ ingenuity-pathway-analysis) were used to facilitate systems-level analyses by enabling the integration, analysis and visualization of gene expression data, in the context of molecular interaction networks and canonical pathways.
Several statistical and analytical tools have been integrated into IPA ® , which were used to rapidly determine canonical pathways that were statistically overrepresented in the transcriptomic experiments of interest. Network analysis of expression changes were performed using NetworkAnalyst (59,60) which is based on curated protein:protein interactions from InnateDB (61), the largest database of interactions curated with respect to genes involved in host responses and provides a major advantage in permitting the creation of substantially enriched networks relevant to integrins, cell migration, and wound repair. Ingenuity ® Pathway Analysis (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ ingenuity-pathway-analysis) was also used to identify transcriptional regulators of integrin α5 and β1 expression with Upstream Regulator Analysis. Upstream Regulator Analysis is a predictive causal analysis tool that identifies upstream molecules or networks of molecular drivers, including transcription factors and endogenous chemicals that may regulate the observed transcriptional differences in the datasets. Specifically, this analysis compares the overlap between known downstream targets of each transcriptional regulator with the differentially expressed (DE) gene lists, and is thus statistically predicted to identify molecules regulating the observed differential expression pattern. Also, IPA ® predicts whether the transcriptional regulators are activated or inhibited, based on known relationships between each regulator and the expression (up-or down-regulation) of the downstream targets within each dataset. These predictions are reported as activation z-scores, and are a second method for ranking predicted upstream transcriptional regulators after the adjusted p-values. Positive z-scores indicate activation and negative z-scores indicate inhibition of the upstream regulator. Absolute activation z-scores ≥2 and adjusted p-values <0.050 were considered significant as recommended by IPA ® .

Evaluation of published transcriptomic datasets
Two published transcriptomic datasets, GSE103166 and doi:10.7303/syn9878922, were selected for validation of our upstream regulator analysis based on utilizing comparable ex vivo pAEC isolation protocols, that is nasal airway samples from children with and without wheeze, or nonasthmatic and asthmatic adults. Although these studies used different transcriptomic platforms, that is Affymetrix Human Gene 2.1 ST Array (39), Agilent ® Genome 4X44K array (16), Illumina ® HiSeq 1000 (17) and Illumina ® HiSeq 2500 (38) as compared to Illumina ® HiSeq 2500 in our study, the cutoffs for differential gene expression analysis were the same (±1.5-fold, adjusted p-values<0.050). Differentially expressed gene lists comparing gene expression in ex vivo pAEC from asthmatic and non-asthmatic adults at baseline and post rhinoviral infection were investigated. Using prior knowledge of protein:protein interactions, minimum networks were generated with NetworkAnalyst and pathway analysis (Reactome) was performed, where datasets were screened for overrepresentation of the PI3K/Akt signaling and integrin pathways.
Weighted gene co-expression network analysis (WGCNA) was utilized to identify gene coexpression networks (modules) or similar function genes that associated with recurrence of respiratory wheeze in the pediatric dataset (39,62). Using WGCNA, gene expression was correlated with sample trait values (gene significance [GS]) and module membership (MM).
Genes with high GS and high MM were considered to be significantly associated with recurrence and modules with possibly common biological processes.

Statistics
Statistical significance (*p-value<0.050) for comparisons between two groups were determined