- Open Access
Role of human rhinovirus in triggering human airway epithelial-mesenchymal transition
© The Author(s). 2017
- Received: 12 April 2017
- Accepted: 24 May 2017
- Published: 30 May 2017
Structural changes in the airways, collectively referred to as airway remodeling, are a characteristic feature of asthma, and are now known to begin in early life. Human rhinovirus (HRV)-induced wheezing illnesses during early life are a potential inciting stimulus for remodeling. Increased deposition of matrix proteins causes thickening of the lamina reticularis, which is a well-recognized component of airway remodeling. Increased matrix protein deposition is believed to be due to the presence of increased numbers of activated mesenchymal cells (fibroblasts/myofibroblasts) in the subepithelial region of asthmatic airways. The origin of these increased mesenchymal cells is not clear, but one potential contributor is the process of epithelial-mesenchymal transition (EMT). We hypothesized that HRV infection may help to induce EMT.
We used the BEAS-2B human bronchial epithelial cells line, which uniformly expresses the major group HRV receptor, to examine the effects of stimulation with HRV alone, transforming growth factor-β1 (TGF-β1), alone, and the combination, on induction of changes consistent with EMT. Western blotting was used to examine expression of epithelial and mesenchymal phenotypic marker proteins and selected signaling molecules. Cell morphology was also examined.
In this study, we show that two different strains of HRV, which use two different cellular receptors, are each capable of triggering phenotypic changes consistent with EMT. Moreover, both HRV serotypes synergistically induced changes consistent with EMT when used in the presence of TGF-β1. Morphological changes were also most pronounced with the combination of HRV and TGF-β1. Viral replication was not essential for phenotypic changes. The synergistic interactions between HRV and TGF-β1 were mediated, at least in part, via activation of mitogen activated protein kinase pathways, and via induction of the transcription factor SLUG.
These data support a role for HRV in the induction of EMT, which may contribute to matrix protein deposition and thickening of the lamina reticularis in airways of patients with asthma.
- Human rhinovirus
- Epithelial-mesenchymal transition
- Transforming growth factor-β1
- MAP kinases
The airways of patients with asthma display a number of characteristic structural changes that are collectively referred to as airway remodeling. These structural changes include an increase in airway smooth muscle mass, goblet cell hyperplasia/metaplasia leading to increased mucus production, epithelial fragility [1, 2], and increased numbers of fibroblasts and myofibroblasts in the subepithelial region that are associated with increased extracellular matrix protein deposition and thickening of the lamina reticularis [3, 4].
Although it was long thought that airway remodeling developed only after years of disease expression, bronchial biopsy studies have now established that components of airway remodeling can be demonstrated in early childhood, even prior to the formal diagnosis of asthma being made . Several studies have demonstrated features of airway remodeling, including thickening of the lamina reticularis, increased smooth muscle mass, increased mucus gland area and angiogenesis in pre-school age children [6–9]. Interestingly, such features were not observed in symptomatic infants with reversible airflow obstruction . This suggests that airway remodeling occurs in response to some inciting stimulus (or stimuli) in early life, and is not a congenital phenomenon.
Human rhinovirus (HRV) infections in early life are a common trigger for childhood wheezing illnesses [11, 12], and HRV-induced wheezing illnesses in the first 3 years of life are a major risk factor for subsequent development of asthma . Longitudinal analysis has shown that pre-school age children have about six HRV infections per year , and serial viral infections can lead to recurrent wheezing episodes . Given the concurrent time frame of recurrent HRV-induced wheezing episodes and the development of AR, it is possible that HRV infections may be one stimulus that could contribute to the initiation and progression of airway remodeling in asthma.
The human airway epithelial cell is the primary site of HRV infection and previous studies have shown that infected epithelial cells release a number of growth factors and proteins linked to airway remodeling [16–18]. The epithelium has also been suggested as a potential source of the increased numbers of mesenchymal cells in the airways during a number of disease conditions through the process of epithelial to mesenchymal transition (EMT), in which typical epithelial phenotypic proteins and morphology are lost and features of mesenchymal cells are acquired [19–22]. In the current studies, we tested the hypothesis that HRV infection of airway epithelial cells could contribute to airway remodeling by inducing EMT. Moreover, because the cytokine transforming growth factor-β (TGF-β) has been described to be a key mediator of EMT in human epithelial cells [20, 23], and is present in increased amounts in asthmatic airways [24, 25], we also examined potential interactions of HRV with TGF-β in inducing EMT.
Epithelial cell culture
Major group HRV species, which use intercellular adhesion molecule-1 (ICAM-1) as a receptor, do not infect more than 5-10% of primary human bronchial epithelial cells in culture . Therefore, we chose to perform the current studies using the BEAS-2B human bronchial epithelial cell line, which uniformly expresses ICAM-1 . This cell line was derived , and kindly provided by Dr. Curtis Harris (NCI, Bethesda, MD). The BEAS-2B cell line has been widely used in the literature to study EMT [23, 29–31], as well as epithelial response to HRV [18, 32–34]. BEAS-2B cells were cultured in Bronchial Epithelial Basal Medium (BEBM) (Lonza, Walkersville, MD) supplemented with bovine pituitary extract, epidermal growth factor, epinephrine, gentamicin and amphotericin B, hydrocortisone, insulin, trans-retinoic acid, transferrin, and triiodothyronine to make Bronchial Epithelial Growth Medium (BEGM) on 6-well culture plates at 37 °C and 5% CO2.
HRV generation and purification
Two serotypes of HRV were used, both of which are members of the HRV-A group. Stocks of major group HRV-16, or of minor group HRV-1A, which uses members of the low-density lipoprotein receptor (LDLR) family for cell entry, were originally obtained from American Type Culture Collection (Rockville, MD). HRV-16 was propagated in WI-38 fetal lung fibroblasts cells (American Type Culture Collection, Rockville, MD), while minor group HRV-1A was propagated in H1-HeLa cells (American Type Culture Collection, Rockville, MD). Both strains of HRV were purified by sucrose density centrifugation to remove ribosomes and soluble factors as previously described [35, 36]. Viral titers were determined by using WI-38 or H1-HeLa cells grown in 96-well plates as previously described .
Treatment of epithelial cell culture
When cells achieved 60% confluence, culture medium was removed and replaced with BEGM from which hydrocortisone had been removed (BEGM no HC), for 24 hours prior to stimulation. Fresh BEGM no HC was then added and BEAS-2B cells were infected with 104.5 50% tissue culture-infective dose (TCID50) U/ml of either purified HRV-16 or purified HRV-1A alone, 10 ng/ml recombinant TGF-β1 (R&D Systems, Minneapolis, MN), or the combination of each HRV and TGF-β1, and subsequently incubated at 34 °C in 5% CO2 for specified time points. In 120-hour experiments, the medium and TGF-β1 were replaced at 48 and 96 hours. We demonstrated that treatment with HRV alone, TGF-β1 alone, or the combination, did not reduce cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability assay (data not shown). For studies examining activation of mitogen activated protein kinase (MAPK) signaling pathways, BEAS-2B cells were pretreated for 24 hours in BEGM no HC then placed in BEBM for one hour prior to experiment treatment to reduce basal activation. BEBM was used for treatment medium.
To render HRV replication-deficient, stocks of purified HRV-16 and HRV-1A were exposed for 5 min to a Spectroline Model XX-15 F high intensity short wavelength (254 nm) ultra-violet (UV) lamp (Spectronics Corp., Westbury, NY) at a distance of 5 cm. It was confirmed that treated virus was replication-deficient by showing that the ability to cause lysis in appropriate host cells (WI-38 or H1-HeLa) was lost. UV-treated virus was used in experiments at doses identical to those for intact (replication competent) HRV in all experiments.
Commercially available pharmacological inhibitors, SB203580 and PD95089 from InvivoGen (San Diego, CA), were used to inhibit p38 MAPK and MEK1/MEK2 (ERK 1/2) MAPK pathways, respectively. For these studies, BEAS-2B cells were pretreated with the inhibitor of interest for 1 hour prior to addition of HRV-16, HRV-1A, TGF-β1, and the combination of each HRV serotype with TGF-β1. Cells were harvested for whole-cell lysates at 24 hours.
Post infection, BEAS-2B cells were lysed in ice-cold lysis buffer (1% Triton X-100 in 1 X MES buffered saline pH 7.4, containing anti-protease tablets, 50 nM sodium orthovanadate, 0.4 M sodium pyrophosphate, and 1 M sodium fluoride). Lysates were analyzed for protein content using a DC Protein Assay (BioRad Laboratories, Mississauga, ON, Canada) as manufacturer recommended.
Equivalent amounts of whole cell lysate were separated by 10% SDS-PAGE, and then transferred to a 0.45 μm nitrocellulose membrane. Membranes were blocked with 5% skim milk, and incubated overnight at 4 °C with the following antibodies: E-cadherin (BD Biosciences, San Jose, CA), cytokeratin-18 and cellular fibronectin (both from Sigma-Aldrich, Oakville, ON, Canada), vimentin, phospho-ERK1/2 mitogen activated protein kinase (MAPK), total-ERK1/2 MAPK, phospho-p38 MAPK, total-p38 MAPK, SLUG, SNAIL, or phospho-SMAD 2/3 (all from Cell Signaling, Danvers, MA). Membranes were then stripped and re-probed for the housekeeping protein, β-tubulin (Sigma-Aldrich, Oakville, ON, Canada).
Densitometry analysis was performed using ImageJ software (version 1.41, National Institute of Health, Bethesda, MD). The fold change in expression of the protein of interest was determined by normalizing the raw pixel count of the protein of interest to the raw pixel count of the respective housekeeping protein. This was then compared to the normalized value for medium control. Data is expressed as fold change over medium control unless specified otherwise.
Assessment of morphological changes
To determine the percentage of cells exposed to each treatment that acquired a mesenchymal morphology, we used ImageJ software to measure the length and width of 75 BEAS-2B cells grown under normal medium conditions. The mean and standard deviation of the length to width ratio was then calculated for these 75 cells. We then calculated the mean length to width ratio + 10 standard deviations and established this number as a threshold that must be exceeded before a cell was considered sufficiently long and thin (spindle-like) to be classified as of a mesenchymal morphology. Using a grid reticule, we then used the same software to count a minimum of 50 cells from each treatment condition in each of three separate experiments and calculate the percentage of cells (mean ± SEM) that showed a mesenchymal phenotype in response to each treatment.
All statistics were performed using GraphPad Prism 6 (GraphPad Software, CA). Data is presented as mean ± standard error of the mean. Non-Gaussian distributed data was analyzed by Kruskal-Wallis one-way analysis of variance (ANOVA) with Dunn’s post hoc analysis. Synergy between groups was determined by summing the values obtained in response to each individual treatment per experiment, and comparing that to the value obtained in response to the combined treatment using Wilcoxon matched-pairs analysis. For all statistical tests, a two-tailed p value of ˂0.05 was considered significant.
Purified HRV-16 causes phenotypic changes characteristic of EMT in BEAS-2B cells, both alone and in combination with TGF-β1
Phenotypic protein alterations occur within 24 h of exposure of BEAS-2B cells to HRV-16, or HRV-16 with TGF-β1
Viral replication is not essential in causing changes associated with EMT
The minor group rhinovirus serotype, HRV-1A, causes phenotypic alterations indicative of EMT
As for HRV-16, HRV-1A alone was not an effective stimulus for induction of mesenchymal markers. Exposure to TGF-β1 alone induced significant expression of fibronectin, and this was synergistically enhanced when TGF-β1 was combined with either HRV-1A or UV-HRV-1A (Fig. 7c and d). Similar data were also observed when expression of vimentin was examined (not shown). Thus, both major and minor group HRV serotypes are able to induce changes consistent with EMT and, in both cases, HRV can synergize with TGF-β1 to trigger changes in marker proteins.
Role of P38 and ERK1/2 MAPK Pathways in EMT
HRV and TGF-β1 both induce expression of the E-cadherin transcriptional repressor, SLUG
Recurrent HRV-induced wheezing illnesses in the first 3 years of life are a major risk factor for subsequent development of asthma . Given that this time frame also coincides with the development of airway remodeling, we hypothesized that HRV infections may contribute to aspects of airway remodeling by altering epithelial cell biology. We, and others, have previously shown that HRV infection of human airway epithelial cells, both in vitro and in vivo, can lead to the induction of a number of growth factors linked to airway remodeling [16–18], as well as of chemotactic agents that can cause migration of mesenchymal cells towards the epithelial layer . We now provide direct evidence that two different serotypes of HRV are also able to drive epithelial phenotypic and morphological changes that are indicative of EMT, further supporting a potential role of HRV infections in airway remodeling. Our data contrast somewhat with a recent report that HRV-39 could induce EMT-like marker protein changes in regenerating, but not normal epithelial cell cultures .
The pleiotropic cytokine, TGF-β1 has been reported to be a key initiator of EMT in profibrotic processes occurring in the airways in asthma [20, 23, 45], and levels of TGF-β1 are elevated in the airways of both children and adults with asthma [24, 25]. Given the central role ascribed to TGF-β1 in triggering EMT, we were surprised to observe that both serotypes of HRV tested were more effective than TGF-β1 at inducing the loss of the epithelial phenotypic marker proteins, E-cadherin and cytokeratin-18. This data contrasts with prior reports of TGF-β1 triggering the loss of epithelial markers in both BEAS-2B cells  and primary human airway epithelial cells . The reasons for this discrepancy are unclear, although differences in culture medium and growth conditions could potentially explain the difference between our data and those of Doerner and colleagues who grew BEAS-2B cells in a keratinocyte culture medium . It is not feasible that BEAS-2B cells used in our studies do not express receptors for TGF-β1, given that this cytokine was clearly effective in inducing mesenchymal markers. Our data were consistent with those of Heijink and coworkers who also found that TGF-β1 had only a modest effect on the loss of epithelial markers . Nonetheless, to examine this issue further we used activin A, another member of the TGF-β superfamily that we have previously shown to be induced in both BEAS-2B and primary airway epithelial cells in response to HRV infection  as an alternative ligand for the TGF-β receptor. Consistent with the data obtained using TGF-β1, activin A also caused only a modest loss of E-cadherin but synergized with HRV-16 in inducing loss of this epithelial marker.
It is not clear why exposure to HRV is so effective in downregulating epithelial markers. One possible explanation relates to observations that, in addition to triggering some of the signal pathways studied in the current work, both the major (ICAM-1) and minor (LDL-receptor) HRV receptors are known to be associated with lipid rafts , which, in turn, are known to associated with the actin cytoskeleton . Indeed, it has been known for some time that ICAM-1 is associated with the actin cytoskeleton . More recently, it has been shown that binding of HRV to ICAM-1 is known to recruit the cytoskeletal linker protein ezrin to ICAM-1 . Thus binding of HRV to it receptor (ICAM-1 or LDLR) likely leads to rearrangements in the actin cytoskeleton and/or microtubules, as has been shown for other picornaviruses . Since the actin cytoskeleton is integral to the retention of an epithelial phenotype, and since cytokeratin and junctional proteins, such as E-cadherin, are closely affiliated with the actin cytoskeleton, this may lead to an increased capacity of HRV to induce loss of these epithelial markers, setting the stage for mesenchymal transition.
In contrast to data for epithelial markers, TGF-β1 alone was more effective than either serotype of HRV alone in inducing expression of the mesenchymal markers, fibronectin and vimentin. There was a clear interaction between the two stimuli, however, as the combination of HRV and TGF-β1 clearly induced synergistic changes in both epithelial and mesenchymal markers. The combination of HRV and TGF-β1 was also most effective in causing morphological changes consistent with EMT. This is the first demonstration of an interaction between HRV and TGF-β1 in causing EMT-like changes, but our data show some similarities to those of Heijink and co-workers, who also found that TGF-β1 had only modest effects on reducing expression of epithelial markers, but synergized with house dust mite to induce changes consistent with EMT . Moreover, TGF-β1 has also been reported to synergize with nicotine to induce EMT .
EMT is generally considered to be a slow and progressive process, with intermediate stages in which cells have the characteristics of both epithelial and mesenchymal cells . It was unexpected, therefore, to find that changes associated with EMT, and interactions with TGF-β1, could already be observed within 24 h of HRV exposure, and to find that viral replication was not required for HRV to exert its effects. This implied a role for early, receptor-dependent signaling, rather than replication dependent signaling through double stranded RNA pattern recognition receptors. Given that both major and minor group HRV serotypes induced phenotypic changes, relevant signals must be sent via either ICAM-1 or LDLR. Because little is known about early signaling through LDLR, we examined activation of the p38 and ERK1/2 MAPK pathways, as these are known to be activated both by TGF-β1 [53, 54], and via ICAM-1 signaling [37, 41, 42], to determine if these may also contribute to signaling via the LDLR. As expected, HRV-16 and TGF-β1 each induced activation of the p38 MAPK pathway and we also provide the first evidence that HRV-1A also induced p38 activation. The combination of each serotype of HRV with TGF-β1 led to an approximately additive activation in each case. Despite a prior report of ERK1/2 activation by TGF-β1 in intestinal epithelial cells , TGF-β1 alone did not induce appreciable activation of the ERK1/2 pathway in BEAS-2B cells. As expected, HRV-16 induced robust activation of ERK1/2 and this was slightly enhanced in the presence of TGF-β1. By contrast, HRV-1A was considerably less effective in activation of ERK1/2 and there was no additional response in the presence of TGF-β1.
To determine the potential role of MAPK pathways in EMT induced by HRV and TGF-β1, selective inhibitors of each pathway were used to examine effects on fibronectin expression. Inhibition of the p38 pathway significantly inhibited fibronectin expression induced by either serotype of HRV alone, and in combination with TGF-β1. Surprisingly, inhibition of this pathway had little effect on responses to TGF-β1 alone. Although TGF-β1 did not induce a marked activation of ERK1/2, inhibition of this pathway significantly reduced fibronectin induction in response to the combination of HRV-16 and TGF-β1 without affecting the modest response to HRV-16 alone. Interestingly, fibronectin induction in response to HRV-1A, either alone or combined with TGF-β1, was enhanced by inhibition of the ERK1/2 pathway. Thus the p38 MAPK pathway appears to play a role in induction of EMT by both major and minor group HRV strains, alone and in conjunction with TGF-β1, but the ERK1/2 pathway exerts different effects depending upon the HRV strain receptor involved.
To further examine the underlying basis of the potential mechanisms by which HRV may interact with TGF-β1 to induce EMT, we examined pathways implicated in TGF-β1 signaling. We provide the first evidence that both HRV-16 and HRV-1A are not only able to induce expression of the E-cadherin transcriptional repressor, SLUG, but that the combination of each HRV serotype with TGF-β1 induces activation of SLUG that is greater than that seen with TGF-β1 alone. This provides a potential mechanism to support the reduced expression of E-cadherin seen with HRV alone, and the synergistic induction observed with the combination of HRV and TGF-β1. Interestingly, consistent with an earlier report , while each HRV serotype appeared to modestly induce activation of a second, related transcriptional repressor SNAIL, the combination of HRV and TGF-β1 did not induce any activation above that observed with TGF-β1 alone. Finally, we showed that neither HRV-16 nor HRV-1A induced activation of the SMAD2/3 pathway, and the combination of either strain of virus with TGF-β1 had no effect above that seen with TGF-β1 alone.
A limitation of this study is the use of the BEAS-2B bronchial epithelial cell line. This was chosen because less than 10% of primary cells can be infected by major group HRV strains , raising concerns that insufficient “signal-to-noise” would be obtained. As noted, the BEAS-2B cell has been commonly used to examine EMT [23, 29–31], and has been shown to reproduce effects observed with HRV in primary cells in a number of studies [16, 55–57]. Nonetheless, it will be important to extend these studies to primary epithelial cells. This should be feasible having established that HRV-1A induced EMT as minor group HRV can infect a greater percentage of primary cells than major group strains . It will also be important to determine if HRV-induced EMT is enhanced in epithelial cells from asthmatic children or adults.
It should be noted that, while EMT is a well-recognized process in both physiological and pathophysiological conditions, it remains unclear what percentage of fibroblasts and myofibroblasts in the asthmatic airways derive from EMT. Studies using genetic fate tracking of lung epithelial cells in a mouse model of allergic asthma reported that approximately 30% of murine lung fibroblasts were derived from epithelial cells that underwent EMT , but it is far from clear if this is the case in humans.
In summary, we provide the first evidence that both HRV-16 and HRV-1A, representing major group and minor group serotypes, respectively, are able to induce phenotypic and morphological changes in bronchial epithelial cells that are consistent with EMT. HRV appears to play a greater role in the loss of epithelial markers, but both serotypes of HRV synergize with TGF-β1 to induce changes in epithelial and mesenchymal markers and to change morphology. The effects of HRV on EMT do not depend on viral replication but appear to involve activation of MAPK pathways, as well as activation of the transcriptional repressor SLUG. Taken together, our data add further support for the concept that repeated HRV infections may contribute to the development and progression of airway remodeling in asthma.
Grant number 43923 from the Canadian Institutes of Health Research (CIHR) supported this work. Dr. Proud is the recipient of a Tier 1 Canada Research Chair in Inflammatory Airway Diseases. The funding body played no role in the design of the study; in the collection, analysis and interpretation of the date; in writing the manuscript; or in the decision to submit for publication.
Availability of data and materials
The authors’ unpublished data are available upon request.
DMM and DP conceived of the project. DMM performed all experiments. DMM and DP both performed data analysis and interpretation. DMM wrote the initial draft of the manuscript. DP performed all final editing. Both authors read and approved the final manuscript for submission.
DMM has no competing interests in this manuscript. DP has received consulting fees from AstraZeneca, Janssen, Pfizer and Procter & Gamble, and has received research grants from AstraZeneca and MedImmune. None of these applied to the current work.
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- Al-Muhsen S, Johnson JR, Hamid Q. Remodeling in asthma. J Allergy Clin Immunol. 2011;128:451–62.View ArticlePubMedGoogle Scholar
- Jeffery PK. Remodeling and inflammation of bronchi in asthma and chronic obstructive pulmonary disease. Proc Am Thorac Soc. 2004;1:176–83.View ArticlePubMedGoogle Scholar
- Benayoun L, Druilhe A, Dombret M-C, Aubier M, Pretolani M. Airway structural alterations selectively associated with severe asthma. Am J Respir Crit Care Med. 2003;167:1360–8.View ArticlePubMedGoogle Scholar
- Brewster CEP, Howarth PH, Djukanovic R, Wilson J, Holgate ST, Roche wR. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol. 1990;3:507–11.View ArticlePubMedGoogle Scholar
- Pohunek P, Warner JO, Turzíková J, Kudrmann J, Roche WR. Markers of eosinophilic inflammation and tissue re-modelling in children before clinically diagnosed bronchial asthma. Pediatr Allergy Immunol. 2005;16:43–51.View ArticlePubMedGoogle Scholar
- Saglani S, Payne DN, Zhu J, Nicholson AG, Bush A, Jeffery PK. Early detection of airway wall remodeling and eosinophilic inflammation in preschool wheezers. Am J Respir Crit Care Med. 2007;176:858–64.View ArticlePubMedGoogle Scholar
- Payne DN, Rogers AV, Adelroth E, Bandi V, Guntupalli KK, Bush A, Jeffery PK. Early thickening of the reticular basement membrane in children with difficult asthma. Am J Respir Crit Care Med. 2003;167:78–82.View ArticlePubMedGoogle Scholar
- O’Reilly R, Ullmann N, Irving S, Bossley CJ, Sonnappa S, Zhu J, Oates T, Banya W, Jeffery PK, Bush A, Saglani S. Increased airway smooth muscle in preschool wheezers who have asthma at school age. J Allergy Clin Immunol. 2013;131:1024–32.View ArticlePubMedGoogle Scholar
- Lezmi G, Gosset P, Deschildre A, Abou-Taam R, Mahut B, Beydon N, de Blic J. Airway remodeling in preschool children with severe recurrent wheeze. Am J Respir Crit Care Med. 2015;192:164–71.View ArticlePubMedGoogle Scholar
- Saglani S, Malmström K, Pelkonen AS, Mamberg P, Lindahl H, Kajosaari M, Turpeinen M, Rogers AV, Payne DN, Bush A, Haahtela T, Mäkelä MJ, Jeffery PK. Airway remodeling and inflammation in symptomatic infants with reversible airflow obstruction. Am J Respir Crit Care Med. 2005;171:722–7.View ArticlePubMedGoogle Scholar
- Lemanske Jr RF, Jackson DJ, Gangnon RE, Evans MD, Li Z, Shult PA, Kirk CJ, Reisdorf E, Roberg KA, Anderson EL, Carlson-Dakes KT, Adler KJ, Gilbertson-White S, Pappas TE, DaSilva DF, Tisler CJ, Gern JE. Rhinovirus illnesses during infancy predict subsequent childhood wheezing. J Allergy Clin Immunol. 2005;116:571–7.View ArticlePubMedGoogle Scholar
- Kotaniemi-Syrjänen A, Vainionpää R, Reijonen TM, Waris M, Korhonen K, Korppi M. Rhinovirus-induced wheezing in infancy-the first sign of childhood asthma? J Allergy Clin Immunol. 2003;111:66–71.View ArticlePubMedGoogle Scholar
- Jackson DJ, Gangnon RE, Evans MD, Roberg KA, Anderson EL, Pappas TE, Printz MC, Lee W-M, Shult PA, Reisdorf E, Carlson-Dakes KT, Salazar LP, DaSilva DF, Tisler CJ, Gern JE, Lemanske Jr RF. Wheezing rhinovirus illnesses in early life predict asthma development in high-risk children. Am J Respir Crit Care Med. 2008;178:667–72.View ArticlePubMedPubMed CentralGoogle Scholar
- Winther B, Hayden FG, Hendley JO. Picornavirus infection in children diagnosed by RT-PCR during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season. J Med Virol. 2006;78:644–50.View ArticlePubMedGoogle Scholar
- Jartti T, Lee W-M, Pappas T, Evans M, Lemanske Jr RF, Gern JE. Serial viral infections in infants with recurrent respiratory illnesses. Eur Respir J. 2008;32:314–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Leigh R, Oyelusi W, Wiehler S, Koetzler R, Zaheer RS, Newton R, Proud D. Human rhinovirus infection enhances airway epithelial cell production of growth factors involved in airway remodeling. J Allergy Clin Immunol. 2008;121:1238–45.View ArticlePubMedGoogle Scholar
- Tacon CE, Wiehler S, Holden NS, Newton R, Proud D, Leigh R. Human rhinovirus infection of airway epithelial cells upregulates MMP-9 production via NF-κB. Am J Respir Cell Mol Biol. 2010;43:201–9.View ArticlePubMedGoogle Scholar
- Psarras S, Volonaki E, Skevaki CL, Xatzipsalti M, Bossios A, Pratsinis H, Tsigkos S, Gourgiotis D, Constantopoulos AG, Papapetropoulos A, Saxoni-Papageorgiou P, Papadopoulos NG. Vascular endothelial growth factor-mediated induction of angiogenesis by human rhinovirus. J Allergy Clin Immunol. 2006;117:291–7.View ArticlePubMedGoogle Scholar
- Iwano M, Plieth D, Danoff TM, Xue C, Okada H, Neilson EG. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest. 2002;110:341–50.View ArticlePubMedPubMed CentralGoogle Scholar
- Hackett T-L, Warner SM, Stafanowicz D, Shaheen F, Pechkovsky DV, Murray LA, Argentieri R, Kicic A, Stick SM, Bai TR, Knight DA. Induction of epithelial-mesenchymal transition in primary airway epithelial cells from patients with asthma by transforming growth factor-β1. Am J Respir Crit Care Med. 2009;180:122–33.View ArticlePubMedGoogle Scholar
- Willis BC, Liebler JM, Luby-Phelps K, Nicholson AG, Crandall ED, du Bois RM, Borok Z. Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transfrming growth factor-beta1: potential role in idiopathic pulmonary fibrosis. Am J Pathol. 2005;166:1321–32.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG, Brumwell AN, Sheppard D, Chapman HA. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by extracellular matrix. Proc Natl Acad Sci U S A. 2006;103:13180–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Doerner AM, Zuraw BL. TGF-β1 induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells is enhanced by IL-1β but not abrogated by corticosteroids. Respir Res. 2009;10:100.View ArticlePubMedPubMed CentralGoogle Scholar
- Redington AE, Madden J, Frew AJ, Djukanovic R, Roche W, Holgate ST, Howarth PH. Transforming growth factor-β1 in asthma. Measurement in bronchoalveolar lavage fluid. Am J Respir Crit Care Med. 1997;156:642–7.View ArticlePubMedGoogle Scholar
- Vignola AM, Chanez P, Chiappara G, Merendino A, Pace E, Rizzo A, la Rocca AM, Bellia V, Bonsignore G, Bousquet J. Transforming growth factor-β expression in mucosal biopsies in asthma and chronic bronchitis. Am J Respir Crit Care Med. 1997;156:591–9.View ArticlePubMedGoogle Scholar
- Mosser AG, Brockman-Schneider R, Amineva S, Burchell L, Sedgewick JB, Busse WW, Gern JE. Similar frequency of rhinovirus-infectable cells in upper and lower airway epithelium. J Infect Dis. 2002;185:734–43.View ArticlePubMedGoogle Scholar
- Subauste MC, Choi D-C, Proud D. Transient exposure of human bronchial epithelial cells to cytokines leads to persistent increased expression of ICAM-1. Inflammation. 2001;25:373–80.View ArticlePubMedGoogle Scholar
- Reddel RR, Ke Y, Gerwin BI, McMenamin MG, Lechner JF, Su RT, Brash DE, Park J-B, Rhim JS, Harris CC. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res. 1988;48:1904–9.PubMedGoogle Scholar
- Hosper NA, van den Berg PP, de Rond S, Popa ER, Wilmer MJ, Masereeuw R, Bank RA. Epithelial-to-mesenchymal transition in fibrosis: collagen type I expression is highly upregulated after EMT, but does not contribute to collagen deposition. Exp Cell Res. 2013;319:3000–9.View ArticlePubMedGoogle Scholar
- Hosoki K, Kainuma K, Toda M, Harada E, Chelakkot-Govindalayathila AL, Roeen Z, Nagao M, D’Alessandro-Gabazza CN, Fujisawa T, Gabazza EC. Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils. Biochem Biophys Res Commun. 2014;449:351–6.View ArticlePubMedGoogle Scholar
- Itoigawa Y, Harada N, Harada S, Katsura Y, Makino F, Ito J, Nurwidya F, Kato M, Takahashi F, Atsuta R, Takahashi K. TWEAK enhances TGF-β-induced epithelial-mesenchymal transition in human bronchial epithelial cells. Respir Res. 2015;16:48.View ArticlePubMedPubMed CentralGoogle Scholar
- Subauste MC, Jacoby DB, Richards SM, Proud D. Infection of a human respiratory epithelial cell line with rhinovirus. Induction of cytokine release and modulation of susceptibility to infection by cytokine exposure. J Clin Invest. 1995;96:549–57.View ArticlePubMedPubMed CentralGoogle Scholar
- Unger BL, Faris AN, Ganesan S, Comstock AT, Hershenson MB, Sajjan US. Rhinovirus attenuates non-typeable Haemophilus influenza-stimulated IL-8 responses via TLR2-dependent degradation of IRAK-1. PLoS Pathog. 2012;8:e1002969.View ArticlePubMedPubMed CentralGoogle Scholar
- Eddleston J, Lee RU, Doerner AM, Herschbach J, Zuraw BL. Cigarette smoke decreases the innate responses of epithelial cells to rhinovirus infection. Am J Respir Cell Mol Biol. 2011;44:118–26.View ArticlePubMedGoogle Scholar
- Gern JE, Dick EC, Lee WM, Murray S, Meyer K, Handzel ZT, Busse WW. Rhinovirus enters but does not replicate inside monocytes and airway macrophages. J Immunol. 1996;156:621–7.PubMedGoogle Scholar
- Sanders SP, Siekierski ES, Porter JD, Richards SM, Proud D. Nitric oxide inhibits rhinovirus-induced cytokine production and viral replication in a human respiratory epithelial cell line. J Virol. 1998;72:934–42.PubMedPubMed CentralGoogle Scholar
- Wang X, Lau C, Wiehler S, Pow A, Mazzulli T, Gutierrez C, Proud D, Chow C-W. Syk is downstream of intercellular adhesion molecule-1 and mediates human rhinovirus activation of p38 MAPK in airway epithelial cells. J Immunol. 2006;177:6859–70.View ArticlePubMedGoogle Scholar
- Lau C, Wang X, Song L, North M, Wiehler S, Proud D, Chow C-W. Syk associates with clathrin and mediates phosphatidylinositol 3-kinase activation during human rhinovirus internalization. J Immunol. 2008;180:870–80.View ArticlePubMedGoogle Scholar
- Bentley JK, Newcomb DC, Goldsmith AM, Jia Y, Sajjan US, Hershenson MB. Rhinovirus activates interleukin-8 expression via a Src/p110β phosphatylinositol 3-kinase pathway in human airway epithelial cells. J Virol. 2007;81:1186–94.View ArticlePubMedGoogle Scholar
- Newcomb DC, Sajjan U, Nanua S, Jia Y, Goldsmith AM, Bentley JK, Hershenson MB. Phosphatidylinositol 3-kinase is required for rhinovirus-induced airway epithelial cell interleukin-8 expression. J Biol Chem. 2005;280:36952–61.View ArticlePubMedGoogle Scholar
- Wiehler S, Proud D. Interleukin-17A modulates human airway epithelial responses to human rhinovirus infection. Am J Physiol Cell Mol Physiol. 2007;293:L505–15.View ArticleGoogle Scholar
- Zaheer RS, Koetzler R, Holden NS, Wiehler S, Proud D. Selective transcriptional down-regulation of human rhinovirus-induced production of CXCL10 from airway epithelial cells via the MEK1 pathway. J Immunol. 2009;182:4854–64.View ArticlePubMedGoogle Scholar
- Shelfoon C, Shariff S, Traves SL, Kooi C, Leigh R, Proud D. Chemokine release from human rhinovirus-infected airway epithelial cells promotes fibroblast migration. J Allergy Clin Immunol. 2016;138:110–22.View ArticleGoogle Scholar
- Faris AN, Ganesan S, Chattoraj A, Chattoraj SS, Comstock AT, Unger BL, Hershenson MB, Sajjan US. Rhinovirus delays cell repolarization in a model of injured/regenerating human airway epithelium. Am J Respir Cell Mol Biol. 2016;55:487–99.View ArticlePubMedGoogle Scholar
- Minshall EM, Leung DYM, Martin RJ, Song YL, Cameron L, Ernst P, Hamid Q. Eosinophil-associated TGF-β1 mRNA expression and airways fibrosis in bronchial asthma. Am J Respir Cell Mol Biol. 1997;17:326–33.View ArticlePubMedGoogle Scholar
- Heijink IH, Postma DS, Noordhoek JA, Broekema M, Kapus A. House dust mite-promoted epithelial to mesenchymal transition in human bronchial epithelium. Am J Respir Cell Mol Biol. 2010;42:69–79.View ArticlePubMedGoogle Scholar
- Blaas D. Viral entry pathways: the example of common cold viruses. Wien Med Wochenscr. 2016;166:211–26.View ArticleGoogle Scholar
- Chichili GR, Rodgers W. Cytoskeleton-membrane interactions in membrane raft structure. Cell Mol Life Sci. 2009;66:2319–28.View ArticlePubMedPubMed CentralGoogle Scholar
- Carpén O, Pallai P, Staunton DE, Springer TA. Association of intercellular adhesion molecule-1 (ICAM-1) with actin-containing cytoskelatin and α-actinin. J Cell Biol. 1992;118:1223–34.View ArticlePubMedGoogle Scholar
- Armer H, Moffat K, Wileman T, Belsham GJ, Jackson T, Duprex WP, Ryan M, Monaghan P. Foot-and-mouth disease virus, but not bovine enterovirus, targets the host cell cytoskeleton via the nonstructuralprotein 3CproV. J Virol. 2008;82:10556–66.View ArticlePubMedPubMed CentralGoogle Scholar
- Zou W, Zou Y, Zhao Z, Li B, Ran P. Nicotine-induced epithelial-mesenchymal transition via Wnt/β-catenin signaling in human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2013;304:L199–209.View ArticlePubMedGoogle Scholar
- Bartis D, Mise N, Mahida RY, Eickelberg O, Thickett DR. Epithelial-mesenchymal transition in lung development and disease: does it exist and is it important? Thorax. 2014;69:760–5.View ArticlePubMedGoogle Scholar
- Bakin AV, Rinehart C, Tomlinson AK, Arteaga CL. p38 mitogen activated protein kinase is required for TGFβ-mediated fibroblastic differentiation and cell migration. J Cell Sci. 2002;115:3193–206.PubMedGoogle Scholar
- Hartsough MT, Mulder KM. Transforming growth factor β activation of p44mapk in proliferating cultures of epithelial cells. J Biol Chem. 1995;270:7117–24.View ArticlePubMedGoogle Scholar
- Hudy MH, Traves SL, Wiehler S, Proud D. Cigarette smoke modulates rhinovirus-induced airway epithelial chemokine production. Eur Respir J. 2010;35:1256–63.View ArticlePubMedGoogle Scholar
- Khaitov MR, Laza-Stanca V, Edwards MR, Walton RP, Rohde G, Contoli M, Papi A, Stanciu LA, Kotenko SV, Johnston SL. Respiratory virus induction of alpha-, beta, and lambda-interferons in bronchial epithelial cells and peripheral blood mononuclear cells. Allergy. 2009;64:375–86.View ArticlePubMedGoogle Scholar
- Wang Q, Nagarkar DR, Bowman ER, Schneider D, Gosangi B, Lei J, Zhao Y, McHenry CL, Burgens RV, Miller DJ, Sajjan US, Hershenson MB. Role of double-stranded RNA pattern recognition receptors in rhinovirus-induced airway epithelial cell responses. J Immunol. 2009;183:6989–97.View ArticlePubMedPubMed CentralGoogle Scholar
- Konno S, Grindle KA, Lee W-M, Schroth MK, Mosser AG, Brockman-Schneider RA, Busse WW, Gern JE. Interferon-γ enhances rhinovirus-induced RANTES secretion by airway epithelial cells. Am J Respir Cell Mol Biol. 2002;26:594–601.View ArticlePubMedGoogle Scholar
- Johnson JR, Roos A, Berg T, Nord M, Fuxe J. Chronic respiratory aeroallergen exposure in mice induces epithelial-mesenchymal transition in the large airways. PLoS One. 2011;6:e16175.View ArticlePubMedPubMed CentralGoogle Scholar