Background
Airway remodelling in asthmatics is defined as structural abnormalities including, epithelial integrity loss, basement membrane thickening, and goblet cell and submucosal gland enlargement [
1]. Airway remodelling may predispose asthmatics to exacerbations [
2]. The airway epithelium consists of multiple distinct cell types; ciliated columnar, goblet, side population, serous and basal cells [
3]. Basal cells are capable of self-renewal and differentiation into mucus-secreting and ciliated cells [
4,
5]. The epithelium of asthmatics displays a greater proportion of basal and fewer ciliated cells [
6], suggesting dysregulated differentiation. Particularly in severe asthma, increased epithelial proliferation contributes to thickened epithelium and
lamina reticularis and hence airway narrowing [
7,
8]. Dysregulated epithelial differentiation therefore plays an important role in the remodelling process in asthma. These abnormalities are also associated with functional aberrations including deficient innate immune responses [
3,
9,
10]. The innate immune function of the epithelium is essential for defence against inhaled pathogens such as viruses [
3,
11‐
13]. The differentiation state of the airway epithelium is also important for innate immunity through the compartmentalization of receptors and mediator production [
4]. Consequently, structural and functional abnormalities in the epithelium may contribute to increased susceptibility of asthmatics to noxious environmental stimuli, including respiratory viruses (e.g. influenza A virus [IAV]).
The IAV H1N1 causes significant morbidity and mortality in annual seasonal epidemics [
14]. This virus damages the epithelium [
15] and triggers inflammation and cell signalling events resulting in additional airway remodelling and potentially exacerbations of asthma [
14].
microRNAs (miRNAs) are small non-coding RNAs which regulate the expression of up to 60% of human genes [
16]. Further, changes in specific miRNAs during human airway epithelial cell differentiation regulates gene and protein expression important for differentiation [
17]. miRNAs are hence crucial in most biological and pathological processes [
18,
19], including severe asthma [
20]. Some miRNAs, such as miR-20a from the miR-17-92 cluster, promote the proliferation of lung epithelial progenitor cells [
21]. Whereas, others such as miR-22, are differentiation specific and suppress different genes responsible for cell proliferation [
10,
22‐
24]. Several miRNAs are also associated with the regulation of innate and adaptive immunity, including miR-132 [
25,
26]. Consequently, miRNAs may play a major role in phenotypic and functional abnormalities of airway epithelial cells. IAV H1N1 infection is also reported to dysregulate the expression of some miRNAs in human lung epithelial cells, affecting immune responses [
27,
28].
In this study, we hypothesized that the expression of miRNAs responsible for proliferation, miR-20a, are elevated, whereas miRNAs associated with differentiation, miR-22, are down-regulated in airway epithelial cells of asthmatics. These defects may form the link between abnormal airway epithelial cell differentiation and remodelling. In addition, IAV H1N1 infection may further dysregulate abnormalities in these miRNAs and hence their targets in the airway epithelial cells of asthmatics. Thus, we assessed the expression and role of these miRNAs in the context of airway remodelling in primary bronchoepithelial cells (pBECs) obtained from asthmatics, cultured as monolayers or differentiated ALI conditions at baseline level and after IAV H1N1 infection.
Discussion
We showed that under basal conditions, the pattern of miR-20a, − 132 and − 22 expression is similar in pBECs cultured in monolayer and differentiated ALI conditions, and are not affected by asthma. Our data support previous reports by Martinez-Nunez et al., who did not observe any changes in expression of these miRNAs in pBECs form non-asthmatics and asthmatics cultured as monolayers [
38], as well as Williams et al., who also reported no significant differences in the expression of miRNAs in airway biopsies obtained from normal and mild asthmatic patients [
39]. Moreover, IAV H1N1 infection did not affect the expression of these miRNAs when cells were cultured in submerged monolayers. In contrast, in cells cultured at the ALI, the kinetics of miR-22 expression was different after IAV H1N1 infection in cells from asthmatics compared with non-asthmatics. Specifically, miR-22 expression increased in pBECs of non-asthmatics but remained unchanged in asthmatics. We then found in non-asthmatics, the patterns of miR-22 expression were concomitantly opposite to CD147 gene, HDAC4 gene and protein, and their downstream MMP-9 gene and protein levels. In contrast, in pBECs from asthmatics, the expression of CD147 increased but miR-22, HDAC4 and MMP-9 expression remained unchanged after infection. To determine the mechanism responsible for IAV H1N1 induced induction of CD147 in cells from asthmatics, we further assessed the effect of infection on other regulatory factors of these candidate target genes. Our data indicate that IAV H1N1 infection induced c-Myc and SP1 gene expression in pBECs of asthmatics which may underpin the increased CD147 levels. In addition, we confirmed CD147 and HDAC4 are targets of miR-22. Comparing the effect of ectopic miR-22 mimic expression on its targets in pBECs of non-asthmatics with asthmatics showed that miR-22 overexpression suppressed CD147 in both cohorts whereas HDAC4 was suppressed at the protein level in non-asthmatics and mRNA level in asthmatics. These data indicate that miR-22 regulates CD147 and HDAC4 expression.
No differences were observed in morphology or physical properties of pBECs between non-asthmatics and asthmatics cultured as monolayers or at ALI. This is in agreement with previously reported data by Hackett et al., who showed that TEER was not different between pBECs from non-asthmatics and asthmatics [
6]. While we did not assess different cell types or tight junction complexes, our TEER data were in contrast with Xiao et al., who showed tight junction and TEER was significantly lower in cultures from asthmatics than from non-asthmatics [
55]. This discrepancy may be due to differences in sample numbers (5 vs 40). Furthermore, we observed similar levels of infection in pBECs from both groups at 24 h post infection which support data reported by Fujimoto et al. [
56]. The virus titres in bronchoalveolar lavage fluid from a mouse model of bronchial asthma remained similar to control up to 2 days post IAV H1N1 infection and reached the highest level after 3 days of infection in an asthma model [
56]. Despite these similarities, the different pattern of miR-22 expression in epithelial cells of adults with severe asthma may indicate the potential involvement of this miRNA in asthma pathogenesis. Our results highlight a novel mechanism of miR-22 mediated suppression of CD147 and HDAC4 in airway epithelial dysregulation that is associated with IAV H1N1 infection in severe asthma.
IAV causes significant morbidity and mortality in annual epidemics [
14]. During the 2009 H1N1 swine flu pandemic in Australia and New Zealand, 33% of individuals who developed severe influenza and were admitted to hospital or ICU had asthma [
57]. While vaccination is the major protective intervention against IAV H1N1 infection, its efficacy is challenged by new strains and therefore new vaccines have to be developed annually. Hence it is critical to understand the underlying mechanisms, such as miRNAs and their targets, responsible for severe responses of asthmatics to this infection to be able to provide more efficient interventions. While a number of miRNAs have been associated with abnormalities in asthmatic epithelium and disease progression, current asthma treatments (e.g. inhaled corticosteroids) show no major effect on them [
39,
58,
59]. Hence, targeting miRNA abnormalities, using specific miRNA antagomirs or mimics, and restoring their normal functions may be an effective new intervention strategy.
The miR-20a is a member of miR-17-92 cluster which has essential roles in maintaining the structural homeostasis of the developing lung epithelium [
21]. miR-20a is reported to be increased in the epithelium and mesenchyme of the embryonic compared with fully developed lung [
21]. Our results, however, showed the same baseline level of miR-20a in pBECs from asthmatics compared with non-asthmatics cultured at both conditions. In addition, showed that miR-20a was unaffected by IAV H1N1 infection in pBECs form asthmatics compared with non-asthmatics in both culture conditions, which indicate this miRNA may not be responsive to the virus or its roles in epithelial cell homeostasis are more prominent during lung development.
Our data also showed that miR-132 is expressed at the same level in epithelial cells of asthmatics and non-asthmatics at baseline and after IAV H1N1 infection which was not dependent on culture conditions. Buggele et al., reported that Infection with IAV A/Udorn/72 H3N2 and A/WSN/33 H1N1 strains reported to increase miR-132 expression in the human lung epithelial cell lines A549 and BEAS-2B cells [
26]. miR-132 has been shown to be induced in cells infected with Kaposi’s sarcoma-associated herpesvirus, and to target the histone acetyltransferase protein, p300, which is required for the production of IFNß [
25]. However, we observed no effect of IAV H1N1 infection on miR-132 in epithelial cells from non-asthmatics or asthmatics in both culture conditions. These discrepancies could be due to the nature of cells; primary versus cell-line, or different strains of IAV A/Auckland/1/2009 H1N1 versus A/Udorn/72 H3N2 and A/WSN/33 H1N1.
Despite similar expression of miR-22 at baseline, our data indicate that after IAV H1N1 infection expression of this miRNA increased in pBECs of non-asthmatics whereas it was not altered in pBECs of asthmatics. miR-22 has been proposed to have tumor suppressive effects by targeting different genes responsible for cell proliferation [
22,
23]. One of the targets of miR-22 is CD147 (EMMPRIN/basigin/HAb18G), which has a key role in tumor progression and metastasis [
53]. CD147 induces the production of matrix metalloproteinases (MMP) such as MMP-9 that has important roles in airway remodeling and inflammation [
48]. Patients with severe asthma are reported to have increased levels and activity of MMP-9 in their sputum compared with mild asthmatics and normal subjects [
60]. Also, MMP-9 was shown to increase in airway epithelial cells in a mouse model of airway remodeling induced by ovalbumin challenge [
52]. Furthermore, Jouneau et al., reported that epithelial cells are the major source for CD147 compared with alveolar and blood monocytes [
48]. Another downstream target of miR-22 is HDAC4 [
47]. HDAC4 is a key member of class IIa HDACs which plays an important role in tissue growth and development [
61]. HDAC4 is known to be involved with TGF-ß1 induced epithelial-mesenchymal transition (EMT) in lung epithelial cells [
49]. EMT is defined as a process that epithelial cells lose polarity and intercellular contacts, and adopt a mesenchymal phenotype. These changes are characterized by repression of epithelial genes, such as E-cadherin and ZO-1, and increased expression of mesenchymal proteins, including fibronectin, vimentin, and α-smooth muscle actin [
33]. EMT can also lead to tissue remodeling and inflammation [
49]. pBECs treated with TGF-ß1 also showed increased MMP-9 activity in cell culture supernatant [
62]. There is evidence that epithelial cells of asthmatics are more prone to EMT [
31]. Blocking HDAC4 reported to suppress TGF-ß1 associated inductions of α-smooth muscle actin, fibronectin and vimentin while restore TGF-ß1 related suppression of E-cadherin and TGF-ß1 induced migration of lung epithelial cells [
49]. In addition, inhibiting HDAC4 suppressed MMP-9 elevation and hence EMT in a mouse model of bleomycin-induced acute lung injury [
50]. Hence, MMP-9 is an important downstream factor of both CD147 and HDAC4 activity that is associated with tissue remodeling directly and via EMT processes.
Our data on MMP-9 expression are particularly interesting. To our knowledge there are no previous reports comparing MMP-9 expression in epithelial cells from asthmatics with non-asthmatics. MMP-9 mRNA and protein were expressed at lower levels in epithelial cells from asthmatics compared with non-asthmatics. However, it was previously shown that significant increases in MMP-9 in bronchoalveolar lavage positively correlated with levels of eosinophils and neutrophils in asthmatics compared with non-asthmatics [
63]. These discrepancies may arise from the absence of other cells and hence lack of cell to cell communication in our model.
We showed that differential patterns of miR-22 expression may be a control factor to regulate CD147 and HDAC4 expression and their downstream MMP-9 expression and hence associated airway remodeling after IAV H1N1 infection in non-asthmatics. After infection, CD147, HDAC4 and MMP-9 mRNA levels reduced over time in association with increased miR-22 expression which prevents CD147 protein induction by maintaing its levels unchanged, and reduces HDAC4 and MMP-9 protein levels after infection in pBECs from non-asthmatics. However, these mechanisms differ in epithelial cells of asthmatics. Although miR-22 expression was not affected by IAV H1N1 in cells from asthmatics, CD147 increased at both gene and protein levels, potentially driven by infection induced c-Myc and SP1 expression, whereas HDAC4 and their downstream target MMP-9 remained unchanged thus, potentially promoting airway remodeling. Overexpression of miR-22 in pBECs of asthmatics significantly suppressed CD147 expression. The miR-22 mimic may therefore offer some potential protective role by reversing CD147 over expression after IAV H1N1 infection in asthmatics to prevent further tissue remodeling.
Interestingly, IAV H1N1 infection showed no effect on miR-22 expression in cells in basal monolayers from asthmatics or non-asthmatics confirming that its effect is associated with differentiated cells. As for why the miR-22 pattern of expression differs in epithelial cells from asthmatics compared with non-asthmatics after infection, further molecular investigations are required and it is necessary to determine the effect of IAV on different cells types between two groups. Our data so far may imply that there is a recovery mechanism after IAV H1N1 infection in differentiated cells from non-asthmatics which leads to increase in miR-22 which is not present in asthmatics.
Due to the nature of exacerbations of asthma, investigating the epigenetic regulatory mechanisms within the airway epithelium is technically difficult, especially in response to specific exposures such as IAV. While the use of pseudostratified ALI epithelial cultures has several advantages over monolayer cultures, our study has limitations. One is that we only compared the pBECs at ALI culture of five subjects with asthma and five non-asthmatics. Regardless of the limitations in patient numbers, they were well-phenotyped. Another limitation is that we only investigated targeted miRNAs with different biological roles. However, miRNAs form networks and many of which have multiple binding partners and therefore affect multiple pathways. Thus, our next approach will be to assess global miRNAs and corresponding mRNA targets with high accuracy in our samples, using next gene sequencing technology. Furthermore, in our model, pBECs at passage 2 were differentiated over 23–25 days and during this period cell media was refreshed frequently. The effect of administered inhaled corticosteroids by patients was washed out during this process. However, to our knowledge there are no reports of the effects of common asthma therapeutics (e.g. corticosteroids) on miRNA expression in airway epithelial cells during IAV infection which warrants future investigations. Another limitation is that we did not assess which cell type was most infected with IAV H1N1 when cells were cultured at ALI conditions since we extracted miRNA, mRNA and protein from entire cell populations. However, this and how it affects miRNA expression in different cells (e.g. ciliated cells vs basal cells) will be interesting to determine. In addition, in our infection model, only MOI of 5 of IAV H1N1 was used. Future assessments on infection with IAV H1N1 at multiple MOIs (e.g. 0.5, 1, 5 and 10) will provide more evidence on regulation of miR-22 and its targets by this infection in pBECs. Also, administration of specific inhibitors of c-Myc or SP-1 during infection in cells from both groups, will determine the regulatory roles of these transcription factors on CD147 and HDAC4 in epithelial cells. Further, we assessed miRNA expression and their functions only in pBECs with or without infection, whereas co-culturing epithelial cells with relevant cells, e.g. neutrophils or macrophages which are also a source of MMP-9 in asthmatics [
48], may affect miRNA expression and/or their targets in epithelial cells due to cell to cell crosstalk. Finally, our time course for IAV H1N1 infection was limited to 24 h.