Asthma is a chronic inflammatory disease affecting more than 300 million people worldwide (1). Acute exacerbations of asthma are the major cause of disease worsening and morbidity, and they are responsible for increasing health care burden (2). Rhinovirus infection is the major trigger for asthma exacerbation (2). However, other respiratory viruses such as respiratory syncytial virus (RSV), influenza A or coronaviruses have been also related to an aggravation of the disease (2). For example, intensive care admissions and mortality of patients with H1N1 influenza A was higher in patients with asthma during 2009 H1N1 pandemic (3).

In December 2019, a novel coronavirus disease, named coronavirus disease-19 (COVID-19), emerged in the Hubei province of China (4). This disease is caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) (5). Although the first epidemiologic studies could not find an association of asthma with an increase risk to suffer from COVID-19, novel data from large cohorts evidenced a higher prevalence of asthma in patients with COVID-19 compared to the general population (6). Asthma was also associated to a longer duration of severe COVID-19 disease (7). Furthermore, several studies demonstrated that severe asthma is a risk factor for developing severe COVID-19 (8, 9). The high infection and mortality rates, in combination with the high economic impact of COVID-19, underpinned an unprecedented global search for efficient treatments and successful developments of vaccines. However, there is still a need for new drug opportunities to treat SARS-CoV-2 infection, especially in patients with a high risk to develop a severe sickness such as asthmatic subjects.

Similar to rhinoviruses, SARS-CoV-2 is a single stranded RNA (ssRNA) virus (10). SARS-CoV-2 mainly infects human cells through the high affinity interaction of the receptor binding domain (RBD) of the spike protein 1 (SP1) with the angiotensin converting enzyme II (ACE2) protein on host cells (11, 12). ACE2 is widely expressed in different cell types and tissues including bronchial and upper airway epithelial cells, representing the main port of entry by SARS-CoV-2 (11, 13–15). In addition to ACE2, the host serine protease TMPRSS2, responsible for fusion of the viral and host cell membranes by cleaving S protein at the S1/S2 and S2´sites, is essential for viral entry (11). Hence, investigating the expression of both ACE2 and TMPRSS2 in the bronchial epithelium may contribute to understand the infectivity and actions of SARS-CoV-2 and assist in the search of pharmacological opportunities to combat COVID-19.

Following cell entry, both rhinovirus and SARS-CoV-2 virus replicate and produce intermediate double stranded RNA (dsRNA) molecules (16, 17). dsRNA molecules activate pattern recognition receptors (PRRs) in host cells including Toll-like Receptor (TLR)3, Melanoma Differentiation-Associated protein 5 (MDA5) and Retinoic Acid-Inducible Gene I (RIG-I) (17, 18). Activation of PRRs results in the activation of two important downstream transcriptional factors, NF-κB and Interferon Regulatory Factors (IRFs) leading to production of cytokines or chemokines (such as IL-6, TNF-α, IL-8) and type-I and type-III interferons (IFN; IFN-β and IFN-λ), respectively (17). These molecules represent early responses to viral infection, followed later by anti-viral T-cell responses (17, 19). In COVID-19, a low induction of type-I and type-III IFNs has been described (20, 21). A dysregulation of IFN pathways at early stages of COVID-19 is thought to contribute to uncontrolled viral replication and subsequent dramatic immune responses at later stages (20–23). In addition, asthmatic patients have been described to have an impaired anti-viral IFN response (24, 25), which could predispose these patients to be more susceptible to viral infections. Therefore, one of the desired treatment strategies in order to improve the outcome of viral infection in these patients is the increase of type I IFN responses in the bronchial epithelium.

A second common feature of asthma exacerbations after viral infection and severe COVID-19 is the exaggerated pro-inflammatory response of the bronchial epithelium to virus (17, 26). In this regard, glucocorticoids are an acknowledged treatment of both asthma and severe COVID-19 due to their broad anti-inflammatory effects (27–29). Indeed, inhaled budesonide administered in the early stages of COVID-19 reduced the need for urgent medical care and improved the recovery time (30). However, due to a potential to decrease the anti-viral type I interferon response (31–34), a high dose of glucocorticoids could be harmful if they are given at the peak of viral replication (34). It is, therefore, when the immunopathological features of the illness become more evident that the glucocorticoid effect could be most beneficial, creating a window of opportunity vital to clinical effect (27, 34). The question arises whether drugs can be found that both improve anti-viral responses and reduce severity of pro-inflammatory responses.

Previous attempts to discover pharmacological opportunities to improve both anti-viral defense and reducing cytokine inflammation have been proven to be difficult to combine in one single molecule [(35), and unpublished observations]. Literature data, however, suggest that imiquimod, an imidazo-quinoline used as an immune response modifier, could be an interesting compound to explore further in regards of combined anti-viral and anti-inflammatory actions. Imiquimod is a TLR7 agonist clinically approved as topical treatment of viral and tumoral skin conditions. Imiquimod may activate the production of type-I and III interferons as well as the NF-κB pathway (36), although results vary (37, 38) and effects on human airway epithelium seem unexplored. Imiquimod has been considered as treatment for allergic diseases like asthma due to its inhibition of allergic type 2 inflammation and favoring of type 1 responses (39, 40). Focusing on potential anti-allergic efficacy, local airway treatment with other TLR7 agonist have been employed in clinical trials in asthma and allergy (41, 42). Interestingly, intranasal administration of imiquimod has been demonstrated to protect against Influenza A virus in mice, reducing viral replication, neutrophil infiltration, and lung dysfunction (37). Based on this observation, and observations on potential anti-viral actions in a variety of cell systems, imiquimod has already been suggested as a potential therapy in early phase COVID-19 disease (43–45). However, studies have not focused on major target cells for primary site of infectivity of SARS-CoV-2, the human airway epithelium. Little is thus known about effects of imiquimod on human primary bronchial epithelial cells (HBECs). Considering the occurrence of reduced anti-viral resistance in asthma, demonstrated as reduced interferon production at viral infection (46), and the increasing evidence demonstrating that severe asthma is a risk factor for COVID-19 related admission to intensive care (8, 9), studies of imiquimod in HBECs from asthmatic patients are of particular interest.

Our study explores effects of imiquimod on ACE2 and TMPRSS2 expression in HBECs stimulated with both the TLR3 agonist poly(I:C) (SARS-CoV-2 replication mimic) or the recombinant SP1 from SARS-CoV-2 (RBD). In addition, effects of imiquimod on anti-viral and anti-inflammatory responses were examined by using multiplex ELISA and gene expression analyses, and non-targeted proteomics. We also used siRNA technology to shed light on the potential mechanisms of action of imiquimod in HBECs.

There is an urgent need of finding new drugs for the treatment of respiratory viral infections in asthma. In this study, several pharmacological effects of the TLR7 agonist imiquimod on HBECs from asthmatics, of interest for the treatment of both viral induced asthma exacerbations and COVID-19, have been demonstrated ( Figure 7 ). On one hand, imiquimod boosts IFN-β response and decreases ACE2 expression in HBECs both at baseline and in the presence of the viral infection mimics poly(I:C) and the spike protein of the SARS-CoV-2, SP1. On this note, multiplex gene expression profiling of poly(I:C)-stimulated HBECs subjected to imiquimod treatment demonstrated change of multiple genes involved in anti-viral immune response. We further observed a decrease of C1QBP, a negative regulator of MDA5 and RIG-I signaling. Proteomic analysis of HBECs also highlighted that the potential protective effect of imiquimod on viral infections involved a down-modulation of proteins implicated in viral mRNA and protein synthesis. On the other hand, our results evidenced a down-modulation of several pro-inflammatory mediators implicated in both viral-induced asthma exacerbations and COVID-19 pathophysiology following imiquimod treatment. This was in part supported by the up-regulation of SIGIRR, a negative regulator of TLR signaling, in poly (I:C)-stimulated HBECs treated with imiquimod.

Increasing evidence demonstrate that severe asthma is a risk factor for COVID-19 related admission to intensive care and death (8, 9). Indeed, a deficient anti-viral interferon response in asthma patients has been previously described, potentially leading to more severe virus-induced exacerbations (25). Yuen et al. have also shown that SARS-CoV-2 directly suppresses the interferon response during infection in vitro (51). Treatment with nebulized IFN-β in COVID-19 patients led to greater odds of improvement compared with patients receiving placebo control (52). The same treatment in asthma patients has been shown to reduce need of additional treatment while boosting innate immunity (53). However, IFN-β therapy was mainly beneficial in patients with severe asthma, whereas side effects occurred in milder asthmatics (53). It is possible that drugs boosting endogenous production of IFN-β may hold the advantage of potentially avoiding flu-like symptoms normally associated with exogenous interferon treatment (54). The present data extend previous observations on imiquimod-induced type I IFN in diverse experimental models to primary HBECs (37, 55, 56). Of particular interest, our data support the feasibility of obtaining drugs that improve airway IFN-β levels both at baseline and during a viral infection. mRNA expression profiling of imiquimod´s effect on poly(I:C)-stimulated HBECs revealed upregulation of multiple genes involved in type-I IFN pathway including STAT1, STAT2, IRF1, and JAK1. In addition, a decrease in C1QBP mRNA expression, a negative regulator of MDA5/RIG-I signaling (57), was found after imiquimod treatment of HBECs also exposed to poly (I:C). Further, siRNA mediated inhibition of MDA5/RIG-I expression in HBECs inhibited the imiquimod-induced effect on IFN-β expression, thus highlighting a potential role of these PRRs in imiquimod mediated IFN upregulation. A similar RIG-I and STAT1-dependent upregulation of type I IFN in response to imiquimod has been previously described in plasmacytoid dendritic cells (pDCs) (56).

Apart from the impaired IFN response in asthma and COVID-19 patients, another possible factor for developing a severe respiratory SARS-CoV-2 infection is the rapid increase of ACE2 expression in the first hours of SARS-CoV-2 infection, probably leading to an exaggerated replication and a rapid spread (58). Supporting the relevance of the present work, we demonstrated that poly(I:C) also increased ACE2 in bronchial epithelial cells from asthma and healthy donors. Given the importance of ACE2 levels in initiation and development of the SARS-CoV-2 infection, it is of interest that pharmacological attenuation of ACE2 expression levels may decrease the infection rates of the virus (58). Finney et al. have shown a glucocorticoid-induced lowering of the levels of ACE2 in the bronchial epithelium, strongly arguing that this action is caused by steroid-induced suppression of type I interferon (28). Previous studies have further shown that ACE2 could be an interferon-stimulated gene in airway epithelium (59). By contrast, our study demonstrates that imiquimod both reduces poly(I:C)-induced ACE2 expression and increases IFN-β in HBECs. These distinct observations are not readily explained by different sources of HBECs, COPD (28) and asthma (this study). Further work is clearly warranted to explain the dual effects on infection resistance of imiquimod, increasing IFN-β and decreasing ACE2, which would co-operate in reducing SARS-CoV-2 infectivity.

Both SARS-CoV-2 infection and viral-induced asthma exacerbations display hyperinflammatory immune responses, indicating a dysfunctional host tolerance to infection (60). In this study, imiquimod decreased gene expression of cytokines of special interest in asthma pathogenesis such as IL-33 and IL-1β. Furthering potential relevance of imiquimod treatment in asthma, Maazi et al. have previously demonstrated that TLR7 activation on pDCs induces the release of IFN-α, which in turn will inhibit the proliferation and cytokine production in ILC2 cells, key mediators of asthma pathogenesis (61). Imiquimod was also suggested to reduce allergen-induced airway inflammation in mice, potentially triggering T1 responses and decreasing T2 inflammation (62). Moreover, IL-33 protein release was also reduced by imiquimod in HBECs after poly (I:C) stimulation in the present study. Stanczak et al., have recently demonstrated that IL-33 expression is increased in COVID-19 seropositive patients after stimulation with SARS-CoV-2 peptides (63). They also evidenced that disease severity and T-cell activation in SARS-CoV-2 infected subjects is correlated with IL-33 production (63). Indeed, IL-33 has been purposed as a key player in the pathogenesis of COVID-19, possibly being involved in early activation of innate immune responses, but also in later stages of the disease by inducing lung fibrosis (64). Other cytokines and chemokines such as IL-1β, IL-6, IL-8, or TNF-α have also been implicated in the pathogenesis of viral-induced exacerbations of asthma and severe COVID-19 (17, 65). We demonstrated that IL-1β, IL-6 and IL-8 protein expression were reduced by imiquimod in HBECs at poly(I:C) stimulation. Imiquimod transiently increased baseline gene expression of TNF-α, but this action was not reflected by protein release; in presence of poly(I:C), TNF-α protein was rather reduced by imiquimod. Taken together, the present data suggest that imiquimod-like drugs targeting SARS-CoV-2 infected airway epithelium are of interest for further development towards tentative treatment of viral induced exacerbation of asthma and COVID-19 both at early stages to avert infection and at later stages associated with airway hyperinflammation. It is not known to what extent the present profile of action of imiquimod is shared with other TLR7 agonists, including two drugs that already have been subjected to clinical trials to determine anti-allergic efficacy by local airway administration in asthma and rhinitis (41, 42).

Imiquimod-induced attenuation of the pro-inflammatory cytokines IL-6 and TNF-α, the neutrophilic chemokine IL-8, or different TH-type cytokines including IL-4, IFN-β, or IL-17, has been previously shown in airway epithelial cells or mouse models of RSV and Influenza A virus infections (37, 38). Of note, Salinas et al. suggested the interference of imiquimod with viral macromolecular synthesis as one potential mechanism of imiquimod-dependent down-regulation of pro-inflammatory cytokines after RSV infection (38). This possibility is in line with our exploratory proteomic analysis on the direct effect of imiquimod on HBECs; our data revealed a possible protective effect of imiquimod on viral infections through down-modulation of proteins implicated in viral mRNA and protein synthesis, including several ribosomal proteins. In addition, multiplex mRNA expression analyses in this study demonstrated an increased expression of the negative regulator of IL-1R signaling SIGIRR in imiquimod treated HBECs, highlighting another potential mechanism of the imiquimod-mediated down-modulation of IL-1β and IL-33 expression (66).

Imiquimod is currently available as a drug, Aldara, for topic treatment of skin conditions, including genital warts, actinic keratosis, and superficial basal cell carcinoma. Delivery of imiquimod has previously been employed in mice against influenza A infection (37), and for reducing airway inflammation in rats (62). Moreover, efficacy, as well as safety profile of intranasal application of imiquimod have been also demonstrated in rhinovirus infection in primates (67). However, this study has demonstrated an unexpected profile of action of imiquimod indicating drug feasibility rather than suggesting that imiquimod would be an ideal drug for nasal or inhalational treatment in COVID-19 or future viral pandemics.

A limitation in this study is the use of poly(I:C) and SP1 as a mimic of SARS-CoV-2 and rhinovirus infection, but not the real virus. Poly(I:C) is a dsRNA analogue and TLR3 agonist that mimics several biological effects of viral infection without being subject to the variation in intensity that may apply to actual infections. Poly(I:C) stimulation of bronchial epithelial cells induces a similar cytokine profile compared to rhinovirus and SARS-CoV-2 infection in bronchial epithelial cells (18, 68, 69). Moreover, poly(I:C) and SP1 administration to mice has been proven to induce cytokine storm syndrome and ARDS (70). Although studies on interaction between imiquimod and actual infection by SARS-CoV-2 are now warranted we argue that our approach employing poly(I:C) has strong guiding merits. In this study we have used submerged and not differentiated human bronchial epithelial cells. Ravindra et al. have previously demonstrated that basal bronchial epithelial cells are infected over the course of SARS-CoV-2 infection (71). We further demonstrate that our undifferentiated bronchial basal cell-like monolayer harbors significant ACE2 receptors and respond well to poly(I:C). Moreover, infectivity by rhinovirus is also particularly pronounced in human bronchial basal cells (72). Furthermore, undifferentiated epithelium commonly occurs in asthmatic bronchi, which is a focus in our study. It reflects both preferential shedding of columnar differentiated cells and actual denudation where the naked basement membrane is promptly covered by basal cell-like repair cells.

In conclusion, we demonstrate that imiquimod increases IFN-β expression, in part targeting MDA5 and RIG-I receptors and involving activation of several anti-viral immune pathways. In addition, we demonstrate, for the first time, that treatment with imiquimod reduced ACE2 expression in the bronchial epithelium. Finally, the drug´s profile would be additionally beneficial through its anti-inflammatory effect, reducing several epithelium-derived cytokines and chemokines implicated in the hyper-inflammatory response in viral-induced asthma exacerbation and severe COVID-19 cases. Taken together, the present demonstration of several epithelial actions of imiquimod in HBECs of potential importance for human respiratory viral infections is both promising and challenging. The promise lies in demonstration of feasibility of finding drugs with multiple desirable actions in virus-induced asthma exacerbations and COVID-19. The challenge is manifold but in part lies in further delineation of mechanisms of action of a drug with the present profile of action.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Proteomic data have been uploaded to the PRIDE database (73) under accession no: PXD029135; Nanostring data has been uploaded to NCBI GEO under accession no: GSE181281.

The studies involving human participants were reviewed and approved by Danish Committee on Health Research Ethics (Ethics: H-16043663 and H-16002008). The patients/participants provided their written informed consent to participate in this study.

JJN-F contributed to the design, acquisition, analysis, and interpretation of data, have drafted and revised the work. ST contributed to the design, acquisition, analysis, and interpretation of data, have drafted and revised the work. SC contributed to the design, acquisition, analysis, and interpretation of data, have drafted and revised the work. AS contributed to the acquisition, analysis, and interpretation of data, and have revised the work. MH contributed to the acquisition, analysis, and interpretation of data, and have revised the work. SR contributed to the acquisition, analysis, and interpretation of data, and have revised the work. MM contributed to acquisition, analysis, and interpretation of data, and have revised the work. AFS contributed to the design of the work, interpretation of data, and revised the work. CP contributed to the design, analysis, and interpretation of data, have drafted and revised the work. LU contributed to the design, acquisition, analysis, and interpretation of data, have drafted and revised the work. All authors contributed to the article and approved the submitted version.

This research has been funded by Vetenskapsrådet (Swedish medical research council) grant numbers: 2020-00922_VR and 2017-00806_VR as well as Swedish Heart and lung foundation grant number: 20180207_HLF.

MH reports personal fees from Astra Zeneca, personal fees from Novartis, outside the submitted work. CP reports grants and personal fees from AZ, grants and personal fees from Novartis, grants and personal fees from GlaxoSmithKline, grants and personal fees from TEVA, grants and personal fees from GSK, grants and personal fees from Chiesi, grants from Pharmaxis, grants and personal fees from Sanofi, outside the submitted work.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.