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EphA2 receptor and its ephrin ligands are involved in virus infection, epithelial permeability, and chemokine secretion. We hypothesized that ephrinA1/ephA2 signaling participates in rhinovirus (RV)-induced antiviral immune response in sinonasal mucosa of patients with chronic rhinosinusitis (CRS). Therefore, we investigated the expression of ephrinA1/ephA2 in normal and inflamed sinonasal mucosa and evaluated whether they regulate chemokine secretion and the production of antiviral immune mediators including interferons (IFNs) in RV-infected human primary sinonasal epithelial cells. For this purpose, the expression and distribution of ephrinA1/ephA2 in sinonasal mucosa were evaluated with RT-qPCR, immunofluorescence, and western blot. Their roles in chemokine secretion and the production of antiviral immune mediators such as type I and III IFNs, and interferon stimulated genes were evaluated by stimulating ephA2 with ephrinA1 and inactivating ephA2 with ephA2 siRNA or inhibitor in cells exposed to RV and poly(I:C). We found that ephrinA1/ephA2 were expressed in normal mucosa and their levels increased in inflamed sinonasal mucosa of CRS patients. RV infection or poly(I:C) treatment induced chemokine secretion which were attenuated by blocking the action of ephA2 with ephA2 siRNA or inhibitor. The production of antiviral immune mediators enhanced by rhinovirus or poly (I:C) is increased by blocking ephA2 compared with that of cells stimulated by either rhinovirus or poly(I:C) alone. In addition, blocking ephA2 attenuated RV replication in cultured cells. Taken together, these results describe a novel role of ephrinA1/ephA2 signaling in antiviral innate immune response in sinonasal epithelium, suggesting their participation in RV-induced development and exacerbations of CRS., Competing Interests: The 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., (Copyright © 2021 Lee, Kang, Han, Kwak, Kim, Lee, Lee and Kim.)

Introduction: Air pollution is a risk factor for respiratory infections and asthma exacerbations. We previously reported impaired Type-I and Type-III interferons (IFN-β/λ) from airway epithelial cells of preschool children with asthma and/or atopy. In this study we analyzed the association between rhinovirus-induced IFN-β/λ epithelial expression and acute exposure to the principal outdoor air pollutants in the same cohort., Methods: We studied 34 children (17asthmatics/17non-asthmatics) undergoing fiberoptic bronchoscopy for clinical indications. Bronchial epithelial cells were harvested by brushing, cultured and experimentally infected with Rhinovirus Type 16 (RV16). RV16-induced IFN-β and λ expression was measured by quantitative real time PCR, as was RV16vRNA. The association between IFNs and the mean exposure to PM10, SO2 and NO 2 in the day preceding bronchoscopy was evaluated using a Generalized Linear Model (GLM) with Gamma distribution., Results: Acute exposure to PM10 and NO 2 was negatively associated to RV16-induced IFNβ mRNA. For each increase of 1ug/m 3 of NO 2 we found a significative decrease of 2.3x10 3 IFN-β mRNA copies and for each increase of 1ug/m 3 of PM10 a significative decrease of 1x10 3 IFN-β mRNA copies. No significant associations were detected between IFN-λ mRNA and NO 2 nor PM10. Increasing levels of NO 2 (but not PM10) were found to be associated to increased RV16 replication., Conclusions: Short-term exposure to high levels of NO 2 and PM10 is associated to a reduced IFN-β expression by the airway epithelium, which may lead to increased viral replication. These findings suggest a potential mechanism underlying the link between air pollution, viral infections and asthma exacerbations., Competing Interests: MS reports grants from Chiesi Farmaceutici, grants from Laboratori Guidotti, grants from Bottega Veneta, grants from University of Padova- Italy, outside the submitted work. AP reports grants, personal fees, non-financial support and other from GlaxoSmithKline, grants, personal fees and non-financial support from AstraZeneca, grants, personal fees, non-financial support and other from Boehringer Ingelheim, grants, personal fees, non-financial support and other from Chiesi Farmaceutici, grants, personal fees, non-financial support and other from TEVA, personal fees, non-financial support and other from Mundipharma, personal fees, non-financial support and other from Zambon, personal fees, non-financial support and other from Novartis, grants, personal fees and non-financial support from Menarini, personal fees, non-financial support and other from Sanofi/Regeneron, personal fees from Roche, grants from Fondazione Maugeri, grants from Fondazione Chiesi, personal fees from Edmondpharma, outside the submitted work. MC reports grants, personal fees and non-financial support from Chiesi, personal fees and non-financial support from AstraZeneca, personal fees and non-financial support from Boehringer Ingelheim, personal fees and non-financial support from Alk-Abello, grants, personal fees and non-financial support from GlaxoSmithKline, personal fees and non-financial support from Novartis, personal fees and non-financial support from Zambon, grants from University of Ferrara - Italy, 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., (Copyright © 2021 Bonato, Gallo, Turrin, Bazzan, Baraldi, Saetta, Gregori, Papi, Contoli and Baraldo.)

Inhaled corticosteroids (ICS) are recommended treatments for all degrees of asthma severity and in combination with bronchodilators are indicated for COPD patients with a history of frequent exacerbations. However, the long-term side effects of glucocorticoids (GCs) may include increased risk of respiratory infections, including viral triggered exacerbations. Rhinovirus (RV) infection is the main trigger of asthma and COPD exacerbations. Thus, we sought to explore the influence of GCs on viral replication. We demonstrate the ICS fluticasone propionate (FP) and two selective non-steroidal (GRT7) and steroidal (GRT10) glucocorticoid receptor (GR) agonists significantly suppress pro-inflammatory (IL-6 and IL-8) and antiviral (IFN-λ1) cytokine production and the expression of the interferon-stimulated genes (ISGs) OAS and viperin in RV-infected bronchial epithelial cells, with a consequent increase of viral replication. We also show that FP, GRT7 and GRT10 inhibit STAT1 Y701 and/or STAT2 Y690 phosphorylation and ISG mRNA induction following cell stimulation with recombinant IFN-β. In addition, we investigated the effects of the ICS budesonide (BD) and the long-acting β2 agonist (LABA) formoterol, alone or as an ICS/LABA combination, on RV-induced ISG expression and viral replication. Combination of BD/formoterol increases the suppression of OAS and viperin mRNA observed with both BD and formoterol alone, but an increase in viral RNA was only observed with BD treatment and not with formoterol. Overall, we provide evidence of an impairment of the innate antiviral immune response by GC therapy and the potential for GCs to enhance viral replication. These findings could have important clinical implications., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Human rhinovirus (hRV) is frequently detected in the upper respiratory tract, and symptomatic infection is associated with an increased nasopharyngeal bacterial load, with subsequent development of secondary bacterial diseases. Nontypeable Haemophilus influenzae (NTHI) is a commensal bacterial species of the human nasopharynx; however, in the context of prior or concurrent upper respiratory tract viral infection, this bacterium commonly causes multiple diseases throughout the upper and lower respiratory tracts. The present study was conducted to determine the mechanism(s) by which hRV infection promotes the development of NTHI-induced diseases. We showed that hRV infection of polarized primary human airway epithelial cells resulted in increased adherence of NTHI, due in part to augmented expression of CEACAM1 and ICAM1, host cell receptors to which NTHI binds via engagement of multiple adhesins. Antibody blockade of these host cell receptors significantly reduced NTHI adherence. With a specific focus on the NTHI type IV pilus (T4P), which we have previously shown binds to ICAM1, an essential adhesin and virulence determinant, we next showed that T4P-directed antibody blockade significantly reduced NTHI adherence to hRV-infected airway cells and, further, that expression of this adhesin was required for the enhanced adherence observed. Collectively, these data provide a mechanism by which "the common cold" promotes diseases due to NTHI, and they add further support for the use of PilA (the majority subunit of T4P) as a vaccine antigen, since antibodies directed against PilA are expected to limit the notably increased bacterial load associated with hRV coinfection and thereby to prevent secondary NTHI-induced diseases of the respiratory tract., (Copyright © 2020 American Society for Microbiology.)

Obesity is an important risk factor for severe asthma exacerbations, which are mainly caused by respiratory infections. Dietary fatty acids, which are increased systemically in obese patients and are further increased after high-fat meals, affect the innate immune system and may contribute to dysfunctional immune responses to respiratory infection. In this study we investigated the effects of dietary fatty acids on immune responses to respiratory infection in pulmonary fibroblasts and a bronchial epithelial cell line (BEAS-2B). Cells were challenged with BSA-conjugated fatty acids (ω-6 polyunsaturated fatty acids [PUFAs], ω-3 PUFAs, or saturated fatty acids [SFAs]) +/- the viral mimic polyinosinic:polycytidylic acid (poly[I:C]) or bacterial compound lipoteichoic acid (LTA), and release of proinflammatory cytokines was measured. In both cell types, challenge with arachidonic acid (AA) (ω-6 PUFA) and poly(I:C) or LTA led to substantially greater IL-6 and CXCL8 release than either challenge alone, demonstrating synergy. In epithelial cells, palmitic acid (SFA) combined with poly(I:C) also led to greater IL-6 release. The underlying signaling pathways of AA and poly(I:C)- or LTA-induced cytokine release were examined using specific signaling inhibitors and IB. Cytokine production in pulmonary fibroblasts was prostaglandin dependent, and synergistic upregulation occurred via p38 mitogen-activated protein kinase signaling, whereas cytokine production in bronchial epithelial cell lines was mainly mediated through JNK and p38 mitogen-activated protein kinase signaling. We confirmed these findings using rhinovirus infection, demonstrating that AA enhances rhinovirus-induced cytokine release. This study suggests that during respiratory infection, increased levels of dietary ω-6 PUFAs and SFAs may lead to more severe airway inflammation and may contribute to and/or increase the severity of asthma exacerbations.

Human rhinovirus (HRV) infections are major contributors to the healthcare burden associated with acute exacerbations of chronic airway disease, such as chronic obstructive pulmonary disease and asthma. Cellular responses to HRV are mediated through pattern recognition receptors that may in part signal from membrane microdomains. We previously found Toll-like receptor signaling is reduced, by targeting membrane microdomains with a specific liposomal phosphatidylserine species, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine (SAPS). Here we explored the ability of this approach to target a clinically important pathogen. We determined the biochemical and biophysical properties and stability of SAPS liposomes and studied their ability to modulate rhinovirus-induced inflammation, measured by cytokine production, and rhinovirus replication in both immortalized and normal primary bronchial epithelial cells. SAPS liposomes rapidly partitioned throughout the plasma membrane and internal cellular membranes of epithelial cells. Uptake of liposomes did not cause cell death, but was associated with markedly reduced inflammatory responses to rhinovirus, at the expense of only modest non-significant increases in viral replication, and without impairment of interferon receptor signaling. Thus using liposomes of phosphatidylserine to target membrane microdomains is a feasible mechanism for modulating rhinovirus-induced signaling, and potentially a prototypic new therapy for viral-mediated inflammation.

Rhinovirus-C (RV-C) were discovered recently using molecular methods. Classical methods failed to detect them since they could not grow in standard cell culture. The complete genome sequences of several RV-C strains are now available but there is little information about their biological characteristics. HRV-C were first grown in organ culture, and more recently, we developed a system for culturing RV-C strains in differentiated epithelial cells of human airway at air-liquid interface (ALI). These cultures supported efficient replication of RV-C strains as determined by quantitative RT-PCR. This system has enabled study of the biological characteristics of RV-C strains, including quantitative research.

Human rhinoviruses (RVs) are a major cause of exacerbations in asthma and other chronic airway diseases. A characteristic feature of asthmatic epithelium is goblet cell metaplasia and mucus hypersecretion. Bronchial epithelium is also an important source of lipid mediators, including pro- and antiinflammatory eicosanoids. By using air-liquid interface cultures of airway epithelium from patients with asthma and nonasthmatic control subjects, we compared RV16 replication-induced changes in mRNA expression of asthma candidate genes and eicosanoid production in the epithelium with or without IL-13-induced mucus metaplasia. Mucus metaplastic epithelium was characterized by a 20-fold less effective replication of RV16 and blunted changes in gene expression; this effect was seen to the same extent in patients with asthma and control subjects. We identified ciliary cells as the main target for RV16 by immunofluorescence imaging and demonstrated that the numbers of ciliary cells decreased in RV16-infected epithelium. RV16 infection of mucociliary epithelium resulted in overexpression of genes associated with bronchial remodeling (e.g., MUC5AC, FGF2, and HBEGF), induction of cyclooxygenase-2, and increased secretion of prostaglandins. These responses were similar in both studied groups. These data indicate that structural changes associated with mucus metaplasia renders airway epithelium less susceptible to RV infection. Thus, exacerbations of the lung disease caused by RV may result from severe impairment in mucociliary clearance or activation of immune defense rather than from preferential infection of mucus metaplastic epithelium. Repeated rhinoviral infections of compromised epithelium may contribute to the remodeling of the airways.

Information about the basic biological properties of human rhinovirus-C (HRV-C) viruses is lacking due to difficulties with culturing these viruses. Our objective was to develop a cell culture system to grow HRV-C. Epithelial cells from human sinuses (HSEC) were differentiated at air-liquid interface (ALI). Differentiated cultures supported 1-2 logs growth of HRV-C15 as detected by quantitative RT-PCR. Two distinguishing features of HRVs are acid lability and optimal growth at 33-34 °C. We used this system to show that HRV-C15 is neutralized by low pH (4.5). In contrast to most HRV types, replication of HRV-C15 and HRV-C41 was similar at 34 and 37°C. The HSEC ALI provides a useful tool for quantitative studies of HRV-C replication. The ability of HRV-C to grow equally well at 34°C and 37°C may contribute to the propensity for HRV-C to cause lower airway illnesses in infants and children with asthma., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Rhinoviruses (RVs) are picornaviruses that are causative agents of the majority of upper respiratory tract infections, or "common colds," in humans. RVs infect both the upper and lower respiratory tract, and in addition to the common cold may also cause pneumonia, complications in patients with chronic lung diseases such as cystic fibrosis, and asthma exacerbations. Convenient animal models are not available to study the pathogenesis of rhinovirus-induced illness. Rhinovirus RV1A replicates poorly in mouse cells; variants with improved replication were selected by serial passage through mouse embryonic fibroblasts and mouse lung epithelial cells. Adaptation for improved growth in mouse cells was mediated by amino acid changes in the RV1a non-structural protein 3A. Mouse cell-adapted RV1A was capable of productively infecting mice in which the airway was subjected to chemical permeabilization. A mouse model for RV infection will permit studies of RV pathogenesis and may identify targets for therapeutic intervention., (Copyright © 2011 Elsevier Inc. All rights reserved.)

HeLa cells are used to study the life cycles of many different viruses, including the human rhinoviruses (HRV) in the family Picornaviridae. Although the natural targets of HRV are human bronchial epithelial cells (hBE), it is generally more difficult to obtain and maintain the relevant primary cell cultures, relative to HeLa cells. Given that the HRV are now identified as a major cause of human asthma exacerbations, it becomes important to document how much of the virus biology learned from HeLa cells is common also to natural primary cells. When compared directly in matched infections using A01a virus, the kinetics of RNA replication, the synthesis and processing of viral proteins and the general subcellular localization of key non-structural proteins were resembled in hBE and HeLa cells. Viral-induced shutoff of host cell processes (e.g. nucleo-cytoplasmic trafficking) was also comparable.

Exposure to cigarette smoke is associated with a significant increase in the risk for respiratory viral infections. The airway epithelium is the primary target for both cigarette smoke and respiratory viral infection. We investigated the effects of cigarette smoke on the response of airway epithelial cells to rhinovirus infection. We found that pre-exposure of BEAS-2B cells or primary normal human bronchial epithelial cells (NHBEs) to cigarette smoke extract (CSE) reduced the induction of mRNA of the chemokines CXCL10 and CCL5 by either the viral mimic polyinosine-polycytidylic acid (Poly I:C) or human rhinovirus 16 (HRV-16) infection. The HRV-16-induced release of CXCL10 and CCL5 was also significantly suppressed by CSE. Activation of the IFN mediator STAT-1 and the activation of JNK by poly I:C and HRV-16 were partially suppressed by pre-exposure to CSE. In contrast, the poly I:C-induced and HRV-16-induced phosphorylation of ERK1/2 was unaffected by CSE. HRV-16-stimulated IFN-β mRNA was also significantly reduced by CSE. Because suppression of the IFN response to viral infection was associated with increased viral production, we assessed HRV-16 RNA concentrations. Exposure to CSE resulted in an increase in HRV-16 RNA at 48 hours after the infection of BEAS-2B cells. These data demonstrate that exposure to CSE alters the response of airway epithelial cells to HRV infection, leading to decreased activation of the IFN-STAT-1 and SAP-JNK pathways, the suppression of CXCL10 and CCL5 production, and increased viral RNA. A diminished, early epithelial-initiated antiviral response to rhinovirus infection could contribute to the increased susceptibility of subjects to prolonged respiratory viral infections after exposure to cigarette smoke.

Human rhinoviruses (HRV) are the primary etiological agents in cold infections, and represent a serious risk to individuals with chronic respiratory disease such as asthma. In order to develop treatment options for HRV infections, murine models are a crucial component in the study of infection mechanisms due to the wide array of reagents and techniques available to study murine immunology. We present here a cell culture system for studying isolated murine epithelial cell responses to HRV. Monolayers of primary mouse airway epithelial cells were maintained in a serial culture system, and the identity and purity of the cell population was confirmed via immunostaining (positive for cytokeratin, negative for vimentin). Infection of these cells with a minor group rhinovirus (HRV-1A) was evidenced by increases in viral RNA, de novo synthesis of viral proteins, and production of infectious virus. This model will be useful in experiments to define mechanisms of viral replication and host/virus interactions within airway epithelial cells.

HeLa and 16HBE14o(-) bronchial epithelium cells infected with human rhinovirus serotype 14 (HRV14) were found to exhibit typical apoptotic morphological alterations, such as cell contraction and nuclear condensation. These events coincided with high-molecular-weight DNA fragmentation, activation of caspase-9 and caspase-3 and poly(ADP-ribose) polymerase cleavage. Caspase activation was preceded by cytochrome c translocation from the mitochondria to the cytoplasm, indicating that apoptosis caused by HRV14 infection was triggered predominantly via the mitochondrial pathway. Apoptosis did not affect HRV14 replication per se, but it facilitated the release of newly formed virus from cells. As apoptosis was fully induced at the time of maximal accumulation of progeny HRV14, it is postulated that apoptosis contributed to the destabilization of the cell and facilitated viral progeny release.

To examine the effects of lansoprazole, a proton pump inhibitor, on rhinovirus infection in airways, human tracheal epithelial cells were infected with a major subgroup of rhinoviruses, type 14 rhinovirus. Rhinovirus increased the mRNA expression of intercellular adhesion molecule-1 (ICAM-1) in the cells, the major rhinovirus receptor, and the content of the soluble form of ICAM-1 (sICAM-1) and cytokines in supernatants. Lansoprazole reduced supernatant titers and RNA of rhinovirus, the susceptibility to rhinovirus infection, the ICAM-1 mRNA production, the number and fluorescence intensity of acidic endosomes in the cells, and supernatants sICAM-1 and cytokine concentrations including interleukin-1beta. Antibody to interleukin-1beta reduced baseline and rhinovirus-induced ICAM-1 production. These results suggest that lansoprazole inhibits rhinovirus infection by reducing ICAM-1 via partly endogenous production of interleukin-1beta, and by blocking the rhinovirus RNA entry into the endosomes. Lansoprazole may modulate airway inflammation by reducing the production of cytokines and ICAM-1 in rhinovirus infection.