Anti-fibrotic effect of ciglitazone in HRV-induced airway remodelling cell model.

Fibrosis is an important phenomenon as it can occur early in the pathogenesis of asthma; it may be associated with disease severity and resistance to therapy. There is a strong evidence that infection caused by human rhinovirus (HRV) contributes to remodelling process, but there is lack of studies clearly explaining this pathway. Synthetic peroxisome proliferator-activated receptor (PPAR) γ presents immunomodulatory and anti-inflammatory features. In this study, we examined immunomodulatory properties of ciglitazone - PPAR-γ agonist, in development and modulation of airway remodelling. Epithelial cells (NHBE) and two lines of fibroblasts (WI-38, HFL1) were stimulated with ciglitazone and rhinovirus. The expression of genes related to airway remodelling process were analysed in the cells; moreover NF-κB, c-Myc and STAT3 were silenced in order to estimate potential pathways involved. Ciglitazone decreased mRNA expression of MMP-9 and TGF-ß. It also modified the expression of α-SMA and collagen after rhinovirus infection. Transcription factors knockdown altered the levels of expression. The results suggest possible anti-fibrotic activity of PPAR-γ agonist in human airway cells. Ciglitazone has been shown to be dependent on NF-κB- and STAT3-related pathways, thus, the PPAR-γ agonist may have therapeutic potential for the treatment of airway remodelling in asthma.

The ICAM-1 ligand HRV-A89 is internalized independently of clathrin-mediated endocytosis and its capsid reaches late endosomes.

The human rhinovirus (HRV) A2 is endocytosed by clathrin-mediated endocytosis (CME) bound to the classical LDL receptor and releases its RNA during its transport to late endosomes. Here it is shown that - presumably due to an effect on virus recycling - a low concentration of the CME inhibitor chlorpromazine present during virus internalization (30 min) did not reduce HRV-A2 infection, but strongly inhibited short-time (5 min) endocytosis of HRV-A2. Chlorpromazine had no effect on the colocalization of the ICAM-1 ligand HRV-A89 with early endosomes, excluding CME as the main endocytosis pathway of this virus. As published for HRV-A2 and HRV-A14, HRV-A89 partially colocalized with lysosome-associated membrane protein 2 and the microtubule inhibitor nocodazole did not reduce virus infection when present only during virus internalization. Together with previous work these data suggest that there are no principal differences between endocytosis pathways of ICAM-1-binding rhinoviruses in different cell types.

Rhinovirus-induced epithelial RIG-I inflammasome suppresses antiviral immunity and promotes inflammation in asthma and COVID-19.

Rhinoviruses and allergens, such as house dust mite are major agents responsible for asthma exacerbations. The influence of pre-existing airway inflammation on the infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. We analyse mechanisms of response to viral infection in experimental in vivo rhinovirus infection in healthy controls and patients with asthma, and in in vitro experiments with house dust mite, rhinovirus and SARS-CoV-2 in human primary airway epithelium. Here, we show that rhinovirus infection in patients with asthma leads to an excessive RIG-I inflammasome activation, which diminishes its accessibility for type I/III interferon responses, leading to their early functional impairment, delayed resolution, prolonged viral clearance and unresolved inflammation in vitro and in vivo. Pre-exposure to house dust mite augments this phenomenon by inflammasome priming and auxiliary inhibition of early type I/III interferon responses. Prior infection with rhinovirus followed by SARS-CoV-2 infection augments RIG-I inflammasome activation and epithelial inflammation. Timely inhibition of the epithelial RIG-I inflammasome may lead to more efficient viral clearance and lower the burden of rhinovirus and SARS-CoV-2 infections.

Eosinophil-mediated suppression and anti-IL-5 enhancement of plasmacytoid dendritic cell interferon responses in asthma.

BACKGROUND: Virus-induced IFN-α secretion by plasmacytoid dendritic cells (pDCs) is negatively impacted by IgE and has been linked to asthma exacerbations. Eosinophils, another contributor to type 2 inflammation, are also associated with asthma severity. OBJECTIVE: We sought to investigate the impact of eosinophils on pDC antiviral interferon responses and determine whether anti-IL-5/5Rα therapy enhances pDC antiviral function. METHODS: Blood pDCs purified from anonymous donors were stimulated in vitro with rhinovirus (RV)-16 in the presence or absence of eosinophils/eosinophil supernatants. IFN-α was measured in supernatants and RNA collected for bulk RNA-sequencing. Next, purified pDCs from 8 individuals with moderate to severe asthma, treated or not treated with anti-IL-5/5Rα therapy, were cultured ex vivo with or without RV; IFN-α secretion and differential gene expression analysis were compared between groups. RESULTS: Exposure to either eosinophils or eosinophil supernatants inhibited RV-induced pDC IFN-α secretion in a dose-dependent manner and did not impact pDC viability. Eosinophil-derived neurotoxin and TGF-ß partially recapitulated pDC IFN-α inhibition. Transcriptome analysis revealed global repression of pDC interferon response patterns by eosinophils, most notably in basal expression of interferon-stimulated genes. Increased RV-induced IFN-α secretion and transcription as well as increased basal interferon-stimulated gene expression was detected in pDCs from participants treated with anti-IL-5/5Rα therapy. CONCLUSIONS: Our findings highlight a novel mechanism through which type 2 inflammation regulates pDC IFN-α responses relevant to RV respiratory infections in the context of eosinophilic airway disease, suggesting a potential mechanism through which eosinophil-depleting therapies may reduce severity of RV illnesses.

Targeting intercellular adhesion molecule-1 (ICAM-1) to reduce rhinovirus-induced acute exacerbations in chronic respiratory diseases.

The chronic respiratory non-communicable diseases, asthma and chronic obstructive pulmonary disease (COPD) are among the leading causes of global mortality and morbidity. Individuals suffering from these diseases are particularly susceptible to respiratory infections caused by bacterial and/or viral pathogens, which frequently result in exacerbation of symptoms, lung function decline, frequent hospital emergency visits and increased socioeconomic burden. Human rhinoviruses (HRV) remain the major viral pathogen group implicated in exacerbations of both asthma and COPD. The rhinoviral entry into the host lung epithelium is facilitated primarily by the adhesion site ("receptor") intercellular adhesion molecule-1 (ICAM-1), coincidentally expressed on the respiratory epithelium in these conditions. Multiple observations of increased airway ICAM-1 protein in asthmatics, smokers and smoking-related COPD have been recorded in the literature. However, the lack of robust therapies for COPD in particular has triggered a renewed interest in assessing receptor antagonism-based anti-viral strategies for treatment of intercurrent viral infections in those with pre-existing chronic lung diseases. Given the crucial role ICAM-1 plays in facilitating HRV adhesion and, thus, transmissibility to the host respiratory system, as well as the up-regulation of ICAM-1 by smoking, we summarize the role of HRV in smoking-induced COPD and especially highlight the role of ICAM-1 in epithelial viral adhesion and chronic lung disease progression. Further, the review also sheds light specifically on evolving precision therapeutic strategies in blocking ICAM-1 for preventing viral adhesion and exacerbations of COPD.

Wogonin inhibits tight junction disruption via suppression of inflammatory response and phosphorylation of AKT/NF-κB and ERK1/2 in rhinovirus-infected human nasal epithelial cells.

OBJECTIVE: The maintenance of tight junction integrity contributes significantly to epithelial barrier function. If barrier function is destroyed, cell permeability increases and the movement of pathogens is promoted, further increasing the susceptibility to secondary infection. Here, we examined the protective effects of wogonin on rhinovirus (RV)-induced tight junction disruption. Additionally, we examined the signaling molecules responsible for anti-inflammatory activities in human nasal epithelial (HNE) cells. METHODS AND RESULTS: Primary HNE cells grown at an air-liquid interface and RPMI 2650 cells were infected apically with RV. Incubation with RV resulted in disruption of tight junction proteins (ZO-1, E-cadherin, claudin-1, and occludin) in the HNE cells. Cell viability of wogonin-treated HNE cells was measured using the MTT assay. Pretreatment with wogonin decreased RV-induced disruption of tight junctions in HNE cells. Furthermore, wogonin significantly decreased RV-induced phosphorylation of Akt/NF-κB and ERK1/2. Additionally, RV-induced generation of reactive oxygen species and RV-induced up-regulation of the production of inflammatory cytokines IL-8 and IL-6 were diminished by wogonin in HNE cells. CONCLUSION: Wogonin inhibits HRV-induced tight junction disruption via the suppression of inflammatory responses and phosphorylation of Akt/NF-κB and ERK1/2 in HNE cells. These finds will facilitate the development of novel therapeutic strategies.

Metabolic Modifications by Common Respiratory Viruses and Their Potential as New Antiviral Targets.

Respiratory viruses are known to be the most frequent causative mediators of lung infections in humans, bearing significant impact on the host cell signaling machinery due to their host-dependency for efficient replication. Certain cellular functions are actively induced by respiratory viruses for their own benefit. This includes metabolic pathways such as glycolysis, fatty acid synthesis (FAS) and the tricarboxylic acid (TCA) cycle, among others, which are modified during viral infections. Here, we summarize the current knowledge of metabolic pathway modifications mediated by the acute respiratory viruses respiratory syncytial virus (RSV), rhinovirus (RV), influenza virus (IV), parainfluenza virus (PIV), coronavirus (CoV) and adenovirus (AdV), and highlight potential targets and compounds for therapeutic approaches.

ICAM-1 induced rearrangements of capsid and genome prime rhinovirus 14 for activation and uncoating.

Most rhinoviruses, which are the leading cause of the common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. To release their genomes, rhinoviruses convert to activated particles that contain pores in the capsid, lack minor capsid protein VP4, and have an altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here, we present the structures of virion of rhinovirus 14 and its complex with ICAM-1 determined to resolutions of 2.6 and 2.4 Å, respectively. The cryo-electron microscopy reconstruction of rhinovirus 14 virions contains the resolved density of octanucleotide segments from the RNA genome that interact with VP2 subunits. We show that the binding of ICAM-1 to rhinovirus 14 is required to prime the virus for activation and genome release at acidic pH. Formation of the rhinovirus 14-ICAM-1 complex induces conformational changes to the rhinovirus 14 capsid, including translocation of the C termini of VP4 subunits, which become poised for release through pores that open in the capsids of activated particles. VP4 subunits with altered conformation block the RNA-VP2 interactions and expose patches of positively charged residues. The conformational changes to the capsid induce the redistribution of the virus genome by altering the capsid-RNA interactions. The restructuring of the rhinovirus 14 capsid and genome prepares the virions for conversion to activated particles. The high-resolution structure of rhinovirus 14 in complex with ICAM-1 explains how the binding of uncoating receptors enables enterovirus genome release.

Interference between rhinovirus and influenza A virus: a clinical data analysis and experimental infection study.

BACKGROUND: During the 2009 pandemic of an emerging influenza A virus (IAV; H1N1pdm09), data from several European countries indicated that the spread of the virus might have been interrupted by the annual autumn rhinovirus epidemic. We aimed to investigate viral interference between rhinovirus and IAV with use of clinical data and an experimental model. METHODS: We did a clinical data analysis and experimental infection study to investigate the co-occurrence of rhinovirus and IAV in respiratory specimens from adults (≥21 years) tested with a multiplex PCR panel at Yale-New Haven Hospital (CT, USA) over three consecutive winter seasons (Nov 1 to March 1, 2016-17, 2017-18, and 2018-19). We compared observed versus expected co-detections using data extracted from the Epic Systems electronic medical record system. To assess how rhinovirus infection affects subsequent IAV infection, we inoculated differentiated primary human airway epithelial cultures with rhinovirus (HRV-01A; multiplicity of infection [MOI] 0·1) or did mock infection. On day 3 post-infection, we inoculated the same cultures with IAV (H1N1 green fluorescent protein [GFP] reporter virus or H1N1pdm09; MOI 0·1). We used reverse transcription quantitative PCR or microscopy to quantify host cell mRNAs for interferon-stimulated genes (ISGs) on day 3 after rhinovirus or mock infection and IAV RNA on days 4, 5, or 6 after rhinovirus or mock infection. We also did sequential infection studies in the presence of BX795 (6 µM), to inhibit the interferon response. We compared ISG expression and IAV RNA and expression of GFP by IAV reporter virus. FINDINGS: Between July 1, 2016, and June 30, 2019, examination of 8284 respiratory samples positive for either rhinovirus (n=3821) or IAV (n=4463) by any test method was used to establish Nov 1 to March 1 as the period of peak virus co-circulation. After filtering for samples within this time frame meeting the inclusion criteria (n=13 707), there were 989 (7·2%) rhinovirus and 922 (6·7%) IAV detections, with a significantly lower than expected odds of co-detection (odds ratio 0·16, 95% CI 0·09-0·28). Rhinovirus infection of cell cultures induced ISG expression and protected against IAV infection 3 days later, resulting in an approximate 50 000-fold decrease in IAV H1N1pdm09 viral RNA on day 5 post-rhinovirus inoculation. Blocking the interferon response restored IAV replication following rhinovirus infection. INTERPRETATION: These findings show that one respiratory virus can block infection with another through stimulation of antiviral defences in the airway mucosa, supporting the idea that interference from rhinovirus disrupted the 2009 IAV pandemic in Europe. These results indicate that viral interference can potentially affect the course of an epidemic, and this possibility should be considered when designing interventions for seasonal influenza epidemics and the ongoing COVID-19 pandemic. FUNDING: National Institutes of Health, National Institute of General Medical Sciences, and the Yale Department of Laboratory Medicine.

Superimposition of Viral Protein Structures: A Means to Decipher the Phylogenies of Viruses.

Superimposition of protein structures is key in unravelling structural homology across proteins whose sequence similarity is lost. Structural comparison provides insights into protein function and evolution. Here, we review some of the original findings and thoughts that have led to the current established structure-based phylogeny of viruses: starting from the original observation that the major capsid proteins of plant and animal viruses possess similar folds, to the idea that each virus has an innate "self". This latter idea fueled the conceptualization of the PRD1-adenovirus lineage whose members possess a major capsid protein (innate "self") with a double jelly roll fold. Based on this approach, long-range viral evolutionary relationships can be detected allowing the virosphere to be classified in four structure-based lineages. However, this process is not without its challenges or limitations. As an example of these hurdles, we finally touch on the difficulty of establishing structural "self" traits for enveloped viruses showcasing the coronaviruses but also the power of structure-based analysis in the understanding of emerging viruses.

Individual subunits of a rhinovirus causing common cold exhibit largely different protein-RNA contact site conformations.

Rhinoviruses cause the common cold. They are icosahedral, built from sixty copies each of the capsid proteins VP1 through VP4 arranged in a pseudo T = 3 lattice. This shell encases a ss(+) RNA genome. Three-D classification of single and oligomeric asymmetric units computationally excised from a 2.9 Å cryo-EM density map of rhinovirus A89, showed that VP4 and the N-terminal extension of VP1 adopt different conformations within the otherwise identical 3D-structures. Analysis of up to sixty classes of single subunits and of six classes of subunit dimers, trimers, and pentamers revealed different orientations of the amino acid residues at the interface with the RNA suggesting that local asymmetry is dictated by disparities of the interacting nucleotide sequences. The different conformations escape detection by 3-D structure determination of entire virions with the conformational heterogeneity being only indicated by low density. My results do not exclude that the RNA follows a conserved assembly mechanism, contacting most or all asymmetric units in a specific way. However, as suggested by the gradual loss of asymmetry with increasing oligomerization and the 3D-structure of entire virions reconstructed by using Euler angles selected in the classification of single subunits, RNA path and/or folding likely differ from virion to virion.

A broad-spectrum virus- and host-targeting peptide against respiratory viruses including influenza virus and SARS-CoV-2.

The 2019 novel respiratory virus (SARS-CoV-2) causes COVID-19 with rapid global socioeconomic disruptions and disease burden to healthcare. The COVID-19 and previous emerging virus outbreaks highlight the urgent need for broad-spectrum antivirals. Here, we show that a defensin-like peptide P9R exhibited potent antiviral activity against pH-dependent viruses that require endosomal acidification for virus infection, including the enveloped pandemic A(H1N1)pdm09 virus, avian influenza A(H7N9) virus, coronaviruses (SARS-CoV-2, MERS-CoV and SARS-CoV), and the non-enveloped rhinovirus. P9R can significantly protect mice from lethal challenge by A(H1N1)pdm09 virus and shows low possibility to cause drug-resistant virus. Mechanistic studies indicate that the antiviral activity of P9R depends on the direct binding to viruses and the inhibition of virus-host endosomal acidification, which provides a proof of concept that virus-binding alkaline peptides can broadly inhibit pH-dependent viruses. These results suggest that the dual-functional virus- and host-targeting P9R can be a promising candidate for combating pH-dependent respiratory viruses.

The Dynamic Life of Virus Capsids.

Protein-shelled viruses have been thought as "tin cans" that merely carry the genomic cargo from cell to cell. However, through the years, it has become clear that viruses such as rhinoviruses and caliciviruses are active and dynamic structures waiting for the right environmental cues to deliver their genomic payload to the host cell. In the case of human rhinoviruses, the capsid has empty cavities that decrease the energy required to cause conformational changes, resulting in the capsids "breathing", waiting for the moment when the receptor binds for it to release its genome. Most strikingly, the buried N-termini of VP1 and VP4 are transiently exposed during this process. A more recent example of a "living" protein capsid is mouse norovirus (MNV). This family of viruses have a large protruding (P) domain that is loosely attached to the shell via a single-polypeptide tether. Small molecules found in the gut, such as bile salts, cause the P domains to rotate and collapse onto the shell surface. Concomitantly, bile alters the conformation of the P domain itself from one that binds antibodies to one that recognizes receptors. In this way, MNV appears to use capsid flexibility to present one face to the immune system and a completely different one to attack the host tissue. Therefore, it appears that even protein-shelled viruses have developed an impressive array of tricks to dodge our immune system and efficiently attack the host.

Human Rhinovirus Inhibition Through Capsid "Canyon" Perturbation: Structural Insights into The Role of a Novel Benzothiophene Derivative.

The challenge in targeting human rhinoviruses (HRV) over the years has been attributed to the wide variety in HRV serotypes. Nonetheless, the search for therapeutic agents against HRV continues unabated. These efforts have been augmented by the recent discovery of the novel benzothiophene derivative shown to inhibit HRV viral replication. Bound to subtype HRV-B14, the compound showed similar inhibitory activity as Pleconaril, a known capsid inhibitor. However, the molecular and structural basis of this inhibition remains unclear. In this in silico report, residue interaction network analysis revealed that the binding of the benzothiophene derivative into the "canyon" region of the active site of HRV-B14 distorts its initially extensively networked and compact residue profile. This was characterized by fewer inter-residue hydrogen bonds, reduced van der Waals interactions, and increased residue flexibility. Interestingly, however, the binding of this benzothiophene derivative decreased the flexibility of the north-south wall around the canyon region possibly impeding the "breathing motion" of HRV-B14, hence its inhibition. Atomistic insights also revealed the cruciality of Tyr152 toward inhibitor binding at HRV-B14. This was justified by the amino acid's high intermolecular interaction with both inhibitors. Findings provide important structural insights in the inhibitory activity the novel benzothiophene derivative, and reaffirm its promising potential as an alternative capsid inhibitor toward common cold therapy upon further experimental validation.

Cryo-EM structure of pleconaril-resistant rhinovirus-B5 complexed to the antiviral OBR-5-340 reveals unexpected binding site.

Viral inhibitors, such as pleconaril and vapendavir, target conserved regions in the capsids of rhinoviruses (RVs) and enteroviruses (EVs) by binding to a hydrophobic pocket in viral capsid protein 1 (VP1). In resistant RVs and EVs, bulky residues in this pocket prevent their binding. However, recently developed pyrazolopyrimidines inhibit pleconaril-resistant RVs and EVs, and computational modeling has suggested that they also bind to the hydrophobic pocket in VP1. We studied the mechanism of inhibition of pleconaril-resistant RVs using RV-B5 (1 of the 7 naturally pleconaril-resistant rhinoviruses) and OBR-5-340, a bioavailable pyrazolopyrimidine with proven in vivo activity, and determined the 3D-structure of the protein-ligand complex to 3.6 Å with cryoelectron microscopy. Our data indicate that, similar to other capsid binders, OBR-5-340 induces thermostability and inhibits viral adsorption and uncoating. However, we found that OBR-5-340 attaches closer to the entrance of the pocket than most other capsid binders, whose viral complexes have been studied so far, showing only marginal overlaps of the attachment sites. Comparing the experimentally determined 3D structure with the control, RV-B5 incubated with solvent only and determined to 3.2 Å, revealed no gross conformational changes upon OBR-5-340 binding. The pocket of the naturally OBR-5-340-resistant RV-A89 likewise incubated with OBR-5-340 and solved to 2.9 Å was empty. Pyrazolopyrimidines have a rigid molecular scaffold and may thus be less affected by a loss of entropy upon binding. They interact with less-conserved regions than known capsid binders. Overall, pyrazolopyrimidines could be more suitable for the development of new, broadly active inhibitors.

Distinct transcriptional modules in the peripheral blood mononuclear cells response to human respiratory syncytial virus or to human rhinovirus in hospitalized infants with bronchiolitis.

Human respiratory syncytial virus (HRSV) is the main cause of bronchiolitis during the first year of life, when infections by other viruses, such as rhinovirus, also occur and are clinically indistinguishable from those caused by HRSV. In hospitalized infants with bronchiolitis, the analysis of gene expression profiles from peripheral blood mononuclear cells (PBMC) may be useful for the rapid identification of etiological factors, as well as for developing diagnostic tests, and elucidating pathogenic mechanisms triggered by different viral agents. In this study we conducted a comparative global gene expression analysis of PBMC obtained from two groups of infants with acute viral bronchiolitis who were infected by HRSV (HRSV group) or by HRV (HRV group). We employed a weighted gene co-expression network analysis (WGCNA) which allows the identification of transcriptional modules and their correlations with HRSV or HRV groups. This approach permitted the identification of distinct transcription modules for the HRSV and HRV groups. According to these data, the immune response to HRSV infection-comparatively to HRV infection-was more associated to the activation of the interferon gamma signaling pathways and less related to neutrophil activation mechanisms. Moreover, we also identified host-response molecular markers that could be used for etiopathogenic diagnosis. These results may contribute to the development of new tests for respiratory virus identification. The finding that distinct transcriptional profiles are associated to specific host responses to HRSV or to HRV may also contribute to the elucidation of the pathogenic mechanisms triggered by different respiratory viruses, paving the way for new therapeutic strategies.

Pelargonium sidoides radix extract EPs 7630 reduces rhinovirus infection through modulation of viral binding proteins on human bronchial epithelial cells.

Bronchial epithelial cells are the first target cell for rhinovirus infection. The course of viral infections in patients with acute bronchitis, asthma and COPD can be improved by oral application of Pelargonium sidoides radix extract; however, the mechanism is not well understood. This study investigated the in vitro effect of Pelargonium sidoides radix extract (EPs 7630) on the expression of virus binding cell membrane and host defence supporting proteins on primary human bronchial epithelial cells (hBEC). Cells were isolated from patients with severe asthma (n = 6), moderate COPD (n = 6) and non-diseased controls (n = 6). Protein expression was determined by Western-blot and immunofluorescence. Rhinovirus infection was determined by immunofluorescence as well as by polymerase chain reaction. Cell survival was determined by manual cell count after live/death immunofluorescence staining. All parameters were determined over a period of 3 days. The results show that EPs 7630 concentration-dependently and significantly increased hBEC survival after rhinovirus infection. This effect was paralleled by decreased expression of the inducible co-stimulator (ICOS), its ligand ICOSL and cell surface calreticulin (C1qR). In contrast, EPs 7630 up-regulated the expression of the host defence supporting proteins ß-defensin-1 and SOCS-1, both in rhinovirus infected and un-infected hBEC. The expression of other virus interacting cell membrane proteins such as MyD88, TRL2/4 or ICAM-1 was not altered by EPs 7630. The results indicate that EPs 7630 may reduce rhinovirus infection of human primary BEC by down-regulating cell membrane docking proteins and up-regulating host defence proteins.

Non-Structural Protein 2B of Human Rhinovirus 16 Activates Both PERK and ATF6 Rather Than IRE1 to Trigger ER Stress.

To understand the underlying mechanisms of endoplasmic reticulum (ER) stress caused by human rhinovirus (HRV) 16 and non-structural transmembrane protein 2B, the expressions of ER chaperone glucose-regulated protein 78 (GRP78) and three signal transduction pathways, including protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1), were evaluated after HRV16 infection and 2B gene transfection. Our results showed that both HRV16 infection and 2B gene transfection increased the expression of ER chaperone GRP78, and induced phosphorylation of PERK and cleavage of ATF6 in a time-dependent manner. Our data also revealed that the HRV16 2B protein was localized to the ER membrane. However, both HRV16 infection and HRV16 2B gene transfection did not induce ER stress through the IRE1 pathway. Moreover, our results showed that apoptosis occurred in H1-HeLa cells infected with HRV16 or transfected with 2B gene accompanied with increased expression of CHOP and cleaved caspase-3. Taken together, non-structural protein 2B of HRV16 induced an ER stress response through the PERK and ATF6 pathways rather than the IRE1 pathway.

Bronchial mucosal IFN-α/ß and pattern recognition receptor expression in patients with experimental rhinovirus-induced asthma exacerbations.

BACKGROUND: The innate immune system senses viral infection through pattern recognition receptors (PRRs), leading to type I interferon production. The role of type I interferon and PPRs in rhinovirus-induced asthma exacerbations in vivo are uncertain. OBJECTIVES: We sought to compare bronchial mucosal type I interferon and PRR expression at baseline and after rhinovirus infection in atopic asthmatic patients and control subjects. METHODS: Immunohistochemistry was used to detect expression of IFN-α, IFN-ß, and the PRRs: Toll-like receptor 3, melanoma differentiation-associated gene 5, and retinoic acid-inducible protein I in bronchial biopsy specimens from 10 atopic asthmatic patients and 15 nonasthmatic nonatopic control subjects at baseline and on day 4 and 6 weeks after rhinovirus infection. RESULTS: We observed IFN-α/ß deficiency in the bronchial epithelium at 3 time points in asthmatic patients in vivo. Lower epithelial IFN-α/ß expression was related to greater viral load, worse airway symptoms, airway hyperresponsiveness, and reductions in lung function during rhinovirus infection. We found lower frequencies of bronchial subepithelial monocytes/macrophages expressing IFN-α/ß in asthmatic patients during infection. Interferon deficiency at baseline was not accompanied by deficient PRR expression in asthmatic patients. Both epithelial and subepithelial PRR expression were induced during rhinovirus infection. Rhinovirus infection-increased numbers of subepithelial interferon/PRR-expressing inflammatory cells were related to greater viral load, airway hyperresponsiveness, and reductions in lung function. CONCLUSIONS: Bronchial epithelial IFN-α/ß expression and numbers of subepithelial IFN-α/ß-expressing monocytes/macrophages during infection were both deficient in asthmatic patients. Lower epithelial IFN-α/ß expression was associated with adverse clinical outcomes after rhinovirus infection in vivo. Increases in numbers of subepithelial cells expressing interferon/PRRs during infection were also related to greater viral load/illness severity.

Involvement and Possible Role of Eosinophils in Asthma Exacerbation.

Eosinophils are involved in the development of asthma exacerbation. Recent studies have suggested that sputum and blood eosinophil counts are important factors for predicting asthma exacerbation. In severe eosinophilic asthma, anti-interleukin (IL)-5 monoclonal antibody decreases blood eosinophil count and asthma exacerbation frequency. However, even in the absence of IL-5, eosinophilic airway inflammation can be sufficiently maintained by the T helper (Th) 2 network, which comprises a cascade of vascular cell adhesion molecule-1/CC chemokines/eosinophil growth factors, including granulocyte-macrophage colony-stimulating factor (GM-CSF). Periostin, an extracellular matrix protein and a biomarker of the Th2 immune response in asthma, directly activates eosinophils in vitro. A major cause of asthma exacerbation is viral infection, especially rhinovirus (RV) infection. The expression of intercellular adhesion molecule (ICAM)-1, a cellular receptor for the majority of RVs, on epithelial cells is increased after RV infection, and adhesion of eosinophils to ICAM-1 can upregulate the functions of eosinophils. The expressions of cysteinyl leukotrienes (cysLTs) and CXCL10 are upregulated in virus-induced asthma. CysLTs can directly provoke eosinophilic infiltration in vivo and activate eosinophils in vitro. Furthermore, eosinophils express the CXC chemokine receptor 3, and CXCL10 activates eosinophils in vitro. Both eosinophils and neutrophils contribute to the development of severe asthma or asthma exacerbation. IL-8, which is an important chemoattractant for neutrophils, is upregulated in some cases of severe asthma. Lipopolysaccharide (LPS), which induces IL-8 from epithelial cells, is also increased in the lower airways of corticosteroid-resistant asthma. IL-8 or LPS-stimulated neutrophils increase the transbasement membrane migration of eosinophils, even in the absence of chemoattractants for eosinophils. Therefore, eosinophils are likely to contribute to the development of asthma exacerbation through several mechanisms, including activation by Th2 cytokines, such as IL-5 or GM-CSF or by virus infection-related proteins, such as CXCL10, and interaction with other cells, such as neutrophils.