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.

Airway-delivered short-chain fatty acid acetate boosts antiviral immunity during rhinovirus infection.

BACKGROUND: Microbiota are recognized to play a major role in regulation of immunity through release of immunomodulatory metabolites such as short-chain fatty acids (SCFAs). Rhinoviruses (RVs) induce upper respiratory tract illnesses and precipitate exacerbations of asthma and chronic obstructive pulmonary disease through poorly understood mechanisms. Local interactions between SCFAs and antiviral immune responses in the respiratory tract have not been previously investigated. OBJECTIVE: We sought to investigate whether pulmonary metabolite manipulation through lung-delivered administration of SCFAs can modulate antiviral immunity to RV infection. METHODS: We studied the effects of intranasal administration of the SCFAs acetate, butyrate, and propionate on basal expression of antiviral signatures, and of acetate in a mouse model of RV infection and in RV-infected lung epithelial cell lines. We additionally assessed the effects of acetate, butyrate, and propionate on RV infection in differentiated human primary bronchial epithelial cells. RESULTS: Intranasal acetate administration induced basal upregulation of IFN-ß, an effect not observed with other SCFAs. Butyrate induced RIG-I expression. Intranasal acetate treatment of mice increased interferon-stimulated gene and IFN-λ expression during RV infection and reduced lung virus loads at 8 hours postinfection. Acetate ameliorated virus-induced proinflammatory responses with attenuated pulmonary mucin and IL-6 expression observed at day 4 and 6 postinfection. This interferon-enhancing effect of acetate was confirmed in human bronchial and alveolar epithelial cell lines. In differentiated primary bronchial epithelial cells, butyrate treatment better modulated IFN-ß and IFN-λ gene expression during RV infection. CONCLUSIONS: SCFAs augment antiviral immunity and reduce virus load and proinflammatory responses during RV infection.

Experimental rhinovirus infection induces an antiviral response in circulating B cells which is dysregulated in patients with asthma.

BACKGROUND: Rhinoviruses are the predominant cause of respiratory viral infections and are strongly associated with asthma exacerbations. While humoral immunity plays an important role during virus infections, cellular aspects of this response are less well understood. Here, we investigated the antiviral response of circulating B cells upon experimental rhinovirus infection in healthy individuals and asthma patients. METHODS: We purified B cells from experimentally infected healthy individuals and patients with asthma and subjected them to total RNA-sequencing. Rhinovirus-derived RNA was measured in isolated B cells using a highly sensitive PCR. B cells were stimulated with rhinovirus in vitro to further study gene expression, expression of antiviral proteins and B-cell differentiation in response rhinovirus stimulation. Protein expression of pro-inflammatory cytokines in response to rhinovirus was assessed using a proximity extension assay. RESULTS: B cells isolated from experimentally infected subjects exhibited an antiviral gene profile linked to IFN-alpha, carried viral RNA in vivo and were transiently infected by rhinovirus in vitro. B cells rapidly differentiated into plasmablasts upon rhinovirus stimulation. While B cells lacked expression of interferons in response to rhinovirus exposure, co-stimulation with rhinovirus and IFN-alpha upregulated pro-inflammatory cytokine expression suggesting a potential new function of B cells during virus infections. Asthma patients showed extensive upregulation and dysregulation of antiviral gene expression. CONCLUSION: These findings add to the understanding of systemic effects of rhinovirus infections on B-cell responses in the periphery, show potential dysregulation in patients with asthma and might also have implications during infection with other respiratory viruses.

Pulmonary Innate Lymphoid Cell Responses during Rhinovirus-induced Asthma Exacerbations In Vivo: A Clinical Trial.

Rationale: Type 2 innate lymphoid cells (ILC2s) are significant sources of type 2 cytokines, which are implicated in the pathogenesis of asthma and asthma exacerbations. The role of ILC2s in virus-induced asthma exacerbations is not well characterized. Objectives: To characterize pulmonary ILC responses following experimental rhinovirus challenge in patients with moderate asthma and healthy subjects. Methods: Patients with moderate asthma and healthy subjects were inoculated with rhinovirus-16 and underwent bronchoscopy at baseline and at Day 3, and Day 8 after inoculation. Pulmonary ILC1s and ILC2s were quantified in bronchoalveolar lavage using flow cytometry. The ratio of bronchoalveolar lavage ILC2:ILC1 was assessed to determine their relative contributions to the clinical and immune response to rhinovirus challenge. Measurements and Main Results: At baseline, ILC2s were significantly higher in patients with asthma than in healthy subjects. At Day 8, ILC2s significantly increased from baseline in both groups, which was significantly higher in patients with asthma than in healthy subjects (all comparisons P < 0.05). In healthy subjects, ILC1s increased from baseline at Day 3 (P = 0.001), while in patients with asthma, ILC1s increased from baseline at Day 8 (P = 0.042). Patients with asthma had significantly higher ILC2:ILC1 ratios at baseline (P = 0.024) and Day 8 (P = 0.005). Increased ILC2:ILC1 ratio in patients with asthma correlated with clinical exacerbation severity and type 2 cytokines in nasal mucosal lining fluid. Conclusions: An ILC2-predominant inflammatory profile in patients with asthma was associated with increased severity and duration of rhinovirus infection compared with healthy subjects, supporting the potential role of ILC2s in the pathogenesis of virus-induced asthma exacerbations.

Virus-induced Volatile Organic Compounds Are Detectable in Exhaled Breath during Pulmonary Infection.

Rationale: Chronic obstructive pulmonary disease (COPD) is a condition punctuated by acute exacerbations commonly triggered by viral and/or bacterial infection. Early identification of exacerbation triggers is important to guide appropriate therapy, but currently available tests are slow and imprecise. Volatile organic compounds (VOCs) can be detected in exhaled breath and have the potential to be rapid tissue-specific biomarkers of infection etiology. Objectives: To determine whether volatile organic compound measurement could distinguish viral from bacterial infection in COPD. Methods: We used serial sampling within in vitro and in vivo studies to elucidate the dynamic changes that occur in VOC production during acute respiratory viral infection. Highly sensitive gas chromatography-mass spectrometry techniques were used to measure VOC production from infected airway epithelial-cell cultures and in exhaled breath samples from healthy subjects experimentally challenged with rhinovirus (RV)-A16 and from subjects with COPD with naturally occurring exacerbations. Measurements and Main Results: We identified a novel VOC signature comprising decane and other long-chain alkane compounds that is induced during RV infection of cultured airway epithelial cells and is also increased in the exhaled breath from healthy subjects experimentally challenged with RV and from patients with COPD during naturally occurring viral exacerbations. These compounds correlated with the magnitude of antiviral immune responses, viral burden, and exacerbation severity but were not induced by bacterial infection, suggesting that they represent a specific virus-inducible signature. Conclusions: Our study highlights the potential for measurement of exhaled breath VOCs as rapid, noninvasive biomarkers of viral infection. Further studies are needed to determine whether measurement of these signatures could be used to guide more targeted therapy with antibiotic/antiviral agents for COPD exacerbations.

Loss of regulatory capacity in Treg cells following rhinovirus infection.

BACKGROUND: Respiratory infections with rhinoviruses (RV) are strongly associated with development and exacerbations of asthma, and they pose an additional health risk for subjects with allergy. OBJECTIVE: How RV infections and chronic allergic diseases are linked and what role RV plays in the breaking of tolerance in regulatory T (Treg) cells is unknown. Therefore, this study aims to investigate the effects of RV on Treg cells. METHODS: Treg cells were isolated from subjects with asthma and controls after experimental infection with the RV-A16 (RV16) and analyzed with next-generation sequencing. Additionally, suppression assays, quantitative PCR assays, and protein quantifications were performed with Treg cells after in vitro RV16 infection. RESULTS: RV16 induced a strong antiviral response in Treg cells from subjects with asthma and controls, including the upregulation of IFI44L, MX1, ISG15, IRF7, and STAT1. In subjects with asthma, the inflammatory response was exaggerated and showed a dysregulated immune response compared with that in the controls. Furthermore, subjects with asthma failed to upregulate several immunosuppressive molecules such as CTLA4 and CD69, and they upregulated the inflammasome-related genes PYCARD and AIM2. Additionally, RV16 reduced the suppressive capacity of Treg cells from healthy subjects and subjects with asthma in vitro and increased TH2 cell-type cytokine production. CONCLUSIONS: Treg cells from healthy subjects and subjects with asthma displayed an antiviral response after RV infection and showed reduced suppressive capacity. These data suggest that Treg cell function might be altered or impaired during RV infections, which might play an important role in the association between RV and the development of asthma and asthma exacerbations.

Bronchial mucosal inflammation and illness severity in response to experimental rhinovirus infection in COPD.

BACKGROUND: Respiratory viral infection causes chronic obstructive pulmonary disease (COPD) exacerbations. We previously reported increased bronchial mucosa eosinophil and neutrophil inflammation in patients with COPD experiencing naturally occurring exacerbations. But it is unclear whether virus per se induces bronchial mucosal inflammation, nor whether this relates to exacerbation severity. OBJECTIVES: We sought to determine the extent and nature of bronchial mucosal inflammation following experimental rhinovirus (RV)-16-induced COPD exacerbations and its relationship to disease severity. METHODS: Bronchial mucosal inflammatory cell phenotypes were determined at preinfection baseline and following experimental RV infection in 17 Global Initiative for Chronic Obstructive Lung Disease stage II subjects with COPD and as controls 20 smokers and 11 nonsmokers with normal lung function. No subject had a history of asthma/allergic rhinitis: all had negative results for aeroallergen skin prick tests. RESULTS: RV infection increased the numbers of bronchial mucosal eosinophils and neutrophils only in COPD and CD8+ T lymphocytes in patients with COPD and nonsmokers. Monocytes/macrophages, CD4+ T lymphocytes, and CD20+ B lymphocytes were increased in all subjects. At baseline, compared with nonsmokers, subjects with COPD and smokers had increased numbers of bronchial mucosal monocytes/macrophages and CD8+ T lymphocytes but fewer numbers of CD4+ T lymphocytes and CD20+ B lymphocytes. The virus-induced inflammatory cells in patients with COPD were positively associated with virus load, illness severity, and reductions in lung function. CONCLUSIONS: Experimental RV infection induces bronchial mucosal eosinophilia and neutrophilia only in patients with COPD and monocytes/macrophages and lymphocytes in both patients with COPD and control subjects. The virus-induced inflammatory cell phenotypes observed in COPD positively related to virus load and illness severity. Antiviral/anti-inflammatory therapies could attenuate bronchial inflammation and ameliorate virus-induced COPD exacerbations.

Inflammation and infections in unreported chronic obstructive pulmonary disease exacerbations.

Purpose: COPD patients often do not report acute exacerbations to healthcare providers - unreported exacerbations. It is not known whether variances in symptoms, airway obstruction, aetiology and inflammatory responses account for differences in reporting of COPD exacerbations. The aims of the study were to compare symptoms, lung function changes, aetiology and inflammatory markers between exacerbations that were reported to healthcare providers or treated, with those that were unreported and untreated. Patients and methods: We recruited a cohort of COPD patients and collected clinical data and blood and airway samples when stable and during acute exacerbations. Virological and bacterial analyses were carried out and inflammatory markers measured. Results: We found no differences in symptoms, lung function, incidence of infection and inflammatory markers between reported and unreported exacerbations. Subjects who reported all exacerbations had higher BODE scores, lower FEV1 and more exacerbations compared with those who did not. Conclusion: The failure to report exacerbations is not related to the severity, aetiology or inflammatory profile of the exacerbation. Patients with less severe COPD and less frequent exacerbations are less likely to report exacerbations. The decision to report an exacerbation is not an objective marker of exacerbation severity and therefore studies that do not count unreported exacerbations will underestimate the frequency of clinically significant exacerbations. A better understanding of the factors that determine non-reporting of exacerbations is required to improve exacerbation reporting. Trial registration: ClinicalTrials.gov Identifier: NCT01376830. Registered June 17, 2011.

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.

Vitamin D attenuates rhinovirus-induced expression of intercellular adhesion molecule-1 (ICAM-1) and platelet-activating factor receptor (PAFR) in respiratory epithelial cells.

Human rhinoviruses commonly cause upper respiratory infections, which may be complicated by secondary bacterial infection. Vitamin D replacement reduces risk of acute respiratory infections in vitamin D-deficient individuals, but the mechanisms by which such protection is mediated are incompletely understood. We therefore conducted experiments to characterise the influence of the major circulating metabolite 25-hydroxyvitamin D (25[OH]D) and the active metabolite 1,25-dihydroxyvitamin D (1,25[OH]2D) on responses of a respiratory epithelial cell line (A549 cells) to infection with a major group human rhinovirus (RV-16). Pre-treatment of A549 respiratory epithelial cells with a physiological concentration (10-7M) of 25(OH)D induced transient resistance to infection with RV-16 and attenuated RV-16-induced expression of the genes encoding intercellular adhesion molecule 1 (ICAM-1, a cell surface glycoprotein that acts as the cellular receptor for major group rhinoviruses) and platelet-activating factor receptor (PAFR, a G-protein coupled receptor implicated in adhesion of Streptococcus pneumoniae to respiratory epithelial cells). These effects were associated with enhanced expression of the genes encoding the NF-κB inhibitor IκBα and the antimicrobial peptide cathelicidin LL-37. Our findings suggest possible mechanisms by which vitamin D may enhance resistance to rhinovirus infection and reduce risk of secondary bacterial infection in vitamin D-deficient individuals.

Biological exacerbation clusters demonstrate asthma and chronic obstructive pulmonary disease overlap with distinct mediator and microbiome profiles.

BACKGROUND: Exacerbations of asthma and chronic obstructive pulmonary disease (COPD) are heterogeneous. OBJECTIVE: We sought to investigate the sputum cellular, mediator, and microbiome profiles of both asthma and COPD exacerbations. METHODS: Patients with severe asthma or moderate-to-severe COPD were recruited prospectively to a single center. Sputum mediators were available in 32 asthmatic patients and 73 patients with COPD assessed at exacerbation. Biologic clusters were determined by using factor and cluster analyses on a panel of sputum mediators. Patterns of clinical parameters, sputum mediators, and microbiome communities were assessed across the identified clusters. RESULTS: The asthmatic patients and patients with COPD had different clinical characteristics and inflammatory profiles but similar microbial ecology. Three exacerbation biologic clusters were identified. Cluster 1 was COPD predominant, with 27 patients with COPD and 7 asthmatic patients exhibiting increased blood and sputum neutrophil counts, proinflammatory mediators (IL-1ß, IL-6, IL-6 receptor, TNF-α, TNF receptors 1 and 2, and vascular endothelial growth factor), and proportions of the bacterial phylum Proteobacteria. Cluster 2 had 10 asthmatic patients and 17 patients with COPD with increased blood and sputum eosinophil counts, type 2 mediators (IL-5, IL-13, CCL13, CCL17, and CCL26), and proportions of the bacterial phylum Bacteroidetes. Cluster 3 had 15 asthmatic patients and 29 patients with COPD with increased type 1 mediators (CXCL10, CXCL11, and IFN-γ) and proportions of the phyla Actinobacteria and Firmicutes. CONCLUSIONS: A biologic clustering approach revealed 3 subgroups of asthma and COPD exacerbations, each with different percentages of patients with overlapping asthma and COPD. The sputum mediator and microbiome profiles were distinct between clusters.

Role of airway glucose in bacterial infections in patients with chronic obstructive pulmonary disease.

BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) have increased susceptibility to respiratory tract infection, which contributes to disease progression and mortality, but mechanisms of increased susceptibility to infection remain unclear. OBJECTIVES: The aim of this study was to determine whether glucose concentrations were increased in airway samples (nasal lavage fluid, sputum, and bronchoalveolar lavage fluid) from patients with stable COPD and to determine the effects of viral infection on sputum glucose concentrations and how airway glucose concentrations relate to bacterial infection. METHODS: We measured glucose concentrations in airway samples collected from patients with stable COPD and smokers and nonsmokers with normal lung function. Glucose concentrations were measured in patients with experimentally induced COPD exacerbations, and these results were validated in patients with naturally acquired COPD exacerbations. Relationships between sputum glucose concentrations, inflammatory markers, and bacterial load were examined. RESULTS: Sputum glucose concentrations were significantly higher in patients with stable COPD compared with those in control subjects without COPD. In both experimental virus-induced and naturally acquired COPD exacerbations, sputum and nasal lavage fluid glucose concentrations were increased over baseline values. There were significant correlations between sputum glucose concentrations and sputum inflammatory markers, viral load, and bacterial load. Airway samples with higher glucose concentrations supported more Pseudomonas aeruginosa growth in vitro. CONCLUSIONS: Airway glucose concentrations are increased in patients with stable COPD and further increased during COPD exacerbations. Increased airway glucose concentrations might contribute to bacterial infections in both patients with stable and those with exacerbated COPD. This has important implications for the development of nonantibiotic therapeutic strategies for the prevention or treatment of bacterial infection in patients with COPD.

Microbiome balance in sputum determined by PCR stratifies COPD exacerbations and shows potential for selective use of antibiotics.

BACKGROUND: While a subgroup of patients with exacerbations of chronic obstructive pulmonary disease (COPD) clearly benefit from antibiotics, their identification remains challenging. We hypothesised that selective assessment of the balance between the two dominant bacterial groups (Gammaproteobacteria (G) and Firmicutes (F)) in COPD sputum samples might reveal a subgroup with a bacterial community structure change at exacerbation that was restored to baseline on recovery and potentially reflects effective antibiotic treatment. METHODS: Phylogenetically specific 16S rRNA genes were determined by quantitative real time PCR to derive a G:F ratio in serial sputum samples from 66 extensively-phenotyped COPD exacerbation episodes. RESULTS: Cluster analysis based on Euclidean distance measures, generated across the 4 visit times (stable and exacerbation day: 0,14 and 42) for the 66 exacerbation episodes, revealed three subgroups designated HG, HF, and GF reflecting predominance or equivalence of the two target bacterial groups. While the other subgroups showed no change at exacerbation, the HG cluster (n = 20) was characterized by G:F ratios that increased significantly at exacerbation and returned to baseline on recovery (p<0.00001); ratios in the HG group also correlated positively with inflammatory markers and negatively with FEV1. At exacerbation G:F showed a significant receiver-operator-characteristic curve to identify the HG subgroup (AUC 0.90, p<0.0001). CONCLUSIONS: The G:F ratio at exacerbation can be determined on a timescale compatible with decisions regarding clinical management. We propose that the G:F ratio has potential for use as a biomarker enabling selective use of antibiotics in COPD exacerbations and hence warrants further clinical evaluation.

Reduced sputum expression of interferon-stimulated genes in severe COPD.

BACKGROUND: Exacerbations of COPD are frequent and commonly triggered by respiratory tract infections. The purpose of our study was to investigate innate immunity in stable COPD patients. METHODS: Induced sputum was collected from 51 stable consecutive COPD patients recruited from the COPD Clinic of CHU Liege and 35 healthy subjects. Expression of interferons beta (IFN-ß) and lambda1 (IL-29), IFN-stimulated genes (ISGs) MxA, OAS, and viperin were measured in total sputum cells by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The presence of Picornaviruses was assessed by RT-PCR, while potential pathogenic microorganisms (PPM) were identified by sputum bacteriology. RESULTS: Expression of IL-29 was found in 16 of 51 COPD patients (31%) and in nine of 35 healthy subjects (26%), while IFN-ß was detected in six of 51 COPD patients (12%) and in two of 35 healthy subjects (6%). ISGs were easily detectable in both groups. In the whole group of COPD patients, OAS expression was decreased (P<0.05), while that of viperin was increased (P<0.01) compared to healthy subjects. No difference was found with respect to MxA. COPD patients from group D of Global Initiative for Chronic Obstructive Lung Disease (GOLD) had reduced expression of all three ISGs (P<0.01 for MxA, P<0.05 for OAS, and P<0.01 for viperin) as compared to those of group B patients. Picornaviruses were detected in eight of 51 (16%) COPD patients vs four of 33 (12%) healthy subjects, while PPM were detected in seven of 39 (18%) COPD patients and associated with raised sputum neutrophil counts. IFN-ß expression was raised when either picornavirus or PPM were detected (P=0.06), but no difference was seen regarding IL-29 or ISGs. CONCLUSION: ISGs expression was reduced in severe COPD that may favor exacerbation and contribute to disease progress by altering response to infection.

Oxidative and Nitrosative Stress and Histone Deacetylase-2 Activity in Exacerbations of COPD.

BACKGROUND: Respiratory virus infections are commonly associated with COPD exacerbations, but little is known about the mechanisms linking virus infection to exacerbations. Pathogenic mechanisms in stable COPD include oxidative and nitrosative stress and reduced activity of histone deacetylase-2 (HDAC2), but their roles in COPD exacerbations is unknown. We investigated oxidative and nitrosative stress (O&NS) and HDAC2 in COPD exacerbations using experimental rhinovirus infection. METHODS: Nine subjects with COPD (Global Initiative for Chronic Obstructive Lung Disease stage II), 10 smokers, and 11 nonsmokers were successfully infected with rhinovirus. Markers of O&NS-associated cellular damage, and inflammatory mediators and proteases were measured in sputum, and HDAC2 activity was measured in sputum and bronchoalveolar macrophages. In an in vitro model, monocyte-derived THP-1 cells were infected with rhinovirus and nitrosylation and activity of HDAC2 was measured. RESULTS: Rhinovirus infection induced significant increases in airways inflammation and markers of O&NS in subjects with COPD. O&NS markers correlated with virus load and inflammatory markers. Macrophage HDAC2 activity was reduced during exacerbation and correlated inversely with virus load, inflammatory markers, and nitrosative stress. Sputum macrophage HDAC2 activity pre-infection was inversely associated with sputum virus load and inflammatory markers during exacerbation. Rhinovirus infection of monocytes induced nitrosylation of HDAC2 and reduced HDAC2 activity; inhibition of O&NS inhibited rhinovirus-induced inflammatory cytokines. CONCLUSIONS: O&NS, airways inflammation, and impaired HDAC2 may be important mechanisms of virus-induced COPD exacerbations. Therapies targeting these mechanisms offer potential new treatments for COPD exacerbations.

Picornavirus-Induced Airway Mucosa Immune Profile in Asymptomatic Neonates.

BACKGROUND: Bacterial airway colonization is known to alter the airway mucosa immune response in neonates whereas the impact of viruses is unknown. The objective was therefore to examine the effect of respiratory viruses on the immune signature in the airways of asymptomatic neonates. METHODS: Nasal aspirates from 571 asymptomatic 1-month-old neonates from the Copenhagen Prospective Studies on Asthma in Childhood 2010 birth cohort were investigated for respiratory viruses. Simultaneously, unstimulated airway mucosal lining fluid was obtained and quantified for levels of 20 immune mediators related to type 1, type 2, type 17, and regulatory immune paths. The association between immune mediator levels and viruses was tested by conventional statistics and partial least square discriminant analysis. RESULTS: Picornaviruses were detected in 58 neonates (10.2%) and other viruses in 10 (1.8%). A general up-regulation of immune mediators was found in the neonates with picornavirus (P < .0001; partial least square discriminant analysis). The association was pronounced for type 1- and type 2-related markers and was unaffected by comprehensive confounder adjustment. Detection of picornavirus and bacteria was associated with an additive general up-regulating effect. CONCLUSIONS: Asymptomatic presence of picornavirus in the neonatal airway is a potent activator of the topical immune response. This is relevant to understanding the immune potentiating effect of early life exposure to viruses.

Soluble major histocompatibility complex class I-related chain B molecules are increased and correlate with clinical outcomes during rhinovirus infection in healthy subjects.

BACKGROUND: Surface major histocompatibility complex class I-related chain (MIC) A and B molecules are increased by IL-15 and have a role in the activation of natural killer group 2 member D-positive natural killer and CD8 T cells. MICA and MICB also exist in soluble forms (sMICA and sMICB). Rhinoviruses (RVs) are the major cause of asthma exacerbations, and IL-15 levels are decreased in the airways of subjects with asthma. The role of MIC molecules in immune responses in the lung has not been studied. Here, we determine the relationship between MICA and MICB and RV infection in vitro in respiratory epithelial cells and in vivo in healthy subjects and subjects with asthma. METHODS: Surface MICA and MICB, as well as sMICA and sMICB, in respiratory epithelial cells were measured in vitro in response to RV infection and exposure to IL-15. Levels of sMICA and sMICB in serum, sputum, and BAL were measured and correlated with blood and bronchoalveolar immune cells in healthy subjects and subjects with asthma before and during RV infection. RESULTS: RV increased MICA and MICB in vitro in epithelial cells. Exogenous IL-15 upregulated sMICB levels in RV-infected epithelial cells. Levels of sMICB molecules in serum were increased in healthy subjects compared with subjects with stable asthma. Following RV infection, airway levels of sMIC are upregulated, and there are positive correlations between sputum MICB levels and the percentage of bronchoalveolar natural killer cells in healthy subjects but not subjects with asthma. CONCLUSIONS: RV infection induces MIC molecules in respiratory epithelial cells in vitro and in vivo. Induction of MICB molecules is impaired in subjects with asthma, suggesting these molecules may have a role in the antiviral immune response to RV infections.

Airway inflammation and illness severity in response to experimental rhinovirus infection in asthma.

BACKGROUND: The nature of bronchial mucosal inflammation and its physiologic and clinical significance in rhinovirus-induced asthma exacerbations is unclear. We investigated bronchial mucosal inflammatory response and its association with physiologic and clinical outcomes in an experimental model of rhinovirus-induced asthma exacerbations. METHODS: We used immunohistochemistry methods to detect phenotypes of inflammatory cells infiltrating the bronchial mucosa before and after experimental rhinovirus infection in 10 subjects with asthma and 15 normal subjects. RESULTS: Compared with baseline, rhinovirus infection significantly increased the number of epithelial (P = .005) and subepithelial (P = .017) neutrophils in subjects with asthma only and subepithelial CD68+ macrophages in both subjects with asthma (P = .009) and normal subjects (P = .018) but more so in those with asthma (P = .021). Numbers of CD45+, CD68+, and CD20+ cells; neutrophils; and eosinophils at day 4 postinfection were positively associated with virus load (r = 0.50-0.72, P = .016-0.03). At acute infection in subjects with asthma, CD4+ cells correlated with chest symptom scores (r = 0.69, P = .029), the fall in the 10% fall in FEV1 (PC10) correlated with neutrophils (r = -0.89, P = .029), the PC10 correlated inversely with CD4+ (r = -0.67, P = .023) and CD8+ cells (r = -0.65, P = .03), the 20% fall in FEV1 was inversely associated with CD20+ cells (r = -0.65, P = .03), and higher epithelial CD8+ cell counts were significantly associated with a greater maximum fall in FEV1 (r = -0.72, P = .03), whereas higher subepithelial mast cell counts were significantly associated with a lower maximum percent fall in peak expiratory flow (r = 0.8, P = .024). CONCLUSIONS: In subjects with asthma, rhinovirus infection induces bronchial mucosal neutrophilia and more severe monocyte/macrophage infiltration than in normal subjects. Airway neutrophils, eosinophils, and T and B lymphocytes during infection are related to virus load and physiologic and clinical severity, whereas mast cells are related to greater lung function.