The role of IL-15 deficiency in the pathogenesis of virus-induced asthma exacerbations.

Rhinovirus infections are the major cause of asthma exacerbations. We hypothesised that IL-15, a cytokine implicated in innate and acquired antiviral immunity, may be deficient in asthma and important in the pathogenesis of asthma exacerbations. We investigated regulation of IL-15 induction by rhinovirus in human macrophages in vitro, IL-15 levels in bronchoalveolar lavage (BAL) fluid and IL-15 induction by rhinovirus in BAL macrophages from asthmatic and control subjects, and related these to outcomes of infection in vivo. Rhinovirus induced IL-15 in macrophages was replication-, NF-κB- and α/ß interferon-dependent. BAL macrophage IL-15 induction by rhinovirus was impaired in asthmatics and inversely related to lower respiratory symptom severity during experimental rhinovirus infection. IL-15 levels in BAL fluid were also decreased in asthmatics and inversely related with airway hyperresponsiveness and with virus load during in vivo rhinovirus infection. Deficient IL-15 production in asthma may be important in the pathogenesis of asthma exacerbations.

Neutrophil adhesion molecules in experimental rhinovirus infection in COPD.

BACKGROUND: COPD exacerbations are associated with neutrophilic airway inflammation. Adhesion molecules on the surface of neutrophils may play a key role in their movement from blood to the airways. We analysed adhesion molecule expression on blood and sputum neutrophils from COPD subjects and non-obstructed smokers during experimental rhinovirus infections. METHODS: Blood and sputum were collected from 9 COPD subjects and 10 smoking and age-matched control subjects at baseline, and neutrophil expression of the adhesion molecules and activation markers measured using flow cytometry. The markers examined were CD62L and CD162 (mediating initial steps of neutrophil rolling and capture), CD11a and CD11b (required for firm neutrophil adhesion), CD31 and CD54 (involved in neutrophil transmigration through the endothelial monolayer) and CD63 and CD66b (neutrophil activation markers). Subjects were then experimentally infected with rhinovirus-16 and repeat samples collected for neutrophil analysis at post-infection time points. RESULTS: At baseline there were no differences in adhesion molecule expression between the COPD and non-COPD subjects. Expression of CD11a, CD31, CD62L and CD162 was reduced on sputum neutrophils compared to blood neutrophils. Following rhinovirus infection expression of CD11a expression on blood neutrophils was significantly reduced in both subject groups. CD11b, CD62L and CD162 expression was significantly reduced only in the COPD subjects. Blood neutrophil CD11b expression correlated inversely with inflammatory markers and symptom scores in COPD subjects. CONCLUSION: Following rhinovirus infection neutrophils with higher surface expression of adhesion molecules are likely preferentially recruited to the lungs. CD11b may be a key molecule involved in neutrophil trafficking in COPD exacerbations.

Role of deficient type III interferon-lambda production in asthma exacerbations.

Rhinoviruses are the major cause of asthma exacerbations, and asthmatics have increased susceptibility to rhinovirus and risk of invasive bacterial infections. Here we show deficient induction of interferon-lambdas by rhinovirus in asthmatic primary bronchial epithelial cells and alveolar macrophages, which was highly correlated with severity of rhinovirus-induced asthma exacerbation and virus load in experimentally infected human volunteers. Induction by lipopolysaccharide in asthmatic macrophages was also deficient and correlated with exacerbation severity. These results identify previously unknown mechanisms of susceptibility to infection in asthma and suggest new approaches to prevention and/or treatment of asthma exacerbations.

Experimental rhinovirus infection as a human model of chronic obstructive pulmonary disease exacerbation.

RATIONALE: Respiratory virus infections are associated with chronic obstructive pulmonary disease (COPD) exacerbations, but a causative relationship has not been proven. Studies of naturally occurring exacerbations are difficult and the mechanisms linking virus infection to exacerbations are poorly understood. We hypothesized that experimental rhinovirus infection in subjects with COPD would reproduce the features of naturally occurring COPD exacerbations and is a valid model of COPD exacerbations. OBJECTIVES: To evaluate experimental rhinovirus infection as a model of COPD exacerbation and to investigate the mechanisms of virus-induced exacerbations. METHODS: We used experimental rhinovirus infection in 13 subjects with COPD and 13 nonobstructed control subjects to investigate clinical, physiologic, pathologic, and antiviral responses and relationships between virus load and these outcomes. MEASUREMENTS AND MAIN RESULTS: Clinical data; inflammatory mediators in blood, sputum, and bronchoalveolar lavage; and viral load in nasal lavage, sputum, and bronchoalveolar lavage were measured at baseline and after infection with rhinovirus 16. After rhinovirus infection subjects with COPD developed lower respiratory symptoms, airflow obstruction, and systemic and airway inflammation that were greater and more prolonged compared with the control group. Neutrophil markers in sputum related to clinical outcomes and virus load correlated with inflammatory markers. Virus load was higher and IFN production by bronchoalveolar lavage cells was impaired in the subjects with COPD. CONCLUSIONS: We have developed a new model of COPD exacerbation that strongly supports a causal relationship between rhinovirus infection and COPD exacerbations. Impaired IFN production and neutrophilic inflammation may be important mechanisms in virus-induced COPD exacerbations.

RSV-induced bronchial epithelial cell PD-L1 expression inhibits CD8+ T cell nonspecific antiviral activity.

Respiratory syncytial virus (RSV) is a major cause of bronchiolitis in infants. It is also responsible for high morbidity and mortality in the elderly. Programmed death ligands (PD-Ls) on antigen-presenting cells interact with receptors on T cells to regulate immune responses. The programmed death receptor-ligand 1/programmed death receptor 1 (PD-L1-PD-1) pathway is inhibitory in chronic viral infections, but its role in acute viral infections is unclear. We hypothesized that bronchial epithelial cell (BEC) expression of PD-Ls would inhibit local effector CD8(+) T cell function. We report that RSV infection of primary human BECs strongly induces PD-L1 expression. In a co-culture system of BECs with purified CD8(+) T cells, we demonstrated that RSV-infected BECs increased CD8(+) T cell activation, proliferation, and antiviral function. Blocking PD-L1 on RSV-infected BECs co-cultured with CD8(+) T cells enhanced CD8(+) T cell IFN-γ, IL-2, and granzyme B production. It also decreased the virus load of the BECs. Based on our findings, we believe therapeutic strategies that target the PD-L1-PD-1 pathway might increase antiviral immune responses to RSV and other acute virus infections.

Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus.

Rhinoviruses are the major trigger of acute asthma exacerbations and asthmatic subjects are more susceptible to these infections. To investigate the underlying mechanisms of this increased susceptibility, we examined virus replication and innate responses to rhinovirus (RV)-16 infection of primary bronchial epithelial cells from asthmatic and healthy control subjects. Viral RNA expression and late virus release into supernatant was increased 50- and 7-fold, respectively in asthmatic cells compared with healthy controls. Virus infection induced late cell lysis in asthmatic cells but not in normal cells. Examination of the early cellular response to infection revealed impairment of virus induced caspase 3/7 activity and of apoptotic responses in the asthmatic cultures. Inhibition of apoptosis in normal cultures resulted in enhanced viral yield, comparable to that seen in infected asthmatic cultures. Examination of early innate immune responses revealed profound impairment of virus-induced interferon-beta mRNA expression in asthmatic cultures and they produced >2.5 times less interferon-beta protein. In infected asthmatic cells, exogenous interferon-beta induced apoptosis and reduced virus replication, demonstrating a causal link between deficient interferon-beta, impaired apoptosis and increased virus replication. These data suggest a novel use for type I interferons in the treatment or prevention of virus-induced asthma exacerbations.

Rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and Th1/2 cytokine and IL-10 production.

Acute exacerbations are the major cause of asthma morbidity, mortality, and health-care costs and are difficult to treat and prevent. The majority of asthma exacerbations are associated with rhinovirus (RV) infection, but evidence supporting a causal relationship is weak and mechanisms are poorly understood. We hypothesized that in asthmatic, but not normal, subjects RV infection would induce clinical, physiologic, and pathologic lower airway responses typical of an asthma exacerbation and that these changes would be related to virus replication and impaired T helper 1 (Th1)/IL-10 or augmented Th2 immune responses. We investigated physiologic, virologic, and immunopathologic responses to experimental RV infection in blood, induced sputum, and bronchial lavage in 10 asthmatic and 15 normal volunteers. RV infection induced significantly greater lower respiratory symptoms and lung function impairment and increases in bronchial hyperreactivity and eosinophilic lower airway inflammation in asthmatic compared with normal subjects. In asthmatic, but not normal, subjects virus load was significantly related to lower respiratory symptoms, bronchial hyperreactivity, and reductions in blood total and CD8(+) lymphocytes; lung function impairment was significantly related to neutrophilic and eosinophilic lower airway inflammation. The same virologic and clinical outcomes were strongly related to deficient IFN-gamma and IL-10 responses and to augmented IL-4, IL-5, and IL-13 responses. This study demonstrates increased RV-induced clinical illness severity in asthmatic compared with normal subjects, provides evidence of strong relationships between virus load, lower airway virus-induced inflammation and asthma exacerbation severity, and indicates augmented Th2 or impaired Th1 or IL-10 immunity are likely important mechanisms.

Protein kinase R, IkappaB kinase-beta and NF-kappaB are required for human rhinovirus induced pro-inflammatory cytokine production in bronchial epithelial cells.

Rhinovirus infections cause the majority of acute exacerbations of airway diseases such as asthma and chronic obstructive pulmonary disease, with increased pro-inflammatory cytokine production by infected bronchial epithelial cells contributing to disease pathogenesis. Theses diseases are a huge cause of morbidity worldwide, and contribute a major economic burden to healthcare costs. Current steroid based treatments are only partially efficient at controlling virus induced inflammation, which remains an unmet therapeutic goal. Although NF-kappaB has been implicated, the precise mechanisms of rhinovirus induction of pro-inflammatory gene expression in bronchial epithelial cells are unclear. We hypothesised that rhinovirus replication and generation of dsRNA was an important process of pro-inflammatory cytokine induction. Using pharmalogical (2-aminopurine and a new small molecule inhibitor) and genetic inhibition of the dsRNA binding kinase protein kinase R, striking inhibition of dsRNA (polyrIC) and rhinovirus induced CCL5, CXCL8 and IL-6 protein was observed. Using confocal microscopy, rhinovirus induced protein kinase R phosphorylation co-located with NF-kappaB p65 nuclear translocation. Focusing on CXCL8, both rhinovirus infection and dsRNA treatment required IkappaB kinase-beta for induction of CXCL8. Analysis of cis-acting sites in the CXCL8 promoter revealed that both rhinovirus infection and dsRNA treatment upregulated CXCL8 promoter activation via NF-kappaB and NF-IL6 binding sites. Together, the results demonstrate the importance of dsRNA in induction of pro-inflammatory cytokines by rhinoviruses, and suggest that protein kinase R is involved in NF-kappaB mediated gene transcription of pro-inflammatory cytokines via IkappaB kinase-beta. These molecules regulating rhinovirus induction of inflammation represent therapeutic targets.

Vitamin D increases the antiviral activity of bronchial epithelial cells in vitro.

BACKGROUND: By modulating the antiviral immune response via vitamin D receptor, the active form of vitamin D (1,25-dihydroxyvitamin D, calcitriol) could play a central role in protection against respiratory virus infections. This in vitro study tested the hypothesis that respiratory viruses modulate vitamin D receptor expression in human bronchial epithelial cells and this modulation affects the antiviral response to exogenous vitamin D. METHODS: Human primary bronchial epithelial cells were infected with rhinoviruses and respiratory syncytial virus in the presence or absence of vitamin D. Expression of vitamin D receptor, 1α-hydroxylase (1α(OH)ase), 24-hydroxylase (24(OH)ase), innate interferons, interferon stimulated genes and cathelicidin were measured by quantitative polymerase chain reaction. The antiviral effect of vitamin D on rhinovirus replication was determined by measurement of virus load. A direct inactivation assay was used to determine the antiviral activity of cathelicidin. RESULTS: Both RV and RSV decreased vitamin D receptor and 24(OH)ase and, in addition, RSV increased 1α(OH)ase expression in epithelial cells. Vitamin D decreased rhinovirus replication and release, and increased rhinovirus-induced interferon stimulated genes and cathelicidin. Furthermore, cathelicidin had direct anti-rhinovirus activity. CONCLUSIONS: Despite lower vitamin D receptor levels in rhinovirus-infected epithelial cells, exogenous vitamin D increased antiviral defences most likely via cathelicidin and innate interferon pathways.

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.

Lymphocyte subsets in experimental rhinovirus infection in chronic obstructive pulmonary disease.

BACKGROUND: COPD is associated with increased numbers of T cells in the lungs, particularly CD8+ T cells. The mechanisms of increased T cells are unknown but may be related to repeated virus infections in COPD patients. We analysed lymphocyte subsets in blood and bronchoalveolar lavage in smokers and COPD subjects during experimental rhinovirus infections. METHODS: Lymphocytes were isolated from blood and bronchoalveolar lavage from COPD subjects and non-obstructed smokers prior to, and following experimental rhinovirus infection. Lymphocyte surface markers and intracellular cytokines were analysed using flow cytometry. RESULTS: Following rhinovirus infection CD4+ and CD8+ T cell numbers in the COPD subjects were significantly reduced in blood and CD3+ and CD8+ T cells increased in bronchoalveolar lavage compared to baseline. T cells did not increase in BAL in the control subjects. CD3+ T cells correlated with virus load. CONCLUSIONS: Following rhinovirus infection T cells move from the circulation to the lung. Repeated virus infections may contribute to T cell accumulation in COPD patients.

Local CD11c+ MHC class II- precursors generate lung dendritic cells during respiratory viral infection, but are depleted in the process.

Increases in numbers of lung dendritic cells (DC) observed during respiratory viral infections are assumed to be due to recruitment from bone marrow precursors. No local production has been demonstrated. In this study, we isolated defined populations of murine lung cells based on CD11c and MHC class II (MHC II) expression. After culture for 12 days with GM-CSF, we analyzed cell numbers, DC surface markers, and Ag-presenting capacity. Only CD11c+ MHC II- cells from naive mice proliferated, yielding myeloid DC, which induced Ag-specific proliferation of naive T cells. After respiratory syncytial virus (RSV) infection, numbers of pulmonary CD11c+ MHC II- precursor cells were significantly reduced and DC could not be generated. Moreover, RSV infection prevented subsequent in vivo expansion of pulmonary DC in response to influenza infection or LPS treatment. These results provide direct evidence of local generation of fully functional myeloid DC in the lung from CD11c+ MHC II(-) precursor cells that are depleted by RSV infection, leading to an inability to expand lung DC numbers in response to subsequent viral infection or exposure to bacterial products. This depletion of local DC precursors in respiratory viral infections may be important in explaining complex interactions between multiple and intercurrent pulmonary infections.

Expression of programmed death-1 ligand (PD-L) 1, PD-L2, B7-H3, and inducible costimulator ligand on human respiratory tract epithelial cells and regulation by respiratory syncytial virus and type 1 and 2 cytokines.

BACKGROUND: Respiratory syncytial virus (RSV) is associated with wheezing illness, and infections can occur repeatedly throughout life. We hypothesized that RSV infection of respiratory tract epithelial cells up-regulates B7 molecules that regulate memory immune responses and that type 1 and 2 cytokines differentially modulate this induction. METHODS: We used flow-cytometric analysis to investigate programmed death-1 ligand (PD-L) 1, PD-L2, B7-H3, and inducible costimulatory ligand (ICOS-L) expression on tracheal (NCI-H292), bronchial (BEAS-2B), and alveolar (A549) epithelial cells; regulation of this expression by RSV, interferon (IFN)- gamma , and interleukin (IL)-4; and the effects of IFN-gamma and IL-4 on RSV-induced expression of these molecules. RESULTS: B7-H3 was strongly expressed, PD-L1 and ICOS-L were moderately expressed, and PD-L2 was weakly expressed on unstimulated tracheal, bronchial, and alveolar epithelial cells. RSV infection up-regulated PD-L1, PD-L2, and B7-H3 expression on all cells and ICOS-L expression on bronchial and alveolar epithelial cells. IL-4 treatment alone had no effect, whereas IFN-gamma treatment alone increased PD-L1 and PD-L2 expression on all cells and decreased B7-H3 expression on bronchial and alveolar epithelial cells. On RSV-infected alevolar epithelial cells, IFN-gamma treatment increased PD-L1 and PD-L2 expression and decreased B7-H3 and ICOS-L expression, and IL-4 treatment increased PD-L2 and B7-H3 expression and decreased ICOS-L expression. CONCLUSIONS: Respiratory tract epithelial cells express a wide range of B7 molecules. RSV infection increases their expression, and this expression is differentially regulated by IFN-gamma and IL-4. These processes may be involved in decreasing T cell antiviral immune responses to RSV and in RSV-associated wheezing.

Rhinovirus replication in human macrophages induces NF-kappaB-dependent tumor necrosis factor alpha production.

Rhinoviruses (RV) are the major cause of acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Rhinoviruses have been shown to activate macrophages, but rhinovirus replication in macrophages has not been reported. Tumor necrosis factor alpha (TNF-alpha) is implicated in the pathogenesis of acute exacerbations, but its cellular source and mechanisms of induction by virus infection are unclear. We hypothesized that rhinovirus replication in human macrophages causes activation and nuclear translocation of NF-kappaB, leading to TNF-alpha production. Using macrophages derived from the human monocytic cell line THP-1 and from primary human monocytes, we demonstrated that rhinovirus replication was productive in THP-1 macrophages, leading to release of infectious virus into supernatants, but was limited in monocyte-derived macrophages, likely due to type I interferon production, which was robust in monocyte-derived but deficient in THP-1-derived macrophages. Similar to bronchial epithelial cells, only small numbers of cells supported complete virus replication. We demonstrated RV-induced activation of NF-kappaB and colocalization of p65/NF-kappaB nuclear translocation with virus replication in both macrophage types. The infection induced TNF-alpha release in a time- and dose-dependent, RV serotype- and receptor-independent manner and was largely (THP-1 derived) or completely (monocyte derived) dependent upon virus replication. Finally, we established the requirement for NF-kappaB but not p38 mitogen-activated protein kinase in induction of TNF-alpha. These data suggest RV infection of macrophages may be an important source of proinflammatory cytokines implicated in the pathogenesis of exacerbations of asthma and COPD. They also confirm inhibition of NF-kappaB as a promising target for development of new therapeutic intervention strategies.

Toll-like receptor 3 is induced by and mediates antiviral activity against rhinovirus infection of human bronchial epithelial cells.

Rhinoviruses (RV) are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. Toll-like receptors (TLRs) are a conserved family of receptors that recognize and respond to a variety of pathogen-associated molecular patterns. TLR3 recognizes double-stranded RNA, an important intermediate of many viral life cycles (including RV). The importance of TLR3 in host responses to virus infection is not known. Using BEAS-2B (a human bronchial epithelial cell-line), we demonstrated that RV replication increased the expression of TLR3 mRNA and TLR3 protein on the cell surface. We observed that blocking TLR3 led to a decrease in interleukin-6, CXCL8, and CCL5 in response to poly(IC) but an increase following RV infection. Finally, we demonstrated that TLR3 mediated the antiviral response. This study demonstrates an important functional requirement for TLR3 in the host response against live virus infection and indicates that poly(IC) is not always a good model for studying the biology of live virus infection.

Rhinovirus viremia in children with respiratory infections.

RATIONALE: Viremia has been implicated in many viral infections; however, viremia due to rhinovirus (RV; rhinoviremia) has been considered not to occur in normal individuals. OBJECTIVE: To evaluate whether RV enters the bloodstream and identify the possible risk factors. METHODS: Nasopharyngeal washes (NPWs) of 221 children with respiratory infections were examined for the presence of RV by reverse transcription-polymerase chain reaction. Blood from 88 children, whose NPW was RV-positive, and 31 of RV-negative control subjects was subsequently examined for the presence of RV in the blood by semi-nested reverse transcription-polymerase chain reaction. Rhinoviremia was then correlated with clinical characteristics of the disease. RESULTS: RV was detected in the blood of 10 out of 88 NPW RV-positive cases (11.4%): 7 of 28 children with asthma exacerbations (25.0%), 2 of 26 with common cold (7.7%), 1 of 25 with bronchiolitis (4.0%), and 0 of 9 with pneumonia (0%). All NPW RV-negative cases were negative in the blood. The proportion of rhinoviremia in children with asthma exacerbation was significantly higher compared with children suffering from the other diseases (25 vs. 5%, p = 0.01). Significant risk factors were: sampling