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1.  Soluble Major Histocompatibility Complex Class I-Related Chain B Molecules Are Increased and Correlate With Clinical Outcomes During Rhinovirus Infection in Healthy Subjects 
Chest  2014;146(1):32-40.
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.
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.
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.
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.
PMCID: PMC4077410  PMID: 24556715
2.  Lymphocyte subsets in experimental rhinovirus infection in chronic obstructive pulmonary disease☆ 
Respiratory Medicine  2014;108(1):78-85.
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.
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.
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.
Following rhinovirus infection T cells move from the circulation to the lung. Repeated virus infections may contribute to T cell accumulation in COPD patients.
PMCID: PMC3969590  PMID: 24099891
Chronic obstructive pulmonary disease; Acute exacerbations of COPD; Respiratory viruses; T lymphocytes
3.  Neutrophil adhesion molecules in experimental rhinovirus infection in COPD 
Respiratory Research  2013;14(1):72.
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.
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.
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.
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.
PMCID: PMC3726453  PMID: 23834268
Chronic obstructive pulmonary disease; Exacerbations; Respiratory viruses; Neutrophils
4.  Correction: The Role of IL-15 Deficiency in the Pathogenesis of Virus-Induced Asthma Exacerbations 
PLoS Pathogens  2012;8(4):10.1371/annotation/43a4a197-1739-4561-8b8d-b13cd6d7009f.
PMCID: PMC3321056
5.  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.
PMCID: PMC3086441  PMID: 21148500
6.  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.
PMCID: PMC3081284  PMID: 20889904
disease exacerbation; respiratory tract infections; COPD; rhinovirus
7.  The Role of IL-15 Deficiency in the Pathogenesis of Virus-Induced Asthma Exacerbations 
PLoS Pathogens  2011;7(7):e1002114.
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/β 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.
Author Summary
We previously reported deficiency in interferon production in asthma, which correlated with disease severity and viral load during experimental rhinovirus infection. Here we show that macrophages produce IL-15 upon rhinovirus infection and that IFN-β plays an important role in IL-15 production. In asthmatic subjects, there is a deficiency in rhinovirus-induced production of IL-15 by macrophages, which indicates immunodeficiency in asthma is surprisingly broad, also involving IL-15, an important cytokine that bridges innate and acquired immunity. These results show that IFN-β therapy in asthma exacerbations could be effective not only due to direct anti-viral effects of IFN-β, but also by inducing IL-15 production. We also show induction of IFN-β and IL-15 to be NF-kB dependent, an important finding which has implications for NF-kB inhibitor drug development programmes as these drugs have potential to worsen rather than improve asthma exacerbation severity, by further enhancing deficiencies of IL-15 and IFN-β. This study investigating the role of IL-15 in rhinovirus infection and asthma has also major implications in other diseases, for example pandemic influenza, where asthma is a major risk factor for severe disease and death, and COPD and cystic fibrosis where IFN-β deficiency is also present.
PMCID: PMC3136447  PMID: 21779162
8.  Rhinovirus Replication in Human Macrophages Induces NF-κB-Dependent Tumor Necrosis Factor Alpha Production 
Journal of Virology  2006;80(16):8248-8258.
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-α) 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-κB, leading to TNF-α 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-κB and colocalization of p65/NF-κB nuclear translocation with virus replication in both macrophage types. The infection induced TNF-α 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-κB but not p38 mitogen-activated protein kinase in induction of TNF-α. 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-κB as a promising target for development of new therapeutic intervention strategies.
PMCID: PMC1563804  PMID: 16873280
9.  Toll-Like Receptor 3 Is Induced by and Mediates Antiviral Activity against Rhinovirus Infection of Human Bronchial Epithelial Cells 
Journal of Virology  2005;79(19):12273-12279.
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.
PMCID: PMC1211516  PMID: 16160153
10.  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-β mRNA expression in asthmatic cultures and they produced >2.5 times less interferon-β protein. In infected asthmatic cells, exogenous interferon-β induced apoptosis and reduced virus replication, demonstrating a causal link between deficient interferon-β, 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.
PMCID: PMC2213100  PMID: 15781584

Results 1-10 (10)