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.
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.
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.
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.
COPD; host defense; infection; viral disease; 3-NT, 3-nitrotyrosine; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; GM-CSF, granulocyte-macrophage colony-stimulating factor; GOLD, Global Initiative for Obstructive Lung Disease; HDAC2, histone deacetylase-2; MMP-9, matrix metalloprotease-9; NAC, N-acetylcysteine; O&NS, oxidative and nitrosative stress; TNF-α, tumor necrosis factor-α
Rationale: Rhinoviruses are the major cause of asthma exacerbations;
however, its underlying mechanisms are poorly understood. We hypothesized that the
epithelial cell–derived cytokine IL-33 plays a central role in exacerbation
pathogenesis through augmentation of type 2 inflammation.
Objectives: To assess whether rhinovirus induces a type 2 inflammatory
response in asthma in vivo and to define a role for IL-33 in this
Methods: We used a human experimental model of rhinovirus infection and
novel airway sampling techniques to measure IL-4, IL-5, IL-13, and IL-33 levels in
the asthmatic and healthy airways during a rhinovirus infection. Additionally, we
cultured human T cells and type 2 innate lymphoid cells (ILC2s) with the supernatants
of rhinovirus-infected bronchial epithelial cells (BECs) to assess type 2 cytokine
production in the presence or absence of IL-33 receptor blockade.
Measurements and Main Results: IL-4, IL-5, IL-13, and IL-33 are all
induced by rhinovirus in the asthmatic airway in vivo and relate to exacerbation
severity. Further, induction of IL-33 correlates with viral load and IL-5 and IL-13
levels. Rhinovirus infection of human primary BECs induced IL-33, and culture of
human T cells and ILC2s with supernatants of rhinovirus-infected BECs strongly
induced type 2 cytokines. This induction was entirely dependent on IL-33.
Conclusions: IL-33 and type 2 cytokines are induced during a
rhinovirus-induced asthma exacerbation in vivo. Virus-induced IL-33 and
IL-33–responsive T cells and ILC2s are key mechanistic links between viral
infection and exacerbation of asthma. IL-33 inhibition is a novel therapeutic
approach for asthma exacerbations.
ILC2; infection; Th2; virus
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.
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.
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).
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.
Rationale: Chronic obstructive pulmonary disease (COPD) exacerbations are associated with virus (mostly rhinovirus) and bacterial infections, but it is not known whether rhinovirus infections precipitate secondary bacterial infections.
Objectives: To investigate relationships between rhinovirus infection and bacterial infection and the role of antimicrobial peptides in COPD exacerbations.
Methods: We infected subjects with moderate COPD and smokers and nonsmokers with normal lung function with rhinovirus. Induced sputum was collected before and repeatedly after rhinovirus infection and virus and bacterial loads measured with quantitative polymerase chain reaction and culture. The antimicrobial peptides secretory leukoprotease inhibitor (SLPI), elafin, pentraxin, LL-37, α-defensins and β-defensin-2, and the protease neutrophil elastase were measured in sputum supernatants.
Measurements and Main Results: After rhinovirus infection, secondary bacterial infection was detected in 60% of subjects with COPD, 9.5% of smokers, and 10% of nonsmokers (P < 0.001). Sputum virus load peaked on Days 5–9 and bacterial load on Day 15. Sputum neutrophil elastase was significantly increased and SLPI and elafin significantly reduced after rhinovirus infection exclusively in subjects with COPD with secondary bacterial infections, and SLPI and elafin levels correlated inversely with bacterial load.
Conclusions: Rhinovirus infections are frequently followed by secondary bacterial infections in COPD and cleavage of the antimicrobial peptides SLPI and elafin by virus-induced neutrophil elastase may precipitate these secondary bacterial infections. Therapy targeting neutrophil elastase or enhancing innate immunity may be useful novel therapies for prevention of secondary bacterial infections in virus-induced COPD exacerbations.
rhinovirus; chronic obstructive pulmonary disease; disease exacerbation; bacteria
Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine associated with acute and chronic inflammatory disorders and corticosteroid insensitivity. Its expression in the airways of patients with chronic obstructive pulmonary disease (COPD), a relatively steroid insensitive inflammatory disease is unclear, however.
Sputum, bronchoalveolar lavage (BAL) macrophages and serum were obtained from non-smokers, smokers and COPD patients. To mimic oxidative stress-induced COPD, mice were exposed to ozone for six-weeks and treated with ISO-1, a MIF inhibitor, and/or dexamethasone before each exposure. BAL fluid and lung tissue were collected after the final exposure. Airway hyperresponsiveness (AHR) and lung function were measured using whole body plethysmography. HIF-1α binding to the Mif promoter was determined by Chromatin Immunoprecipitation assays.
MIF levels in sputum and BAL macrophages from COPD patients were higher than those from non-smokers, with healthy smokers having intermediate levels. MIF expression correlated with that of HIF-1α in all patients groups and in ozone-exposed mice. BAL cell counts, cytokine mRNA and protein expression in lungs and BAL, including MIF, were elevated in ozone-exposed mice and had increased AHR. Dexamethasone had no effect on these parameters in the mouse but ISO-1 attenuated cell recruitment, cytokine release and AHR.
MIF and HIF-1α levels are elevated in COPD BAL macrophages and inhibition of MIF function blocks corticosteroid-insensitive lung inflammation and AHR. Inhibition of MIF may provide a novel anti-inflammatory approach in COPD.
In animal models, resident memory CD8+ T (Trm) cells assist in respiratory virus elimination but their importance in man has not been determined. Here, using experimental human respiratory syncytial virus (RSV) infection, we investigate systemic and local virus-specific CD8+ T-cell responses in adult volunteers. Having defined the immunodominance hierarchy, we analyse phenotype and function longitudinally in blood and by serial bronchoscopy. Despite rapid clinical recovery, we note surprisingly extensive lower airway inflammation with persistent viral antigen and cellular infiltrates. Pulmonary virus-specific CD8+ T cells display a CD69+CD103+ Trm phenotype and accumulate to strikingly high frequencies into convalescence without continued proliferation. While these have a more highly differentiated phenotype, they express fewer cytotoxicity markers than in blood. Nevertheless, their abundance before infection correlates with reduced symptoms and viral load, implying that CD8+ Trm cells in the human lung can confer protection against severe respiratory viral disease when humoral immunity is overcome.
Respiratory syncytial virus (RSV) is a common cause of respiratory tract infections. Here the authors analyse cellular immune responses of individuals experimentally infected with RSV and reveal the presence of high frequencies of virus- specific resident memory CD8+ T cells in the airway, which correlate with improved viral control.
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.
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.
disease exacerbation; respiratory tract infections; COPD; rhinovirus
Asthma exacerbations represent a significant disease burden and are commonly caused by rhinovirus (RV), which is sensed by Toll-like receptors (TLR) such as TLR7. Some asthmatics have impaired interferon (IFN) responses to RV, but the underlying mechanisms of this clinically relevant observation are poorly understood.
To investigate the importance of intact TLR7 signalling in vivo during RV exacerbation using mouse models of house dust mite (HDM)-induced allergic airways disease exacerbated by a superimposed RV infection.
Wild-type and TLR7-deficient (Tlr7−/−) BALB/c mice were intranasally sensitised and challenged with HDM prior to infection with RV1B. In some experiments, mice were administered recombinant IFN or adoptively transferred with plasmacytoid dendritic cells (pDC).
Allergic Tlr7−/− mice displayed impaired IFN release upon RV1B infection, increased virus replication and exaggerated eosinophilic inflammation and airways hyper reactivity. Treatment with exogenous IFN or adoptive transfer of TLR7-competent pDCs blocked these exaggerated inflammatory responses and boosted IFNγ release in the absence of host TLR7 signalling. TLR7 expression in the lungs was suppressed by allergic inflammation and by interleukin (IL)-5-induced eosinophilia in the absence of allergy. Subjects with moderate-to-severe asthma and eosinophilic but not neutrophilic airways inflammation, despite inhaled steroids, showed reduced TLR7 and IFNλ2/3 expression in endobronchial biopsies. Furthermore, TLR7 expression inversely correlated with percentage of sputum eosinophils.
This implicates IL-5-induced airways eosinophilia as a negative regulator of TLR7 expression and antiviral responses, which provides a molecular mechanism underpinning the effect of eosinophil-targeting treatments for the prevention of asthma exacerbations.
Asthma; Respiratory Infection; Pulmonary eosinophilia; Innate Immunity
Influenza is a disease with global impact that causes enormous morbidity and mortality on an annual basis. It primarily infects the respiratory tract and causes a broad range of illness ranging from symptomless infection to fulminant primary viral and secondary bacterial pneumonia. The severity of infection depends on both the virus strain and a number of host factors, primarily age and the presence of comorbid conditions such as cardiopulmonary disease. The mortality and utilization of healthcare resources associated with influenza is concentrated in the elderly and those with coexisting disease such as chronic obstructive pulmonary disease (COPD). Increasing use of vaccination and the development of new antiviral drugs hold out hope that the burden of disease associated with influenza can be reduced. However the constant emergence of new influenza strains and the current risk of avian influenza pandemic serve as warnings that influenza will remain a serious pathogen for the foreseeable future.
COPD; influenza; exacerbations
Rhinovirus infections are the dominant cause of asthma exacerbations, and deficient virus induction of IFN-α/β/λ in asthmatic patients is important in asthma exacerbation pathogenesis. Mechanisms causing this interferon deficiency in asthmatic patients are unknown.
We sought to investigate the expression of suppressor of cytokine signaling (SOCS) 1 in tissues from asthmatic patients and its possible role in impaired virus-induced interferon induction in these patients.
We assessed SOCS1 mRNA and protein levels in vitro, bronchial biopsy specimens, and mice. The role of SOCS1 was inferred by proof-of-concept studies using overexpression with reporter genes and SOCS1-deficient mice. A nuclear role of SOCS1 was shown by using bronchial biopsy staining, overexpression of mutant SOCS1 constructs, and confocal microscopy. SOCS1 levels were also correlated with asthma-related clinical outcomes.
We report induction of SOCS1 in bronchial epithelial cells (BECs) by asthma exacerbation–related cytokines and by rhinovirus infection in vitro. We found that SOCS1 was increased in vivo in bronchial epithelium and related to asthma severity. SOCS1 expression was also increased in primary BECs from asthmatic patients ex vivo and was related to interferon deficiency and increased viral replication. In primary human epithelium, mouse lung macrophages, and SOCS1-deficient mice, SOCS1 suppressed rhinovirus induction of interferons. Suppression of virus-induced interferon levels was dependent on SOCS1 nuclear translocation but independent of proteasomal degradation of transcription factors. Nuclear SOCS1 levels were also increased in BECs from asthmatic patients.
We describe a novel mechanism explaining interferon deficiency in asthmatic patients through a novel nuclear function of SOCS1 and identify SOCS1 as an important therapeutic target for asthma exacerbations.
Rhinovirus; asthma; asthma exacerbation; atopy; interferon; innate immunity; cytokine; TH2 inflammation; suppressor of cytokine signaling; AA, Atopic asthma; BAL, Bronchoalveolar lavage; BEC, Bronchial epithelial cell; CISH, Cytokine-inducible SH2-containing protein; GFP, Green fluorescent protein; ISG, Interferon-stimulated gene; ISRE, Interferon-stimulated response element; KC, Keratinocyte-derived chemokine; LIX, LPS-induced CXC chemokine; NANA, Nonatopic nonasthmatic; NF-κB, Nuclear factor κB; NLS, Nuclear localization sequence; polyI:C, Polyinosinic-polycytidylic acid; SOCS, Suppressor of cytokine signaling; SOCS1wt, Full-length wild-type human SOCS1; STAT, Signal transducer and activator of transcription; STRA, Severe therapy-resistant atopic asthma
Viral exacerbations of chronic obstructive pulmonary disease (COPD), commonly caused by rhinovirus (RV) infections, are poorly controlled by current therapies. This is due to a lack of understanding of the underlying immunopathological mechanisms. Human studies have identified a number of key immune responses that are associated with RV-induced exacerbations including neutrophilic inflammation, expression of inflammatory cytokines and deficiencies in innate anti-viral interferon. Animal models of COPD exacerbation are required to determine the contribution of these responses to disease pathogenesis. We aimed to develop a short-term mouse model that reproduced the hallmark features of RV-induced exacerbation of COPD. Evaluation of complex protocols involving multiple dose elastase and lipopolysaccharide (LPS) administration combined with RV1B infection showed suppression rather than enhancement of inflammatory parameters compared with control mice infected with RV1B alone. Therefore, these approaches did not accurately model the enhanced inflammation associated with RV infection in patients with COPD compared with healthy subjects. In contrast, a single elastase treatment followed by RV infection led to heightened airway neutrophilic and lymphocytic inflammation, increased expression of tumour necrosis factor (TNF)-α, C-X-C motif chemokine 10 (CXCL10)/IP-10 (interferon γ-induced protein 10) and CCL5 [chemokine (C-C motif) ligand 5]/RANTES (regulated on activation, normal T-cell expressed and secreted), mucus hypersecretion and preliminary evidence for increased airway hyper-responsiveness compared with mice treated with elastase or RV infection alone. In summary, we have developed a new mouse model of RV-induced COPD exacerbation that mimics many of the inflammatory features of human disease. This model, in conjunction with human models of disease, will provide an essential tool for studying disease mechanisms and allow testing of novel therapies with potential to be translated into clinical practice.
The present study describes a new short-term mouse model of rhinovirus (RV)-induced exacerbation of COPD (chronic obstructive pulmonary disease) which will facilitate insight into disease mechanisms and could provide a more efficient tool to test novel therapies with potential to be translated into clinical practice.
chronic obstructive pulmonary disease; exacerbation; inflammation; mouse models; rhinovirus; AHR, airway hyper-responsiveness; BAL, bronchoalveolar lavage; CCL5, chemokine (C-C motif) ligand 5; COPD, chronic obstructive pulmonary disease; CXCL10, C-X-C motif chemokine 10; FRC, functional residual capacity; H&E, haematoxylin and eosin; IFN, interferon; IL, interleukin; IP-10, interferon γ-induced protein 10; LPS, lipopolysaccharide; MIP-2, macrophage inflammatory protein 2; MPO, myeloperoxidase; PAS, periodic acid–Schiff; PFA, paraformaldehyde; RANTES, regulated on activation, normal T-cell expressed and secreted; RV, rhinovirus; TLC, total lung capacity; TNF, tumour necrosis factor
Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive lung
disease of unknown cause that leads to respiratory failure and death within 5 years
of diagnosis. Overt respiratory infection and immunosuppression carry a high
morbidity and mortality, and polymorphisms in genes related to epithelial integrity
and host defense predispose to IPF.
Objectives: To investigate the role of bacteria in the pathogenesis and
progression of IPF.
Methods: We prospectively enrolled patients diagnosed with IPF according
to international criteria together with healthy smokers, nonsmokers, and subjects
with moderate chronic obstructive pulmonary disease as control subjects. Subjects
underwent bronchoalveolar lavage (BAL), from which genomic DNA was isolated. The
V3–V5 region of the bacterial 16S rRNA gene was amplified, allowing
quantification of bacterial load and identification of communities by 16S rRNA
quantitative polymerase chain reaction and pyrosequencing.
Measurements and Main Results: Sixty-five patients with IPF had double
the burden of bacteria in BAL fluid compared with 44 control subjects. Baseline
bacterial burden predicted the rate of decline in lung volume and risk of death and
associated independently with the rs35705950 polymorphism of the MUC5B mucin gene, a
proven host susceptibility factor for IPF. Sequencing yielded 912,883 high-quality
reads from all subjects. We identified Haemophilus,
Streptococcus, Neisseria, and
Veillonella spp. to be more abundant in cases than control
subjects. Regression analyses indicated that these specific operational taxonomic
units as well as bacterial burden associated independently with IPF.
Conclusions: IPF is characterized by an increased bacterial burden in
BAL that predicts decline in lung function and death. Trials of antimicrobial therapy
are needed to determine if microbial burden is pathogenic in the disease.
idiopathic pulmonary fibrosis; Muc5b; bacteria; microbiome
The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion.
IMPORTANCE The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual nonstructural proteins. We demonstrate that individual nonstructural HRV16 proteins, when expressed in HeLa cells, can both fragment the Golgi apparatus and block secretion, whereas viral infection fragments the Golgi apparatus without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from the expression of single viral proteins.
Rhinoviruses are the most common cause of virally-induced asthma exacerbations which continue to account for the greatest burden in terms of morbidity, mortality and cost associated with this disease. IL-25 activates type-2-driven inflammation and is potentially important in virally-induced asthma exacerbations. Rhinovirus-infected cultured asthmatic bronchial epithelial cells exhibited a heightened intrinsic capacity for IL-25 expression which correlated with donor atopic status. In vivo human IL-25 expression was greater in asthmatics at baseline and during experimental rhinovirus infection. In mice rhinovirus infection induced IL-25 expression and augmented allergen-induced IL-25. Blockade of the IL-25 receptor reduced many RV-induced exacerbation-specific responses including type-2 cytokine expression, mucus production and recruitment of eosinophils, neutrophils, basophils, T and non-T type-2 cells. We have identified that asthmatic epithelial cells possess increased intrinsic capacity for expression of a pro-type-2 cytokine in response to a viral infection and identify IL-25 as a key mediator in RV-induced exacerbations of pulmonary inflammation.
Rhinoviruses are the most common virus to infect man causing a range of serious respiratory diseases including exacerbations of asthma and COPD. Type I IFN and IL-15 are thought to be required for antiviral immunity however their function during rhinovirus infection in vivo is undefined. In RV infected human volunteers, IL-15 protein expression in fluid from the nasal mucosa and in bronchial biopsies was increased. In mice, RV induced type I IFN-dependent expression of IL-15 and IL-15Rα which in turn were required for NK- and CD8+ T-cell responses. Treatment with IL-15-IL-15Rα complexes (IL-15c) boosted RV-induced expression of IL-15, IL-15Rα, IFN-γ, CXCL9 and CXCL10 followed by recruitment of activated, IFN-γ expressing NK, CD8+ and CD4+ T cells. Treating infected IFNAR1−/− mice with IL-15c similarly increased IL-15, IL-15Rα, IFN-γ and CXCL9 (but not CXCL10) expression also followed by NK-, CD8+- and CD4+-T cell recruitment and activation. We have demonstrated that type I IFN induced IFN-γ and cellular immunity to RV was mediated by IL-15 and IL-15Rα. Importantly we also show that IL-15 could be induced via a type I IFN-independent mechanism by IL-15 complex treatment which in turn was sufficient to drive IFN-γ expression and lymphocyte responses.
Ex vivo, bronchial epithelial cells from people with asthma are more
susceptible to rhinovirus infection caused by deficient induction of the antiviral
protein, IFN-β. Exogenous IFN-β restores antiviral activity.
Objectives: To compare the efficacy and safety of inhaled IFN-β
with placebo administered to people with asthma after onset of cold symptoms to
prevent or attenuate asthma symptoms caused by respiratory viruses.
Methods: A total of 147 people with asthma on inhaled corticosteroids
(British Thoracic Society Steps 2–5), with a history of virus-associated
exacerbations, were randomized to 14-day treatment with inhaled IFN-β (n =
72) or placebo (n = 75) within 24 hours of developing cold symptoms and were
assessed clinically, with relevant samples collected to assess virus infection and
Measurements and Main Results: A total of 91% of randomized patients
developed a defined cold. In this modified intention-to-treat population, asthma
symptoms did not get clinically significantly worse (mean change in six-item Asthma
Control Questionnaire <0.5) and IFN-β treatment had no significant effect
on this primary endpoint, although it enhanced morning peak expiratory flow recovery
(P = 0.033), reduced the need for additional treatment, and
boosted innate immunity as assessed by blood and sputum biomarkers. In an exploratory
analysis of the subset of more difficult-to-treat, Step 4-5 people with asthma (n
= 27 IFN-β; n = 31 placebo), Asthma Control Questionnaire-6 increased
significantly on placebo; this was prevented by IFN-β (P
Conclusions: Although the trial did not meet its primary endpoint, it
suggests that inhaled IFN-β is a potential treatment for virus-induced
deteriorations of asthma in difficult-to-treat people with asthma and supports the
need for further, adequately powered, trials in this population.
Clinical trial registered with www.clinicaltrials.gov (NCT
innate immunity; treatment; respiratory virus
Rhinoviruses (RVs) are a major cause of common colds and induce exacerbations of asthma and chronic inflammatory lung diseases.
We expressed and purified recombinant RV coat proteins VP1-4, non-structural proteins as well as N-terminal fragments of VP1 from four RV strains (RV14, 16, 89, C) covering the three known RV groups (RV-A, RV-B and RV-C) and measured specific IgG-subclass-, IgA- and IgM-responses by ELISA in subjects with different severities of asthma or without asthma before and after experimental infection with RV16.
Before infection subjects showed IgG1 > IgA > IgM > IgG3 cross-reactivity with N-terminal fragments from the representative VP1 proteins of the three RV groups. Antibody levels were higher in the asthmatic group as compared to the non-asthmatic subjects. Six weeks after infection with RV16, IgG1 antibodies showed a group-specific increase towards the N-terminal VP1 fragment, but not towards other capsid and non-structural proteins, which was highest in subjects with severe upper and lower respiratory symptoms.
Our results demonstrate that increases of antibodies towards the VP1 N-terminus are group-specific and associated with severity of respiratory symptoms and suggest that it may be possible to develop serological tests for identifying causative RV groups.
•Increases of rhinovirus-specific antibodies are surrogate markers for severity of rhinovirus-induced respiratory symptoms.•Serological tests based on recombinant rhinovirus coat protein fragments identify the culprit rhinovirus strain.•Identification of the most relevant RV strains by serology should allow the rational design of RV vaccines.
RV, Rhinovirus; COPD, Chronic obstructive pulmonary disease; ICAM-1, Intercellular adhesion molecule 1; LDL-R, Low density lipoprotein receptor; ICS, Inhaled corticosteroids; SABA, Short-acting β2 agonists; PEF, Peak expiratory flow; MALDI–TOF, Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry; ELISA, Enzyme-linked immunosorbent assay; HRP, Horseradish peroxidase; O.D, Optical density; HSA, Human serum albumin; MBP, Maltose binding protein; TCID50, Tissue culture 50% infective dose; Rhinovirus; Asthma; Recombinant rhinovirus coat protein; Antibody response; Serological test
Bacterial infection of the lower respiratory tract in chronic obstructive pulmonary disease (COPD) patients is common both in stable patients and during acute exacerbations. The most frequent bacteria detected in COPD patients is Haemophilus influenzae, and it appears this organism is uniquely adapted to exploit immune deficiencies associated with COPD and to establish persistent infection in the lower respiratory tract. The presence of bacteria in the lower respiratory tract in stable COPD is termed colonization; however, there is increasing evidence that this is not an innocuous phenomenon but is associated with airway inflammation, increased symptoms, and increased risk for exacerbations. In this review, we discuss host immunity that offers protection against H. influenzae and how disturbance of these mechanisms, combined with pathogen mechanisms of immune evasion, promote persistence of H. influenzae in the lower airways in COPD. In addition, we examine the role of H. influenzae in COPD exacerbations, as well as interactions between H. influenzae and respiratory virus infections, and review the role of treatments and their effect on COPD outcomes. This review focuses predominantly on data derived from human studies but will refer to animal studies where they contribute to understanding the disease in humans.
chronic obstructive pulmonary disease; Haemophilus influenzae; nontypeable Haemophilus influenzae; respiratory viruses; vaccination
Rationale: Rhinovirus infection is followed by significantly increased frequencies of positive, potentially pathogenic sputum cultures in chronic obstructive pulmonary disease (COPD). However, it remains unclear whether these represent de novo infections or an increased load of organisms from the complex microbial communities (microbiome) in the lower airways.
Objectives: To investigate the effect of rhinovirus infection on the airway bacterial microbiome.
Methods: Subjects with COPD (n = 14) and healthy control subjects with normal lung function (n = 17) were infected with rhinovirus. Induced sputum was collected at baseline before rhinovirus inoculation and again on Days 5, 15, and 42 after rhinovirus infection and DNA was extracted. The V3–V5 region of the bacterial 16S ribosomal RNA gene was amplified and pyrosequenced, resulting in 370,849 high-quality reads from 112 of the possible 124 time points.
Measurements and Main Results: At 15 days after rhinovirus infection, there was a sixfold increase in 16S copy number (P = 0.007) and a 16% rise in numbers of proteobacterial sequences, most notably in potentially pathogenic Haemophilus influenzae (P = 2.7 × 10-20), from a preexisting community. These changes occurred only in the sputum microbiome of subjects with COPD and were still evident 42 days after infection. This was in contrast to the temporal stability demonstrated in the microbiome of healthy smokers and nonsmokers.
Conclusions: After rhinovirus infection, there is a rise in bacterial burden and a significant outgrowth of Haemophilus influenzae from the existing microbiota of subjects with COPD. This is not observed in healthy individuals. Our findings suggest that rhinovirus infection in COPD alters the respiratory microbiome and may precipitate secondary bacterial infections.
rhinovirus; chronic obstructive pulmonary disease; bacteria; microbiome
Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.
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.
Asthma exacerbations are frequently triggered by rhinovirus infections. Both asthma and respiratory tract infection can activate haemostasis. Therefore we hypothesized that experimental rhinovirus-16 infection and asthmatic airway inflammation act in synergy on the haemostatic balance.
28 patients (14 patients with mild allergic asthma and 14 healthy non-allergic controls) were infected with low-dose rhinovirus type 16. Venous plasma and bronchoalveolar lavage fluid (BAL fluid) were obtained before and 6 days after infection to evaluate markers of coagulation activation, thrombin-antithrombin complexes, von Willebrand factor, plasmin-antiplasmin complexes, plasminogen activator inhibitor type-1, endogenous thrombin potential and tissue factor-exposing microparticles by fibrin generation test, in plasma and/or BAL fluid. Data were analysed by nonparametric tests (Wilcoxon, Mann Whitney and Spearman correlation).
13 patients with mild asthma (6 females, 19-29 y) and 11 healthy controls (10 females, 19-31 y) had a documented Rhinovirus-16 infection. Rhinovirus-16 challenge resulted in a shortening of the fibrin generation test in BAL fluid of asthma patients (t = -1: 706 s vs. t = 6: 498 s; p = 0.02), but not of controls (t = -1: 693 s vs. t = 6: 636 s; p = 0.65). The fold change in tissue factor-exposing microparticles in BAL fluid inversely correlated with the fold changes in eosinophil cationic protein and myeloperoxidase in BAL fluid after virus infection (r = -0.517 and -0.528 resp., both p = 0.01).
Rhinovirus-16 challenge led to increased plasminogen activator inhibitor type-1 levels in plasma in patients with asthma (26.0 ng/mL vs. 11.5 ng/mL in healthy controls, p = 0.04). Rhinovirus-16 load in BAL showed a linear correlation with the fold change in endogenous thrombin potential, plasmin-antiplasmin complexes and plasminogen activator inhibitor type-1.
Experimental rhinovirus infection induces procoagulant changes in the airways of patients with asthma through increased activity of tissue factor-exposing microparticles. These microparticle-associated procoagulant changes are associated with both neutrophilic and eosinophilic inflammation. Systemic activation of haemostasis increases with Rhinoviral load.
This trial was registered at the Dutch trial registry (http://www.trialregister.nl): NTR1677.
Rhinovirus; Coagulation; Fibrinolysis; Asthma; Microparticles; Inflammation
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.
Chronic obstructive pulmonary disease; Acute exacerbations of COPD; Respiratory viruses; T lymphocytes
Lysosomes play important roles in multiple aspects of physiology, but the problem of how the transcription of lysosomal genes is coordinated remains incompletely understood. The goal of this study was to illuminate the physiological contexts in which lysosomal genes are coordinately regulated and to identify transcription factors involved in this control.
As transcription factors and their target genes are often co-regulated, we performed meta-analyses of array-based expression data to identify regulators whose mRNA profiles are highly correlated with those of a core set of lysosomal genes. Among the ~50 transcription factors that rank highest by this measure, 65% are involved in differentiation or development, and 22% have been implicated in interferon signaling. The most strongly correlated candidate was Stat6, a factor commonly activated by interleukin-4 (IL-4) or IL-13. Publicly available chromatin immunoprecipitation (ChIP) data from alternatively activated mouse macrophages show that lysosomal genes are overrepresented among Stat6-bound targets. Quantification of RNA from wild-type and Stat6-deficient cells indicates that Stat6 promotes the expression of over 100 lysosomal genes, including hydrolases, subunits of the vacuolar H+ ATPase and trafficking factors. While IL-4 inhibits and activates different sets of lysosomal genes, Stat6 mediates only the activating effects of IL-4, by promoting increased expression and by neutralizing undefined inhibitory signals induced by IL-4.
The current data establish Stat6 as a broadly acting regulator of lysosomal gene expression in mouse macrophages. Other regulators whose expression correlates with lysosomal genes suggest that lysosome function is frequently re-programmed during differentiation, development and interferon signaling.