Patrick Mallia

Role of deficient type III interferon-λ 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-λs 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.

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-γ 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.

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.

Asthma exacerbations: Origin, effect, and prevention

Asthma is the most common chronic respiratory disease, affecting up to 10% of adults and 30% of children in the Western world. Despite advances in asthma management, acute exacerbations continue to occur and impose considerable morbidity on patients and constitute a major burden on health care resources. Respiratory tract viruses have emerged as the most frequent triggers for exacerbations in both children and adults; however, the mechanisms underlying these remain poorly understood. More recently, it has become increasingly clear that interactions might exist between viruses and other triggers, increasing the likelihood of an exacerbation. In this article we begin with an overview of the health, economic, and social burden that exacerbations of asthma carry with them. This is followed by a review of the pathogenesis of asthma exacerbations, highlighting the various triggers responsible and multiple interactions that exist between them. The final section first addresses what preventative measures are currently available for asthma exacerbations and subsequently examines which of the new treatments in development might lessen the burden of exacerbations in the future.

IL-33–Dependent Type 2 Inflammation during Rhinovirus-induced Asthma Exacerbations In Vivo

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 pathway. 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.

Outgrowth of the Bacterial Airway Microbiome after Rhinovirus Exacerbation of Chronic Obstructive Pulmonary Disease

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.

The Role of Bacteria in the Pathogenesis and Progression of Idiopathic Pulmonary Fibrosis

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.

How viral infections cause exacerbation of airway diseases

Exacerbations of asthma and COPD are major causes of morbidity, mortality, and health-care costs. Over the last decade, studies using new molecular diagnostic techniques have established that respiratory viruses are a major cause of exacerbations of both asthma and COPD. The most prevalent viruses detected during exacerbations are the rhinoviruses. Despite the burden of disease associated with exacerbations, little is known about the mechanisms of virus-induced exacerbations of airway diseases. Exacerbations are associated with increased airway inflammation in patients with both asthma and COPD, but many questions remain unanswered regarding the key inflammatory cells and mediators involved. Identifying the key inflammatory mediators involved in exacerbations holds the promise of developing diagnostic and prognostic markers of exacerbation. In addition, such studies can identify new therapeutic targets for the development of novel drugs for the prevention and treatment of exacerbations.

Rhinovirus Infection Induces Degradation of Antimicrobial Peptides and Secondary Bacterial Infection in Chronic Obstructive Pulmonary Disease

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 16–induced IFN-α and IFN-β are deficient in bronchoalveolar lavage cells in asthmatic patients

BACKGROUND Asthmatic patients have defective rhinovirus-induced IFN-β and IFN-λ production from bronchial epithelial cells and IFN-λ from bronchoalveolar lavage (BAL) cells. Whether bronchoalveolar lavage cells have defective type I interferon responses to rhinovirus is unknown, as are mechanisms explaining defective rhinovirus interferon induction in asthmatic patients. OBJECTIVE We sought to investigate rhinovirus induction of type I interferons in BAL and blood mononuclear cells from asthmatic patients and healthy subjects and to investigate mechanisms of any deficiency observed. METHODS BAL and blood mononuclear cells from atopic asthmatic patients and healthy subjects were infected with rhinovirus ex vivo. Interferon proteins were analyzed by using ELISA. mRNA expression of key components of interferon induction pathways were analyzed by using quantitative PCR. RESULTS Rhinovirus induction of type I interferon protein was delayed and deficient in BAL cells from asthmatic patients, and lower interferon levels were associated with greater airway hyperresponsiveness and skin prick test response positivity. Expression of Toll-like receptor (TLR) 3, TLR7, TLR8, retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), TIR domain-containing adapter-inducing IFN-β (TRIF), myeloid differentiation primary response gene 88 (MyD88), caspase recruitment domain adaptor inducing IFN-β (CARDIF), IL-1 receptor-associated kinase 4 (IRAK4), IκB kinase β (IKKB), IκB kinase ι (IKKI), interferon regulatory factors 3 and 7, and rhinovirus induction of expression of the virus-inducible molecules TLR3, TLR7, RIG-I, and MDA-5 were not impaired in these interferon-deficient BAL cells in asthmatic patients. Defective rhinovirus interferon induction was not observed in blood mononuclear cells. CONCLUSIONS Rhinovirus induction of type I interferons in BAL cells is delayed and deficient and might be a marker of more severe asthma. Defective rhinovirus interferon induction in asthmatic patients was not accompanied by differences in the expression or induction of key molecules implicated in viral induction of interferons.