Julia Aniscenko

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.

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.

Association of bacteria and viruses with wheezy episodes in young children: prospective birth cohort study

Objective To study the association between wheezy symptoms in young children and the presence of bacteria in the airways. Design Birth cohort study. Setting Clinical research unit in Copenhagen. Participants Children of asthmatic mothers, from age 4 weeks to 3 years, with planned visits and acute admissions to the research clinic. Main outcome measure Frequency of bacteria and virus carriage in airway aspirates during wheezy episodes and at planned visits without respiratory symptoms. Results 984 samples (361 children) were analysed for bacteria, 844 (299 children) for viruses, and 696 (277 children) for both viruses and bacteria. Wheezy episodes were associated with both bacterial infection (odds ratio 2.9, 95% confidence interval 1.9 to 4.3; P<0.001) and virus infection (2.8, 1.7 to 4.4; P<0.001). The associations of bacteria and viruses were independent of each other. Conclusion Acute wheezy episodes in young children were significantly associated with bacterial infections similar to but independent of the association with virus infections.

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.

Defining critical roles for NF-κB p65 and type I interferon in innate immunity to rhinovirus.

The importance of NF‐κB activation and deficient anti‐viral interferon induction in the pathogenesis of rhinovirus‐induced asthma exacerbations is poorly understood. We provide the first in vivo evidence in man and mouse that rhinovirus infection enhanced bronchial epithelial cell NF‐κB p65 nuclear expression, NF‐κB p65 DNA binding in lung tissue and NF‐κB‐regulated airway inflammation. In vitro inhibition of NF‐κB reduced rhinovirus‐induced pro‐inflammatory cytokines but did not affect type I/III interferon induction. Rhinovirus‐infected p65‐deficient mice exhibited reduced neutrophilic inflammation, yet interferon induction, antiviral responses and virus loads were unaffected, indicating that NF‐κB p65 is required for pro‐inflammatory responses, but redundant in interferon induction by rhinoviruses in vivo. Conversely, IFNAR1−/− mice exhibited enhanced neutrophilic inflammation with impaired antiviral immunity and increased rhinovirus replication, demonstrating that interferon signalling was critical to antiviral immunity. We thus provide new mechanistic insights into rhinovirus infection and demonstrate the therapeutic potential of targeting NF‐κB p65 (to suppress inflammation but preserve anti‐viral immunity) and type I IFN signalling (to enhance deficient anti‐viral immunity) to treat rhinovirus‐induced exacerbations of airway diseases.

Rhinovirus infection induces expression of airway remodelling factors in vitro and in vivo

Background and objective:  A hallmark of asthma is airway remodelling, which includes increased deposition of extracellular matrix (ECM) protein. Viral infections may promote the development of asthma and are the most common causes of asthma exacerbations. We evaluated whether rhinovirus (RV) infection induces airway remodelling, as assessed by ECM deposition.

Host DNA released by NETosis promotes rhinovirus-induced type-2 allergic asthma exacerbation

Respiratory viral infections represent the most common cause of allergic asthma exacerbations. Amplification of the type-2 immune response is strongly implicated in asthma exacerbation, but how virus infection boosts type-2 responses is poorly understood. We report a significant correlation between the release of host double-stranded DNA (dsDNA) following rhinovirus infection and the exacerbation of type-2 allergic inflammation in humans. In a mouse model of allergic airway hypersensitivity, we show that rhinovirus infection triggers dsDNA release associated with the formation of neutrophil extracellular traps (NETs), known as NETosis. We further demonstrate that inhibiting NETosis by blocking neutrophil elastase or by degrading NETs with DNase protects mice from type-2 immunopathology. Furthermore, the injection of mouse genomic DNA alone is sufficient to recapitulate many features of rhinovirus-induced type-2 immune responses and asthma pathology. Thus, NETosis and its associated extracellular dsDNA contribute to the pathogenesis and may represent potential therapeutic targets of rhinovirus-induced asthma exacerbations.

A short-term mouse model that reproduces the immunopathological features of rhinovirus-induced exacerbation of COPD

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.

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

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

IL-15 complexes induce NK- and T-cell responses independent of type I IFN signaling during rhinovirus infection

Rhinoviruses are among the most common viruses 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 expressions 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.