Nicolas Regamey

The Airway Epithelium: Soldier in the Fight against Respiratory Viruses

SUMMARY The airway epithelium acts as a frontline defense against respiratory viruses, not only as a physical barrier and through the mucociliary apparatus but also through its immunological functions. It initiates multiple innate and adaptive immune mechanisms which are crucial for efficient antiviral responses. The interaction between respiratory viruses and airway epithelial cells results in production of antiviral substances, including type I and III interferons, lactoferrin, β-defensins, and nitric oxide, and also in production of cytokines and chemokines, which recruit inflammatory cells and influence adaptive immunity. These defense mechanisms usually result in rapid virus clearance. However, respiratory viruses elaborate strategies to evade antiviral mechanisms and immune responses. They may disrupt epithelial integrity through cytotoxic effects, increasing paracellular permeability and damaging epithelial repair mechanisms. In addition, they can interfere with immune responses by blocking interferon pathways and by subverting protective inflammatory responses toward detrimental ones. Finally, by inducing overt mucus secretion and mucostasis and by paving the way for bacterial infections, they favor lung damage and further impair host antiviral mechanisms.

Pediatric severe asthma is characterized by eosinophilia and remodeling without TH2 cytokines

BACKGROUND The pathology of pediatric severe therapy-resistant asthma (STRA) is little understood. OBJECTIVES We hypothesized that STRA in children is characterized by airway eosinophilia and mast cell inflammation and is driven by the T(H)2 cytokines IL-4, IL-5, and IL-13. METHODS Sixty-nine children (mean age, 11.8 years; interquartile range, 5.6-17.3 years; patients with STRA, n = 53; control subjects, n = 16) underwent fiberoptic bronchoscopy, bronchoalveolar lavage (BAL), and endobronchial biopsy. Airway inflammation, remodeling, and BAL fluid and biopsy specimen T(H)2 cytokines were quantified. Children with STRA also underwent symptom assessment (Asthma Control Test), spirometry, exhaled nitric oxide and induced sputum evaluation. RESULTS Children with STRA had significantly increased BAL fluid and biopsy specimen eosinophil counts compared with those found in control subjects (BAL fluid, P < .001; biopsy specimen, P < .01); within the STRA group, there was marked between-patient variability in eosinophilia. Submucosal mast cell, neutrophil, and lymphocyte counts were similar in both groups. Reticular basement membrane thickness and airway smooth muscle were increased in patients with STRA compared with those found in control subjects (P < .0001 and P < .001, respectively). There was no increase in BAL fluid IL-4, IL-5, or IL-13 levels in patients with STRA compared with control subjects, and these cytokines were rarely detected in induced sputum. Biopsy IL-5(+) and IL-13(+) cell counts were also not higher in patients with STRA compared with those seen in control subjects. The subgroup (n = 15) of children with STRA with detectable BAL fluid T(H)2 cytokines had significantly lower lung function than those with undetectable BAL fluid T(H)2 cytokines. CONCLUSIONS STRA in children was characterized by remodeling and variable airway eosinophil counts. However, unlike in adults, there was no neutrophilia, and despite the wide range in eosinophil counts, the T(H)2 mediators that are thought to drive allergic asthma were mostly absent.

The Th17 pathway in cystic fibrosis lung disease.

RATIONALE Cystic fibrosis (CF) is characterized by bronchoalveolar neutrophilia and submucosal lymphocytosis. We hypothesized that Th17 lymphocytes are part of this submucosal infiltrate. OBJECTIVES Quantification and phenotyping of the lymphocytic infiltrate in the bronchial submucosa of patients with CF (n = 53, of which 20 were newly diagnosed), non-CF bronchiectasis (n = 17), and healthy control subjects (n = 13). METHODS We measured IL-17 levels in bronchoalveolar lavage and CD4(+), CD8(+), and IL-17(+) cell counts in endobronchial biopsies. Correlations were made with infection status and other inflammatory markers. Potential cellular sources of IL-17 were determined by double staining. MEASUREMENTS AND MAIN RESULTS IL-17(+) cell counts (median [interquartile range] cells/mm(2)) were significantly higher in patients with established CF (205 [115-551]) and non-CF bronchiectasis (245 [183-436]) than in control subjects (53 [12-82]) (P < 0.01 for both). Patients with newly diagnosed CF had intermediate counts (171 [91-252]). IL-17-positive CD4(+) T cells, γδT cells, natural killer T cells, and neutrophils were identified. Bronchoalveolar lavage IL-17 levels (pg/ml) were highest in established CF (14.6 [2.2-38.4]), low in newly diagnosed CF and control subjects (1.7 [1.7-1.74]; 1.7 [1.7-3]), and intermediate in non-CF bronchiectasis (9.1 [1.7-34] pg/ml) (Kruskal-Wallis P = 0.001). There was a significant correlation between IL-17 and neutrophil counts (P < 0.001, R = 0.6) as well as IL-4 (P < 0.001, R = 0.84). CONCLUSIONS Th17 lymphocytes are present in the airway submucosa in CF, even in a young, newly diagnosed group. Other IL-17(+) cells include neutrophils, γδ T cells, and natural killer T cells.

New Respiratory Enterovirus and Recombinant Rhinoviruses among Circulating Picornaviruses

Increased genomic diversity of these viruses is demonstrated.

Increased Airway Smooth Muscle Mass in Children with Asthma, Cystic Fibrosis, and Non-Cystic Fibrosis Bronchiectasis

RATIONALE Structural alterations to airway smooth muscle (ASM) are a feature of asthma and cystic fibrosis (CF) in adults. OBJECTIVES We investigated whether increase in ASM mass is already present in children with chronic inflammatory lung disease. METHODS Fiberoptic bronchoscopy was performed in 78 children (median age [IQR], 11.3 [8.5-13.8] yr): 24 with asthma, 27 with CF, 16 with non-CF bronchiectasis (BX), and 11 control children without lower respiratory tract disease. Endobronchial biopsy ASM content and myocyte number and size were quantified using stereology. MEASUREMENTS AND MAIN RESULTS The median (IQR) volume fraction of subepithelial tissue occupied by ASM was increased in the children with asthma (0.27 [0.12-0.49]; P < 0.0001), CF (0.12 [0.06-0.21]; P < 0.01), and BX (0.16 [0.04-0.21]; P < 0.01) compared with control subjects (0.04 [0.02-0.05]). ASM content was related to bronchodilator responsiveness in the asthmatic group (r = 0.66, P < 0.01). Median (IQR) myocyte number (cells per mm(2) of reticular basement membrane) was 8,204 (5,270-11,749; P < 0.05) in children with asthma, 4,504 (2,838-8,962; not significant) in children with CF, 4,971 (3,476-10,057; not significant) in children with BX, and 1,944 (1,596-6,318) in control subjects. Mean (SD) myocyte size (mum(3)) was 3,344 (801; P < 0.01) in children with asthma, 3,264 (809; P < 0.01) in children with CF, 3,177 (873; P < 0.05) in children with BX, and 1,927 (386) in control subjects. In all disease groups, the volume fraction of ASM in subepithelial tissue was related to myocyte number (asthma: r = 0.84, P < 0.001; CF: r = 0.81, P < 0.01; BX: r = 0.95, P < 0.001), but not to myocyte size. CONCLUSIONS Increases in ASM (both number and size) occur in children with chronic inflammatory lung diseases that include CF, asthma, and BX.

Airway remodelling in children with cystic fibrosis.

Background: The relationship between airway structural changes and inflammation is unclear in early cystic fibrosis (CF) lung disease. A study was undertaken to determine changes in airway remodelling in children with CF compared with appropriate disease and healthy controls. Methods: Bronchoalveolar lavage and endobronchial biopsy were performed in a cross-sectional study of 43 children with CF (aged 0.3–16.8 years), 7 children with primary ciliary dyskinesia (PCD), 26 with chronic respiratory symptoms (CRS) investigated for recurrent infection and/or cough and 7 control children with no lower airway symptoms. Inflammatory cells, cytokines, proteases and matrix constituents were measured in bronchoalveolar lavage fluid (BALF). Reticular basement membrane (RBM) thickness was measured on biopsy specimens using light microscopy. Results: Increased concentrations of elastin, glycosaminoglycans and collagen were found in BALF from children with CF compared with the CRS group and controls, each correlating positively with age, neutrophil count and proteases (elastase activity and matrix metalloproteinase-9 (MMP-9) concentration). There were significant negative correlations between certain of these and pulmonary function (forced expiratory volume in 1 s) in the CF group (elastin: r = −0.45, p<0.05; MMP-9:TIMP-1 ratio: r = −0.47, p<0.05). Median RBM thickness was greater in the CF group than in the controls (5.9 μm vs 4.0 μm, p<0.01) and correlated positively with levels of transforming growth factor-β1 (TGF-β1; r = 0.53, p = 0.01), although not with other inflammatory markers or pulmonary function. Conclusions: This study provides evidence for two forms of airway remodelling in children with CF: (1) matrix breakdown, related to inflammation, proteolysis and impaired pulmonary function, and (2) RBM thickening, related to TGF-β1 concentration but independent of other markers of inflammation.

Viral etiology of acute respiratory infections with cough in infancy: a community-based birth cohort study

Background: Acute respiratory infections (ARI) are a major cause of morbidity in infancy worldwide, with cough and wheeze being alarming symptoms to parents. We aimed to analyze in detail the viral aetiology of ARI with such symptoms in otherwise healthy infants, including rhinoviruses and recently discovered viruses such as human metapneumovirus (HMPV), coronavirus NL63 and HKU1, and human bocavirus (HBoV). Methods: We prospectively followed 197 unselected infants during their first year of life and assessed clinical symptoms by weekly standardized interviews. At the first ARI with cough or wheeze, we analyzed nasal swabs by sensitive individual real time polymerase chain reaction assays targeting 16 different respiratory viruses. Results: All 112 infants who had an ARI had cough, and 39 (35%) had wheeze. One or more respiratory viruses were found in 88 of 112 (79%) cases. Fifteen (17%) dual and 3 (3%) triple infections were recorded. Rhino- (23% of all viruses) and coronaviruses (18%) were most common, followed by parainfluenza viruses (17%), respiratory syncytial virus (RSV) (16%), HMPV (13%), and HBoV (5%). Together rhinoviruses, coronaviruses, HMPV, and HBoV accounted for 60% (65 of 109) of viruses. Although symptom scores and need for general practitioner (GP) consultations were highest in infants infected with RSV, they were similar in infants infected with other viruses. Viral shedding at 3 weeks occurred in 20% of cases. Conclusions: Rhinoviruses, coronaviruses, HMPV, and HBoV are common pathogens associated with respiratory symptoms in otherwise healthy infants. They should be considered in the differential diagnosis of the aetiology of ARI in this age group.

Sputum inflammatory phenotypes are not stable in children with asthma

Background Two distinct, stable inflammatory phenotypes have been described in adults with asthma: eosinophilic and non-eosinophilic. Treatment strategies based on these phenotypes have been successful. This study evaluated sputum cytology in children with asthma to classify sputum inflammatory phenotypes and to assess their stability over time. Methods Sputum induction was performed in 51 children with severe asthma and 28 with mild to moderate asthma. Samples were classified as eosinophilic (>2.5% eosinophils), neutrophilic (>54% neutrophils); mixed granulocytic (>2.5% eosinophils, >54% neutrophils); or paucigranulocytic (≤2.5% eosinophils, ≤54% neutrophils). Sputum induction was repeated every 3 months in children with severe asthma (n=42) over a 1-year period and twice in mild to moderate asthma (n=17) over 3–6 months. Results 62 children (78%) had raised levels of inflammatory cells in at least one sputum sample. In the longitudinal analysis 37 of 59 children (63%) demonstrated two or more phenotypes. Variability in sputum inflammatory phenotype was observed in both the severe and the mild to moderate asthma groups. Change in phenotype was not related to change in inhaled corticosteroid (ICS) dose or asthma control, nor was it reflected in a change in exhaled nitric oxide (FENO). 24 children (41%) fulfilled the criteria for non-eosinophilic asthma on one occasion and eosinophilic on another. There were no differences in severity, asthma control, atopy, ICS dose or forced expiratory volume in 1 s between those who were always non-eosinophilic and those always eosinophilic. Conclusion Raised levels of inflammatory cells were frequently found in children with asthma of all severities. Sputum inflammatory phenotype was not stable in children with asthma.

Use of sputum eosinophil counts to guide management in children with severe asthma

Background Previous studies in adults with asthma incorporating the control of sputum eosinophils into management strategies have shown significant reductions in exacerbations. A study was undertaken to investigate whether this strategy would be successful in children with severe asthma. Methods 55 children (7–17 years) with severe asthma were randomised to either a conventional symptom-based management strategy or to an inflammation-based strategy (principally sputum eosinophils). Children were seen 3-monthly over a 1-year period. Results The annual rate of total and major exacerbations (courses of oral corticosteroids) was non-significantly lower in the inflammatory management group compared with the symptom management group (3.6 vs 4.8, incident rate ratio (IRR) 0.75, 95% CI 0.54 to 1.04, p=0.082; and 1.9 vs 2.7 IRR 0.73, 95% CI 0.42 to 1.28, p=0.274 for total and major exacerbations, respectively). Significantly fewer subjects in the inflammatory management group experienced an exacerbation within 28 days of a study visit. There were small non-significant differences in measures of asthma control (symptom-free days and short-acting β agonist use) favouring the inflammatory management group. There was no significant difference in the inhaled corticosteroid dose prescribed over the course of the study. Conclusion Incorporating the control of sputum eosinophils into the management algorithm did not significantly reduce overall exacerbations or improve asthma control. Exacerbations were reduced in the short term, suggesting that more frequent measurements would be needed for a clinically useful effect and that controlling inflammation may have a role to play in subgroups of children with severe asthma.

Isolation of human bocavirus from Swiss infants with respiratory infections.

Human bocavirus (HBoV) is a novel agent associated with respiratory symptoms in adults and children. We studied prospectively the first acute respiratory infection in a birth cohort of healthy neonates in Switzerland. HBoV was identified in 5 (4.5%) of 112 infants as young as 3 months of age. In 4 of the 5 infants, HBoV was associated with other respiratory viruses. We conclude that HBoV circulates in the community and is acquired early in life.