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Presence of eosinophils in the airways is a hallmark of allergic asthma but several other asthmatic phenotypes exist including severe disease with a predominance of neutrophils. In addition to wheezing and dyspnea, there is also an increased risk of pneumoccal infections, in particular during severe asthma. Eosinophils can be recruited to the airways by chemokines including eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26, RANTES/CCL5, and MEC/CCL28 that activate CCR3 on these cells. Several of these chemokines also have defensin-like antibacterial properties. Severe asthma is associated with high expression of the glycoprotein osteopontin in the airways. Osteopontin itself has neutrophil-recruiting properties and we hypothesized that this molecule can play roles in skewing the inflammatory profile during the course of asthma. OPN bound all eosinophil-recruiting chemokines with high affinity but less so to TSLP. In OVA-sensitized mice, administration of OPN had no direct effect on airway eosinophilia. The eosinophil-recruiting chemokines all displayed bactericidal activity against the common airway pathogen S. pneumoniae , but only CCL26 and CCL28 retained high antibacterial activity in the presence of sodium chloride (140 mM). Preincubation of the chemokines with osteopontin at eqimolar amounts strongly inhibited the antibacterial activity. The results suggest that osteopontin may impair host defense activities of the chemokines, increasing the vulnerability to acquire bacterial infections. In addition, osteopontin may lead to a different inflammatory subtype during the course of asthma through its ability to recruit neutrophils.

Rationale: The airway microbiota is altered in asthma, with less Bacteriodetes and more Proteobacteria than in healthy individuals. AHR to mannitol is related to eosinophilic airway inflammation and mast cell activation, but the relationship between AHR to mannitol and changes in airway microbiota is currently unknown. Aim: To examine the relative contribution of microbiota, eosinophilic inflammation and mast cell changes to AHR to mannitol in subjects with asthma. Methods: In 20 steroid-free, non-smoking subjects with asthma, AHR to mannitol was examined and bacterial DNA was extracted from broncheo-alveolar lavage (BAL) and subjected to Illumina MiSeq sequencing of the 16S rDNA V4 region. Eosinophils and mast cells (tryptase+/chymase- and trytase+/chymase+) were quantified by immunohistochemistry and computerized image analysis. Results: The relative abundance of Bacteroidetes was lower in asthmatics with AHR to mannitol compared to those without (median [IQR]: 18.4% [7.4] vs. 30.3% [16.8], p=0.006), whereas the relative abundance of Proteobacteria was higher (32.7% [11.4] vs. 24.8% [11.4], p=0.018). The degree of AHR to mannitol negatively correlated with the proportion of Bacteroidetes, rho(s): -0.61, p=0.002 and positively correlated with the proportion of Proteobacteria, rho(s): 0.54, p=0.008. The level of Bacteroidetes alone could explain 57% of the variation in AHR to mannitol independently of eosinophilic inflammation or mast cell phenotype. Conclusion: AHR to mannitol is associated with changes in the airway microbiota, independently of eosinophilic inflammation and mast cell phenotype, suggesting that the airway microbiota may constitute a driver of AHR in asthma.

Rationale: Although COPD is regarded as a non-eosinophilic and Th2-low disease, a significant proportion of the patients display elevated blood and sputum eosinophils. The nature of the eosinophilia in COPD and presence of basophils in lung tissues remains unexplored. Objectives: This study maps eosinophil and basophil tissue infiltration and associated Th2 immunity in major lung compartments across COPD severities. Methods: Eosinophils, basophils and GATA3+ cells were investigated in lung tissues and lymph nodes obtained from 30 COPD patients divided into GOLD I, II-III, and IV categories. Never-smoking and smoking non-COPD subjects (n=15) served as controls. Bronchi, bronchioles, pulmonary vessels, alveolar regions, and lymph nodes from > 140 tissue blocks underwent automated immunohistochemical staining and quantitative image analysis. Results: In COPD, the overall tissue density of eosinophils and basophils were increased (p 0.0001) and co-localization studies revealed a strong regional spatial relationship between eosinophils, basophils as well as GATA3+ cells. Conclusions: This study reveals that a surprisingly large proportion of COPD patients have focal lung infiltrations of eosinophils and basophils, which are likely caused by highly regionalized and still unexplored Th2 responses.

Rationale: Whether antigen presenting dendritic cell (DC) subsets are differently altered across asthma phenotypes remains unknown. This study explores multiple DC subsets in bronchial and alveolar tissues from patients with different phenotypes of asthma. Methods: Endobronchial biopsies (n=185) were obtained from healthy controls (n=8), patients with mild-moderate controlled (n=12) or uncontrolled (n=12) atopic asthma, and severe asthma (n=25). Transbronchial biopsies (n = 160) were obtained from healthy controls and mild-moderate asthmatics. Automatized immunohistochemistry was used for visualizing DC identification markers (CD1a, langerin, CD11c, BDCA2), confounding non-DCs (CD68, CD163), and DC phenotype markers (S-100, CD83 and RUNX3). Analysis was performed by tissue slide scanning techniques and computerized image analysis. Results: In healthy controls, langerin+ and CD11c+CD68-CD163- DCs were the most frequent DC populations within the bronchi. Epithelial langerin+ DCs decreased in severe asthmatics compared with controls (p Conclusions: This study shows that DC subsets are differentially altered in asthmatic patients and where severe asthma is associated with a particularly marked DC reduction which is likely caused by the high doses of steroids in this patient group.

Rationale: IL-17A plays a crucial role in lung inflammation. However, the major source for IL-17 in lung diseases remains unknown. The aim of this study is to identify major IL-17A expressing cell populations in healthy and diseased lungs. Methods: Surgical resections as well as nasal, bronchial and transbronchial biopsies were used to explore IL-17 in healthy controls(n=18), COPD(n=40), cystic fibrosis(n=5), allergic rhinitis(n=12), and asthma(n=40). Simultaneous visualization of IL17A and cell identification markers was performed by triple immunofluorescencetechniques. IL-17A+ cells were identified and levels of IL-17 were quantified in individual cells by combined virtual microscopy and computerized image analysis. Results: T-cells, mast cells and neutrophils were identified as IL-17A+ cells. Irrespective of disease, mast cells displayed significantly higher expression levels compared to neutrophils(p