BackgroundMolecular microbiological analysis of airway samples in asthma has demonstrated an altered microbiome in comparison to healthy controls. Such changes may have relevance to treatment-resistant severe asthma, particularly those with neutrophilic airway inflammation, as bacteria might be anticipated to activate the innate immune response, a process that is poorly steroid responsive. An understanding of the relationship between airway bacterial presence and dominance in severe asthma may help direct alternative treatment approaches.ObjectiveWe aimed to use a culture independent analysis strategy to describe the presence, dominance and abundance of bacterial taxa in induced sputum from treatment resistant severe asthmatics and correlate findings with clinical characteristics and airway inflammatory markers.MethodsInduced sputum was obtained from 28 stable treatment-resistant severe asthmatics. The samples were divided for supernatant IL-8 measurement, cytospin preparation for differential cell count and Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling for bacterial community analysis.ResultsIn 17/28 patients, the dominant species within the airway bacterial community was Moraxella catarrhalis or a member of the Haemophilus or Streptococcus genera. Colonisation with these species was associated with longer asthma disease duration (mean (SD) 31.8 years (16.7) vs 15.6 years (8.0), p = 0.008), worse post-bronchodilator percent predicted FEV1 (68.0% (24.0) vs 85.5% (19.7), p = 0.025) and higher sputum neutrophil differential cell counts (median (IQR) 80% (67–83) vs 43% (29–67), p = 0.001). Total abundance of these organisms significantly and positively correlated with sputum IL-8 concentration and neutrophil count.ConclusionsAirway colonisation with potentially pathogenic micro-organisms in asthma is associated with more severe airways obstruction and neutrophilic airway inflammation. This altered colonisation may have a role in the development of an asthma phenotype that responds less well to current asthma therapies.

Background Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that strongly activates dendritic cells and can initiate allergic inflammation. Since exposure to rhinovirus or double-stranded (ds) RNA (a surrogate of viral infection) induces TSLP expression in bronchial epithelial cells (BECs), this cytokine may link innate antiviral responses and the type 2 adaptive immune response. Objective As BECs from donors with asthma have a deficient interferon (IFN) response to rhinovirus infection, a study was undertaken to test the hypothesis that their antiviral response shows a bias towards TSLP production. Methods Primary BECs were grown from subjects with asthma and healthy volunteers. After exposure to dsRNA, interleukin (IL)-8, IFNb and TSLP mRNA and protein expression were measured by RT-qPCR and ELISA, respectively. Results dsRNA dose-dependently increased IL-8 expression in BECs with no significant difference between the groups. However, BECs from subjects with asthma expressed less IFNb and more TSLP mRNA and protein in response to dsRNA than BECs from those without asthma (median (IQR) 57 (38e82) pg/ml vs 106 (57e214) pg/ml for IFNb (p<0.05) and 114 (86e143) pg/ml vs 65 (32e119) pg/ml for TSLP (p<0.05) in response to 10 mg/ml dsRNA for 24 h).

Background Tissue factor (TF), a primary initiator of blood coagulation, also plays a pivotal role in angiogenesis. TF expression in the airways is associated with asthma, a disease characterized in part by subepithelial angiogenesis. Objectives To determine potential sources of TF and the mechanisms of its availability in the lung microenvironment. Methods Normal Human Bronchial Epithelial (NHBE) cells grown in air-liquid interface (ALI) culture were subjected to a compressive stress of 30 cmH2O; this is comparable to that generated in the airway epithelium during bronchoconstriction in asthma. Conditioned media and cells were harvested to measure TF mRNA and TF protein. We also tested bronchoalveolar lavage fluid (BALF) and airway biopsies from asthmatics and healthy controls for TF. Results TF mRNA was upregulated 2.2-fold after 3 hours of stress compared to unstressed cells. Intracellular and secreted TF proteins were enhanced 1.6-fold and over 50-fold, respectively, compared to that of control cells after onset of compression. The amount TF in BALF from patients with asthma was found at mean concentrations that were 5 times greater than that of healthy controls. Immunohistochemical staining of endobronchial biopsies identified epithelial localization of TF with increased expression in asthma. Exosomes isolated from the conditioned media of NHBECs and BALF of asthmatic subjects by ultracentrifugation contained TF. Conclusions Our in vitro and in vivo studies show that mechanically-stressed bronchial epithelial cells are a source of secreted TF and that exosomes are potentially a key carrier of the TF signal.