Potentially pathogenic airway bacteria and neutrophilic inflammation in treatment resistant severe asthma.

BACKGROUND: Molecular 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. OBJECTIVE: We 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. METHODS: Induced 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. RESULTS: In 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. CONCLUSIONS: Airway 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.

Transforming growth factor-beta promotes rhinovirus replication in bronchial epithelial cells by suppressing the innate immune response.

Rhinovirus (RV) infection is a major cause of asthma exacerbations which may be due to a deficient innate immune response in the bronchial epithelium. We hypothesized that the pleiotropic cytokine, TGF-ß, influences interferon (IFN) production by primary bronchial epithelial cells (PBECs) following RV infection. Exogenous TGF-ß(2) increased RV replication and decreased IFN protein secretion in response to RV or double-stranded RNA (dsRNA). Conversely, neutralizing TGF-ß antibodies decreased RV replication and increased IFN expression in response to RV or dsRNA. Endogenous TGF-ß(2) levels were higher in conditioned media of PBECs from asthmatic donors and the suppressive effect of anti-TGF-ß on RV replication was significantly greater in these cells. Basal SMAD-2 activation was reduced when asthmatic PBECs were treated with anti-TGF-ß and this was accompanied by suppression of SOCS-1 and SOCS-3 expression. Our results suggest that endogenous TGF-ß contributes to a suppressed IFN response to RV infection possibly via SOCS-1 and SOCS-3.

Exogenous IFN-ß has antiviral and anti-inflammatory properties in primary bronchial epithelial cells from asthmatic subjects exposed to rhinovirus.

BACKGROUND: Rhinoviruses are the major cause of asthma exacerbations. Previous studies suggest that primary bronchial epithelial cells (PBECs) from asthmatic subjects are more susceptible to rhinovirus infection because of deficient IFN-ß production. Although augmenting the innate immune response might provide a novel approach for treatment of virus-induced asthma exacerbations, the potential of IFN-ß to modulate antiviral and proinflammatory responses in asthmatic epithelium is poorly characterized. OBJECTIVES: We sought to compare responses of PBECs from nonasthmatic and asthmatic subjects to exogenous IFN-ß and test the inflammatory effects of IFN-ß in response to rhinovirus infection. METHODS: PBECs were treated with IFN-ß and infected with a low inoculum of human rhinovirus serotype 1B to simulate a natural viral infection. Expression of interferon-responsive genes and inflammatory responses were analyzed by using reverse transcription-quantitative real-time PCR, cytometric bead arrays, or both; viral titers were assessed by using the 50% tissue culture infection dose. RESULTS: Expression of IFN-ß-stimulated antiviral genes was comparable in PBECs from nonasthmatic or asthmatic donors. Exogenous IFN-ß significantly protected PBECs from asthmatic donors against rhinovirus infection by suppressing viral replication. Interferon-inducible protein 10 (IP-10), RANTES, and IL-6 release in response to rhinovirus infection was triggered only in PBECs from asthmatic donors. Although exogenous IFN-ß alone stimulated some release of IP-10 (but not IL-6 or RANTES), it significantly reduced rhinovirus-induced IP-10, RANTES, and IL-6 expression when tested in combination with rhinovirus. CONCLUSIONS: PBECs from asthmatic donors have a normal antiviral response to exogenous IFN-ß. The ability of IFN-ß to suppress viral replication suggests that it might limit virus-induced exacerbations by shortening the duration of the inflammatory response.

Bacterial activity in cystic fibrosis lung infections.

BACKGROUND: Chronic lung infections are the primary cause of morbidity and mortality in Cystic Fibrosis (CF) patients. Recent molecular biological based studies have identified a surprisingly wide range of hitherto unreported bacterial species in the lungs of CF patients. The aim of this study was to determine whether the species present were active and, as such, worthy of further investigation as potential pathogens. METHODS: Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiles were generated from PCR products amplified from 16S rDNA and Reverse Transcription Terminal Restriction Fragment Length Polymorphism (RT-T-RFLP) profiles, a marker of metabolic activity, were generated from PCR products amplified from 16S rRNA, both extracted from the same CF sputum sample. To test the level of activity of these bacteria, T-RFLP profiles were compared to RT-T-RFLP profiles. RESULTS: Samples from 17 individuals were studied. Parallel analyses identified a total of 706 individual T-RF and RT-T-RF bands in this sample set. 323 bands were detected by T-RFLP and 383 bands were detected by RT-T-RFLP (statistically significant; P < or = 0.001). For the group as a whole, 145 bands were detected in a T-RFLP profile alone, suggesting metabolically inactive bacteria. 205 bands were detected in an RT-T-RFLP profile alone and 178 bands were detected in both, suggesting a significant degree of metabolic activity. Although Pseudomonas aeruginosa was present and active in many patients, a low occurrence of other species traditionally considered to be key CF pathogens was detected. T-RFLP profiles obtained for induced sputum samples provided by healthy individuals without CF formed a separate cluster indicating a low level of similarity to those from CF patients. CONCLUSION: These results indicate that a high proportion of the bacterial species detected in the sputum from all of the CF patients in the study are active. The widespread activity of bacterial species in these samples emphasizes the potential importance of these previously unrecognized species within the CF lung.