C. M. Burke

Disordered Microbial Communities in Asthmatic Airways

Background A rich microbial environment in infancy protects against asthma [1], [2] and infections precipitate asthma exacerbations [3]. We compared the airway microbiota at three levels in adult patients with asthma, the related condition of COPD, and controls. We also studied bronchial lavage from asthmatic children and controls. Principal Findings We identified 5,054 16S rRNA bacterial sequences from 43 subjects, detecting >70% of species present. The bronchial tree was not sterile, and contained a mean of 2,000 bacterial genomes per cm2 surface sampled. Pathogenic Proteobacteria, particularly Haemophilus spp., were much more frequent in bronchi of adult asthmatics or patients with COPD than controls. We found similar highly significant increases in Proteobacteria in asthmatic children. Conversely, Bacteroidetes, particularly Prevotella spp., were more frequent in controls than adult or child asthmatics or COPD patients. Significance The results show the bronchial tree to contain a characteristic microbiota, and suggest that this microbiota is disturbed in asthmatic airways.

Rapid dendritic cell recruitment to the bronchial mucosa of patients with atopic asthma in response to local allergen challenge

BACKGROUND Airway dendritic cells (DC) play an important role in chronic allergic airway inflammation in experimental animals, but a similar role for DC in human allergic asthma has been difficult to define. This pilot study was undertaken to elucidate the role of DC in allergic asthma by examining their potential to migrate to the lower airways in response to bronchial challenge with specific allergen. METHODS Bronchial biopsy specimens were obtained from seven patients with allergic asthma before and 4–5 hours after allergen challenge. Multicolour immunofluorescence staining was performed on mucosal cryosections to identify changes in the number and phenotypes of DC. RESULTS A dramatic increase in the number of CD1c+HLA-DR+ DC were observed in the lamina propria after challenge compared with baseline (22.4v 7.8 cells/mm2). The rapid accumulation (within 4–5 hours) of these cells strongly suggests that they were directly recruited from peripheral blood. CONCLUSION We have shown for the first time that a specific DC subset rapidly emigrates into the human bronchial mucosa during allergic inflammation. While this study is based on relatively few patients, the consistency of the overall results strongly suggests that the rapid population dynamics of human airway DC closely parallel those in animal models of acute inflammation. These findings support suggestions that DC have an important role in human airway allergy.

Lung immunopathology in cases of sudden asthma death

The histopathology of airway inflammation in rare cases of sudden asphyxic asthma death (SAAD) is unclear. This study examines, for the first time, the relative disposition of lymphocyte and macrophage subsets and eosinophils in proximal and distal tissues of such cases. Multiple resection specimens from five cases of SAAD were studied. Tissue blocks were obtained at necroscopy and immediately frozen in liquid nitrogen within 18 hours of death (death occurring within 1 h of the onset of an unprovoked asphyxic asthma attack). After immunohistological staining, frozen sections underwent semi-quantitative analysis (cell counts per unit area) for T-cells, macrophages and eosinophils using computerized imaging systems. Subsets of T-cells and macrophages were estimated using double immunofluorescence techniques. Variability within samples, between samples and between cases was compared. These cases of fatal asthma showed infiltrates of T-cells, macrophages and eosinophils within peribronchial tissues. Distinct from stable asthma, a CD8+ T-cell dominance was found. A high proportion of eosinophils were activated (EG2+), whereas the relative proportion of antigen-presenting cells (RFD1+) did not seem to be abnormal, although numbers of these cells were high. These features were seen both in proximal and distal tissues. The variability of these parameters within an individual was 9.4-15.2%, however, the variability between individual cases was greater. Sudden asphyxic asthma is associated with inflammatory infiltrates both of proximal and distal lung tissues. In contrast to stable asthma, this infiltrate contains large numbers of CD8+ T-cells, suggesting distinct qualitative as well as quantitative characteristics in the immunopathology of sudden asthma death.

Pathways Activated during Human Asthma Exacerbation as Revealed by Gene Expression Patterns in Blood

Background Asthma exacerbations remain a major unmet clinical need. The difficulty in obtaining airway tissue and bronchoalveolar lavage samples during exacerbations has greatly hampered study of naturally occurring exacerbations. This study was conducted to determine if mRNA profiling of peripheral blood mononuclear cells (PBMCs) could provide information on the systemic molecular pathways involved during asthma exacerbations. Methodology/Principal Findings Over the course of one year, gene expression levels during stable asthma, exacerbation, and two weeks after an exacerbation were compared using oligonucleotide arrays. For each of 118 subjects who experienced at least one asthma exacerbation, the gene expression patterns in a sample of peripheral blood mononuclear cells collected during an exacerbation episode were compared to patterns observed in multiple samples from the same subject collected during quiescent asthma. Analysis of covariance identified genes whose levels of expression changed during exacerbations and returned to quiescent levels by two weeks. Heterogeneity among visits in expression profiles was examined using K-means clustering. Three distinct exacerbation-associated gene expression signatures were identified. One signature indicated that, even among patients without symptoms of respiratory infection, genes of innate immunity were activated. Antigen-independent T cell activation mediated by IL15 was also indicated by this signature. A second signature revealed strong evidence of lymphocyte activation through antigen receptors and subsequent downstream events of adaptive immunity. The number of genes identified in the third signature was too few to draw conclusions on the mechanisms driving those exacerbations. Conclusions/Significance This study has shown that analysis of PBMCs reveals systemic changes accompanying asthma exacerbation and has laid the foundation for future comparative studies using PBMCs.

Immunological/physiological relationships in asthma: potential regulation by lung macrophages

There is now a consensus that T-cell-mediated inflammation and eosinophil activation in the bronchial wall contribute to the pathogenesis of asthma. However, the relationship between these immunopathological mechanisms and the observed physiological aberrations remain unclear. Here, Len Poulter and colleagues identify the links between T-cell-mediated inflammation and bronchial hyperresponsiveness, and propose a hypothesis for asthma pathogenesis in which the combination of immunological and physiological abnormalities may result in the promotion of disease. Furthermore, they suggest that an integral factor in the prevention of this process is the regulation of bronchial T-cell reactivity by a population of immunosuppressive macrophages.

Allergen-induced CD30 expression on T cells of atopic asthmatics

Background The importance of TH2‐type T cell cytokines in atopic disease is widely accepted. CD30, a member of the TNF/NGF receptor superfamily, is expressed on a proportion of activated CD45RO+ T cells and has been proposed as a marker for TH2 phenotype. CD30 ligand‐CD30 interaction has been shown to positively influence development of the TH2 phenotype, and serum levels of soluble CD30 (sCD30) have been used as prognostic markers in HIV, SLE, Epstein‐Barr Virus infection and Hodgkins Lymphoma but not as yet in allergic disease.

Dietary nitrate supplementation in COPD: An acute, double-blind, randomized, placebo-controlled, crossover trial.

BACKGROUND The acute consumption of dietary nitrate has been shown to improve exercise capacity in athletes, healthy adults and subjects with peripheral vascular disease. Many COPD patients have reduced exercise capacity. We hypothesized that acute nitrate consumption might increase incremental shuttle walk test (ISWT) distance in COPD subjects. METHODS Eleven COPD subjects were randomly assigned to consume either a high nitrate or a matched, low nitrate beverage in a double-blind, randomized, placebo-controlled, crossover design. ISWT distance was measured both before and 3 h after the beverage and change was recorded. After a 7-day washout, ISWT distances were re-measured before and 3 h after the alternate beverage and changes were recorded. RESULTS We observed an increase in ISWT distance after consuming the high nitrate juice (25 m) compared with a reduction after the low nitrate juice (14 m) (p < 0.01). This improvement in exercise capacity was associated with significant increases in serum nitrate (p < 0.000005) and nitrite (p < 0.01) levels and a significant lowering of resting blood pressure (<0.05). CONCLUSIONS In patients with stable COPD, the acute consumption of dietary nitrate increased serum nitrate/nitrite levels and exercise capacity and was associated with a decrease in resting blood pressure. Nitrate consumption might alter exercise capacity in COPD patients.

Release of inflammatory mediators from eosinophils following a hyperosmolar stimulus

Airway dehydration and subsequent hyperosmolarity of periciliary fluid are considered critical events in exercise-induced bronchoconstriction (EIB). It has been shown that an in vitro hyperosmolar stimulation of basophils and mast cells with mannitol can induce the release of histamine and leukotrienes. The aim of this study was to establish if a hyperosmolar challenge could trigger activation of eosinophils to release chemokines and lipid mediators. Peripheral blood eosinophils were isolated from seven asthmatic and six non-asthmatic subjects. Hyperosmolar stimulation of eosinophils with mannitol (0.7 M), resulted in a significant increase in LTC4 levels compared to baseline in both asthmatic (15.2+/-4.6 vs. 70.1+/-9.5; P = 0.0002) and control subjects (14.3+/-4.0 vs. 55.6+/-5.6; P = 0.0001). ECP levels did not increase significantly above baseline following mannitol stimulation in either group. This study shows that eosinophils can be activated by a hyperosmolar stimulus. Therefore it seems reasonable to suggest that eosinophils could contribute to EIB.

T-cell cytokines may control the balance of functionally distinct macrophage populations.

As monocytes differentiate into mature macrophages, subsets emerge that exhibit stimulatory, suppressive or phagocytic potential. These functionally distinct subsets can be discriminated using monoclonal antibodies RFD1 and RFD7. As examples of all these subsets have been repeatedly identified within the macrophage pool in a variety of organs the overall functional capacity of this pool will depend on the relative balance of these subpopulations. This study investigates whether this balance present in mature macrophage populations can be regulated by the local influence of T‐cell‐derived cytokines. The dose‐dependent effect of cytokines interferon‐γ (IFN‐γ), interleukins (IL) IL‐2, IL‐4 and IL‐10 on the phenotype and function of monocyte‐derived macrophages was determined. Subsets of mature cells were quantified by identifying RFD1+ RFD7− stimulatory cells (D1+); RFD1− RFD7+ phagocytes (D7+) and RFD1+ RFD7+ suppressive cells (D1/D7+). IFN‐γ and IL‐4 increased the relative proportions of D1+ stimulatory cells and upregulated HLA‐DR expression. IFN‐γ also increased the capacity of the mature macrophage pool to stimulate T‐cell proliferation. IL‐10 reduced the proportions of D1+ stimulatory cells while promoting the differentiation of D7+ phagocytes and D1/D7+ suppressive cells. IL‐10 induced changes also reduced the functional capacity of the mature populations to stimulate T cells in auto and allogenic mixed lymphocyte reactions (MLR). IL‐2 had no effect on differentiation of monocytes. Thus IL‐4 and IFN‐γ are seen to induce the development of stimulatory macrophages while IL‐10 promotes differentiation of monocytes to mature phagocytes and suppressive macrophages. It is concluded that mature macrophage phenotype is ‘plastic’ and under the control of T‐cell‐derived mediators. Furthermore, within the differentiating monocytes, phenotypic change appears to carry with it functional change, thus retaining the relationship between antigen expression and activity in the mature macrophage populations.

Fluticasone propionate induced alterations to lung function and the immunopathology of asthma over time

BACKGROUND Inhaled corticosteroids are the most widely used treatment for asthma, a disease characterised by both functional and immunopathological abnormalities. This study investigated the relative effects of inhaled corticosteroids on these two features of asthma over time. METHODS A randomised, double blind, placebo controlled, parallel group study with inhaled fluticasone propionate, (FP 2 mg daily) was conducted in 27 patients with asthma. Following baseline analysis, the study tested the effects of short term (two weeks) and longer term (eight weeks) treatment. At each time point (0, 2, and 8 weeks) lung function tests were performed and endobronchial biopsy specimens obtained to determine the distribution and number of lymphocyte, macrophage and eosinophil subsets using immunohistological analysis. Twenty three patients completed the study, 11 on FP and 12 placebo. RESULTS FEV1, ΔFEV1, FEF25–75, and FEV1/FVC all improved after two weeks of FP treatment. This improvement was maintained but not increased after eight weeks. PC20FEV1 showed a trend to increase but was not significantly improved at eight weeks. No significant changes were seen in the placebo group. The numbers of T cells, macrophages, and eosinophils in the bronchial wall were reduced by two weeks of treatment with FP but were unaltered by placebo. The improvement offered by FP continued over the eight week period. Reductions in CD4:CD8 ratio and numbers of activated (EG2+) eosinophils were only significant after eight weeks of treatment. CONCLUSIONS These results reveal that FP influences both functional and immunopathological parameters of asthma. Temporal relationships suggest that these are parallel but not necessarily interrelated effects. While short term treatment is effective in “normalising” the functional abnormalities in asthma, the impact of FP on bronchial inflammation appears to be progressive, taking up to eight weeks and more.