Professor Jodie Simpson

Differential cell counts is a challenging task when applying computer vision algorithms to pathology. Existing approaches to train cell recognition require high availability of multi-class segmentation and/or bounding box annotations and suffer in performance when objects are tightly clustered. We present differential count network (¿DCNet¿), an annotation efficient modality that utilises keypoint detection to locate in brightfield images the centre points of cells (not nuclei) and their cell class. The single centre point annotation for DCNet lowered burden for experts to generate ground truth data by 77.1% compared to bounding box labeling. Yet centre point annotation still enabled high accuracy when training DCNet on a multi-class algorithm on whole cell features, matching human experts in all 5 object classes in average precision and outperforming humans in consistency. The efficacy and efficiency of the DCNet end-to-end system represents a significant progress toward an open source, fully computationally approach to differential cell count based diagnosis that can be adapted to any pathology need.

Add-on azithromycin (AZM) results in a significant reduction in exacerbations among adults with persistent uncontrolled asthma. The aim of this study was to assess the cost-effectiveness of add-on AZM in terms of healthcare and societal costs. The AMAZES trial randomly assigned 420 participants to AZM or placebo. Healthcare use and asthma exacerbations were measured during the treatment period. Healthcare use included all prescribed medicine and healthcare contacts. Costs of antimicrobial resistance (AMR) were estimated based on overall consumption and published estimates of costs. The value of an avoided exacerbation was based on published references. Differences in cost between the two groups were related to differences in exacerbations in a series of net monetary benefit estimates. Societal costs included lost productivity, over the counter medicines, steroid induced morbidity and AMR costs. Add-on AZM resulted in a reduction in healthcare costs (mean (95% CI)) including nights in hospital (AUD 433.70 (AUD 48.59 818.81) or EUR 260.22 (EUR 29.15 491.29)), unplanned healthcare visits (AUD 20.25 (AUD 5.23 35.27) or EUR 12.15 (EUR 3.14 21.16)), antibiotic costs (AUD 14.88 (AUD 7.55 22.21) or EUR 8.93 (EUR 4.53 13.33)) and oral corticosteroid costs (AUD 4.73 (AUD 0.82 8.64) or EUR 2.84 (EUR 0.49 5.18)); all p<0.05. Overall healthcare and societal costs were lower (AUD 77.30 (EUR 46.38) and AUD 256.22 (EUR 153.73) respectively) albeit not statistically significant. The net monetary benefit of add-on AZM was estimated to be AUD 2072.30 (95% CI AUD 1348.55 2805.23) or EUR 1243.38 (EUR 809.13 1683.14) assuming a willingness to pay per exacerbation avoided of AUD 2651 (EUR 1590.60). Irrespective of the sensitivity analysis applied, the net monetary benefit for total, moderate and severe exacerbations remained positive and significant. Add-on AZM therapy in poorly controlled asthma was a cost-effective therapy. Costs associated with AMR did not influence estimated cost-effectiveness.

Purpose: Systemic inflammatory biomarkers can improve diagnosis and assessment of chronic obstructive pulmonary disease (COPD) and asthma. We aimed to validate an airway disease biomarker panel of 4 systemic inflammatory biomarkers, a2-macroglobulin, ceruloplasmin, haptoglobin and hemopexin, to establish their relationship to airway disease diagnosis and inflammatory phenotypes and to identify an optimized biomarker panel for disease differentiation. Methods: Participants with COPD or asthma were classified by inflammatory phenotypes. Immunoassay methods were used to measure levels of validation biomarkers in the sera of participants with disease and non-respiratory disease controls. Markers were analyzed individually and in combination for disease differentiation and compared to established biomarkers (C-reactive protein, interleukin-6, and white blood cell/blood eosinophil count). Results: The study population comprised of 141 COPD, 127 severe asthma, 54 mild-moderate asthma and 71 control participants. Significant differences in ceruloplasmin, haptoglobin and hemopexin levels between disease groups and between systemic inflammatory phenotypes were observed. However, no differences were found between airway inflammatory phenotypes. Hemopexin was the best performing individual biomarker and could diagnose COPD versus control participants (area under the curve [AUC], 98.3%; 95% confidence interval [CI], 96.7%-99.9%) and differentiate COPD from asthmatic participants (AUC, 97.0%; 95% CI, 95.4%-98.6%), outperforming established biomarkers. A biomarker panel, including hemopexin, haptoglobin and other established biomarkers, could diagnose asthma versus control participants (AUC, 87.5%; 95% CI, 82.8%-92.2%). Conclusions: Hemopexin can be a novel biomarker with superior diagnostic ability in differentiating COPD and asthma. We propose an anti-inflammatory axis between the airways and systemic circulation, in which hemopexin is a protective component in airway disease.

Background: Asthma, a heterogeneous disease, can be divided into 4 inflammatory phenotypes using induced sputum cell counts¿eosinophilic asthma (EA), neutrophilic asthma (NA), mixed granulocytic asthma, and paucigranulocytic asthma (PGA). Although research has focused on EA and NA, there is little known about PGA. Objective: To study the heterogeneity of PGA and identify possible PGA clusters to guide clinical treatment. Methods: Patients with PGA were grouped by hierarchical cluster analysis and enrolled into a prospective cohort study to validate the clusters, relative to future risk of asthma exacerbations in a real-world setting. Clusters were validated by tree analysis in a separate population. Finally, we explored PGA stability. Results: Cluster analysis of 145 patients with PGA identified 3 clusters: cluster 1 (n = 110, 75.9%) was ¿mild PGA,¿ cluster 2 (n = 20, 13.8%) was ¿PGA with psychological dysfunction and rhinoconjunctivitis and other allergic diseases,¿ and cluster 3 (n = 15, 10.3%) was ¿smoking-associated PGA.¿ Cluster 3 had significantly increased risk of severe exacerbation (relative risk [RR] = 6.43, P =.01), emergency visit (RR = 8.61, P =.03), and hospitalization (RR = 12.94, P <.01). Results of the cluster analysis were successfully validated in an independent PGA population classified using decision tree analysis. Although PGA can transform into or develop from other phenotypes, 70% were stable over time. Conclusions: Among 3 identified PGA clusters, cluster 3 had a higher risk of severe exacerbation. PGA heterogeneity indicates the requirement of novel targeted interventions.

Background and objective: Long-term data on children with PBB has been identified as a research priority. We describe the 5-year outcomes for children with PBB to ascertain the presence of chronic respiratory disease (bronchiectasis, recurrent PBB and asthma) and identify the risk factors for these. Methods: Prospective cohort study was undertaken at the Queensland Children's Hospital, Brisbane, Australia, of 166 children with PBB and 28 controls (undergoing bronchoscopy for symptoms other than chronic wet cough). Monitoring was by monthly contact via research staff. Clinical review, spirometry and CT chest were performed as clinically indicated. Results: A total of 194 children were included in the analysis. Median duration of follow-up was 59 months (IQR: 50¿71 months) post-index PBB episode, 67.5% had ongoing symptoms and 9.6% had bronchiectasis. Significant predictors of bronchiectasis were recurrent PBB in year 1 of follow-up (ORadj = 9.6, 95% CI: 1.8¿50.1) and the presence of Haemophilus influenzae in the BAL (ORadj = 5.1, 95% CI: 1.4¿19.1). Clinician-diagnosed asthma at final follow-up was present in 27.1% of children with PBB. A significant BDR (FEV1 improvement >12%) was obtained in 63.5% of the children who underwent reversibility testing. Positive allergen-specific IgE (ORadj = 14.8, 95% CI: 2.2¿100.8) at baseline and bronchomalacia (ORadj = 5.9, 95% CI: 1.2¿29.7) were significant predictors of asthma diagnosis. Spirometry parameters were in the normal range. Conclusion: As a significant proportion of children with PBB have ongoing symptoms at 5 years, and outcomes include bronchiectasis and asthma, they should be carefully followed up clinically. Defining biomarkers, endotypes and mechanistic studies elucidating the different outcomes are now required.

Background: Multiple early reports of patients admitted to hospital with COVID-19 showed that patients with chronic respiratory disease were significantly under-represented in these cohorts. We hypothesised that the widespread use of inhaled glucocorticoids among these patients was responsible for this finding, and tested if inhaled glucocorticoids would be an effective treatment for early COVID-19. Methods: We performed an open-label, parallel-group, phase 2, randomised controlled trial (Steroids in COVID-19; STOIC) of inhaled budesonide, compared with usual care, in adults within 7 days of the onset of mild COVID-19 symptoms. The trial was done in the community in Oxfordshire, UK. Participants were randomly assigned to inhaled budsonide or usual care stratified for age (=40 years or >40 years), sex (male or female), and number of comorbidities (=1 and =2). Randomisation was done using random sequence generation in block randomisation in a 1:1 ratio. Budesonide dry powder was delivered using a turbohaler at a dose of 400 µg per actuation. Participants were asked to take two inhalations twice a day until symptom resolution. The primary endpoint was COVID-19-related urgent care visit, including emergency department assessment or hospitalisation, analysed for both the per-protocol and intention-to-treat (ITT) populations. The secondary outcomes were self-reported clinical recovery (symptom resolution), viral symptoms measured using the Common Cold Questionnare (CCQ) and the InFLUenza Patient Reported Outcome Questionnaire (FLUPro), body temperature, blood oxygen saturations, and SARS-CoV-2 viral load. The trial was stopped early after independent statistical review concluded that study outcome would not change with further participant enrolment. This trial is registered with ClinicalTrials.gov, NCT04416399. Findings: From July 16 to Dec 9, 2020, 167 participants were recruited and assessed for eligibility. 21 did not meet eligibility criteria and were excluded. 146 participants were randomly assigned¿73 to usual care and 73 to budesonide. For the per-protocol population (n=139), the primary outcome occurred in ten (14%) of 70 participants in the usual care group and one (1%) of 69 participants in the budesonide group (difference in proportions 0·131, 95% CI 0·043 to 0·218; p=0·004). For the ITT population, the primary outcome occurred in 11 (15%) participants in the usual care group and two (3%) participants in the budesonide group (difference in proportions 0·123, 95% CI 0·033 to 0·213; p=0·009). The number needed to treat with inhaled budesonide to reduce COVID-19 deterioration was eight. Clinical recovery was 1 day shorter in the budesonide group compared with the usual care group (median 7 days [95% CI 6 to 9] in the budesonide group vs 8 days [7 to 11] in the usual care group; log-rank test p=0·007). The mean proportion of days with a fever in the first 14 days was lower in the budesonide group (2%, SD 6) than the usual care group (8%, SD 18; Wilcoxon test p=0·051) and the proportion of participants with at least 1 day of fever was lower in the budesonide group when compared with the usual care group. As-needed antipyretic medication was required for fewer proportion of days in the budesonide group compared with the usual care group (27% [IQR 0¿50] vs 50% [15¿71]; p=0·025) Fewer participants randomly assigned to budesonide had persistent symptoms at days 14 and 28 compared with participants receiving usual care (difference in proportions 0·204, 95% CI 0·075 to 0·334; p=0·003). The mean total score change in the CCQ and FLUPro over 14 days was significantly better in the budesonide group compared with the usual care group (CCQ mean difference -0·12, 95% CI -0·21 to -0·02 [p=0·016]; FLUPro mean difference -0·10, 95% CI -0·21 to -0·00 [p=0·044]). Blood oxygen saturations and SARS-CoV-2 load, measured by cycle threshold, were not different between the groups. Budesonide was safe, with only five (7%) participants reporting...

Background and objective: Severe asthma is responsible for a disproportionate burden of illness and healthcare costs spent on asthma. This study analyses sputum transcriptomics to investigate the mechanisms and novel treatment targets of severe asthma. Methods: Induced sputum samples were collected in a cross-sectional study from participants with severe asthma (n = 12, defined as per GINA criteria), non-severe uncontrolled (n = 21) and controlled asthma (n = 21) and healthy controls (n = 15). Sputum RNA was extracted and transcriptomic profiles were generated (Illumina HumanRef-8 V2) and analysed (GeneSpring). Sputum protein lysates were analysed for p38 activation in a validation study (n = 24 asthma, n = 8 healthy) by western blotting. Results: There were 2166 genes differentially expressed between the four groups. In severe asthma, the expression of 1875, 1308 and 563 genes was altered compared to healthy controls, controlled and uncontrolled asthma, respectively. Of the 1875 genes significantly different to healthy controls, 123 were >2-fold change from which four networks were identified. Thirty genes (>2-fold change) were significantly different in severe asthma compared to both controlled asthma and healthy controls. There was enrichment of genes in the p38 signalling pathway that were associated with severe asthma. Phosphorylation of p38 was increased in a subset of severe asthma samples, correlating with neutrophilic airway inflammation. Conclusion: Severe asthma is associated with substantial differences in sputum gene expression that underlie unique cellular mechanisms. The p38 signalling pathway may be important in the pathogenesis of severe asthma, and future investigations into p38 inhibition are warranted as a ¿non-Th2¿ therapeutic option.

Background: Whether blood eosinophil counts and exhaled nitric oxide (FeNO) are associated with important outcomes in mild asthma is unclear. In this prespecified subgroup analysis of a previously published open-label clinical trial, we aimed to assess associations between blood eosinophil counts and FeNO with outcomes and response to asthma treatment. Methods: In the previously reported 52-week, open-label, randomised controlled trial, people with mild asthma receiving only ß agonist reliever inhalers were enrolled at one of 16 clinical trials units in New Zealand, the UK, Italy, or Australia. Eligible participants were randomly assigned (1:1:1, stratified by country), to receive inhalers to take as-needed salbutamol (two inhalations of 100 µg in a pressurised metered dose inhaler), maintenance budesonide (200 µg twice per day by inhaler) plus as-needed salbutamol (two inhalations of 100 µg), or as-needed budesonide¿formoterol (one inhalation of 200 µg budesonide and 6µg formoterol by inhaler). The primary outcome was the annual rates of asthma exacerbations per patient, and in this prespecified subgroup analysis, we assessed whether annual exacerbation rates in each treatment group were significantly different depending on levels of blood eosinophil count, FeNO, or a composite score of both. Analyses were done for patients with available biomarker measurements The study was registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12615000999538. Findings: 675 participants were enrolled between March 17, 2016, and Aug 29, 2017, of whom 656 had results for blood eosinophil analysis and 668 had results for FeNO. Of the patients who received as-needed salbutamol, the proportion of patients having a severe exacerbation increased progressively with increasing blood eosinophil count (two [4%] of 49 participants with <0·15 × 109/L, six [6%] of 93 with 0·15 to <0·3 × 109/L, and 15 [19%] of 77 with =0·3 × 109/L; p=0·014). There were no significant interactions between blood eosinophil count or FeNO level and the effect of as-needed budesonide¿formoterol compared with as-needed salbutamol for either exacerbations or severe exacerbations. However, there were significant interactions between blood eosinophil count subgroups and the effect of maintenance budesonide plus as-needed salbutamol compared with as-needed salbutamol, both for exacerbations (p=0·0006) and severe exacerbations (p=0·0007). Maintenance budesonide plus as-needed salbutamol was more effective than as-needed salbutamol in patients with blood eosinophil counts of 0·3 × 109/L or more, both for exacerbations (rate ratio 0·13 [95% CI 0·05¿0·33]) and severe exacerbations (risk odds ratio 0·11 [0·03¿0·45]). This difference was not seen for blood eosinophil counts of less than 0·15 × 109/L (1·15 [0·51¿1·28] for exacerbations and 5·72 [0·97¿33·60] for severe exacerbations). There was no consistent interaction between treatment response and FeNO or the composite score. Interpretation: In patients with mild asthma, the effects of as-needed budesonide¿formoterol on exacerbations are independent of biomarker profile, whereas the benefits of maintenance inhaled budesonide are greater in patients with high blood eosinophil counts than in patients with low counts. Funding: AstraZeneca, Health Research Council of New Zealand.

Background: Both the six gene signature (6GS: CPA3, DNASE1L3, CLC, IL1B, ALPL, and CXCR2) and T-helper 2 signature (TH2S: CLCA1, SERPINB2, and POSTN) are proposed as biomarkers in the identification of inflammatory phenotypes of asthma in induced sputum and epithelial brushings, respectively. The aim of this study was to explore patterns of gene expression of known signatures, 6GS and TH2S in endobronchial biopsies. Methods: This was an exploratory cross-sectional study of gene expression in endobronchial biopsies of 55 adults with asthma and 9 healthy controls (HC). The expression of the 6GS and TH2S was determined by quantitative polymerase chain reaction. Correlations with clinical and cellular characteristics were performed, and receiver operating characteristic was utilized to assess signatures' ability to predict asthma from HC and inflammatory phenotypes. Results: Gene expression of DNASE1L3 (P =.045) was upregulated in asthma compared with HC, and IL1B (P =.017) was upregulated in neutrophilic asthma compared with non-neutrophilic asthma. In asthma, the expression of CPA3 was negatively associated with ICS daily dose (r = -.339; P =.011), IL1B expression was positively associated with bronchial lavage fluid (BLF) total cell count (r =.340; P =.013) and both CLC and POSTN expression were associated with lymphocytes percentage in BLF (r = -.355, P =.009; r = -.300, P =.025, respectively). Both 6GS (area under curve [AUC] = 86.3%; P =.017) and TH2S (AUC = 72.7%; P =.037) could significantly predict asthma from HC. In addition, 6GS can identify neutrophilic (AUC = 93.2%; P =.005) and TH2S identifies eosinophilic (AUC = 62.7%; P =.033) asthma. Conclusions and Clinical Relevance: There was increased expression of DNASE1L3 in asthma and IL1B in neutrophilic asthma. These results show similar upregulated patterns of expression in two genes of the 6GS in endobronchial biopsies, previously identified in sputum. The upregulation of DNASE1L3 and IL1B suggests that common mechanisms may be at play throughout the airway.

Background: The 6 gene expression signature (6GS) predicts in¿ammatory phenotype, exacerbation risk, and corticosteroid responsiveness in asthma. In COPD, patterns of airway in¿ammation are similar, suggesting the 6GS may be useful. This study determines the diagnostic and prognostic ability of 6GS in predicting in¿ammatory phenotypes and exacerbation risk in COPD. Methods: We performed 2 studies: a cross-sectional phenotype prediction study in stable COPD (total N=132; n=34 eosinophilic (E)-COPD, n=42 neutrophilic (N)-COPD, n=39 paucigranulocytic (PG)-COPD, n=17 mixed-granulocytic (MG)-COPD) that assessed 6GS ability to discriminate phenotypes (eosinophilia=3%; neutrophilia=61%); and a prospective cohort study (total n=54, n=8 E-COPD; n=18 N-COPD; n=20 PG-COPD; n=8 MG-COPD, n=21 exacerbation prone (=2/year)) that investigated phenotype and exacerbation prediction utility. 6GS was measured by qPCR and evaluated using multiple logistic regression and area under the curve (AUC). Short-term reproducibility (intra-class correlation) and phenotyping method agreement (¿ statistic) were assessed. Results: In the phenotype prediction study, 6GS could accurately identify and discriminate patients with E-COPD from N-COPD (AUC=96.4%; p<0.0001), PG-COPD (AUC=88.2%; p<0.0001) or MG-COPD (AUC=86.2%; p=0.0001), as well as N-COPD from PG-COPD (AUC=83.6%; p<0.0001) or MG-COPD (AUC=87.4%; p<0.0001) and was reproducible. In the prospective cohort study, 6GS had substantial agreement for neutrophilic in¿ammation (82%, ¿=0.63,p<0.001)and moderate agreement foreosinophilici n¿ammation(78%, ¿=0.42,p<0.001). 6GS could signi¿cantly discriminate exacerbationprone patients (AUC=77.2%; p=0.034). Higher IL1B levels were associated with poorer lung function and increased COPD severity. Conclusion: 6GS can signi¿cantly and reproducibly discriminate COPD in¿ammatory phenotypes and predict exacerbation prone patients and may become a useful molecular diagnostic tool assisting COPD management..

Background: Improved diagnostic tools for predicting future exacerbation frequency in asthmatic patients are required. A sputum gene expression signature of 6 biomarkers (6-gene signature [6GS], including Charcot-Leyden crystal galectin [CLC]; carboxypeptidase 3 [CPA3]; deoxyribonuclease 1-like 3 [DNASE1L3]; alkaline phosphatase, liver/bone/kidney [ALPL]; CXCR2; and IL1B) predicts inflammatory and treatment response phenotypes in patients with stable asthma. Recently, we demonstrated that azithromycin (AZM) add-on treatment in patients with uncontrolled moderate-to-severe asthma significantly reduced asthma exacerbations (AMAZES clinical trial). Objectives: We sought to test whether the 6GS predicts future exacerbation and inflammatory phenotypes in a subpopulation of AMAZES and to test the effect of AZM therapy on 6GS expression and prognostic capacity. Methods: One hundred forty-two patients (73 placebo-treated and 69 AZM-treated patients) had sputum stored for quantitative PCR of 6GS markers at baseline and after 48 weeks of treatment. Logistic regression and receiver operating characteristic and area under the curve (AUC) determination were performed on baseline measures, and in an exploratory analysis the predictive value of the 6GS was compared with conventional biomarkers for exacerbation and inflammatory phenotypes. Results: The 6GS significantly predicted all future exacerbation phenotypes tested. Calculated AUCs for the 6GS were significantly greater than AUCs for peripheral blood eosinophil counts, sputum neutrophil counts, and combined sputum eosinophil and neutrophil counts. 6GS AUCs were also numerically but not significantly greater than those for fractional exhaled nitric oxide values and sputum eosinophil counts. AZM treatment altered neither 6GS expression nor the predictive capacity of the 6GS for future exacerbation phenotypes. The 6GS was a significant predictor of airway inflammatory phenotype in this population. Conclusion: We demonstrate that a sputum gene signature can predict future exacerbation phenotypes of asthma, with the greatest biomarker performance in identifying those who would experience frequent severe exacerbations. AZM therapy did not modify 6GS expression or biomarker performance, suggesting the therapeutic action of AZM is independent of 6GS-related inflammatory pathways.

Background: Dysfunction of the bronchial epithelium plays an important role in asthma; however, its measurement is challenging. Columnar epithelial cells are often quantified, yet rarely analysed, in induced sputum studies. Objective: We aimed to test whether sputum columnar epithelial cell proportion and count are altered in asthma, and whether they are associated with clinical and inflammatory variables. We aimed to test whether sputum-based measures could provide a relatively non-invasive means through which to monitor airway epithelial activation status. Methods: We examined the relationship of sputum columnar epithelial cells with clinical and inflammatory variables of asthma in a large retrospective cross-sectional cohort (901 participants with asthma and 138 healthy controls). In further studies, we used flow cytometry, microarray, qPCR and ELISA to characterize sputum columnar epithelial cells and their products. Results: Multivariate analysis and generation of 90th centile cut-offs (=11% or =18.1¿×¿104/mL) to identify columnar epithelial cell ¿high¿ asthma revealed a significant relationship between elevated sputum columnar cells and male gender, severe asthma and non-neutrophilic airway inflammation. Flow cytometry showed viable columnar epithelial cells were present in all sputum samples tested. An epithelial gene signature (SCGB3A1, LDLRAD1, FOXJ1, DNALI1, CFAP157, CFAP53) was detected in columnar epithelial cell-high sputum. CLCA1 mRNA and periostin protein, previously identified biomarkers of IL-13-mediated epithelial activation, were elevated in columnar epithelial cell-high sputum samples, but only when accompanied by eosinophilia. Conclusions & clinical relevance: Sputum columnar epithelial cells are related to important clinical and inflammatory variables in asthma. Measurement of epithelial biomarkers in sputum samples could allow non-invasive assessment of altered bronchial epithelium status in asthma.

Purpose: To characterize neutrophils in obstructive airway disease by measuring their surface adhesion molecules and oxidative burst along with characterizing them into different subsets as per their adhesion molecule expression. Patients and methods: Peripheral blood from adults with COPD (n=17), asthma (n=20), and healthy participants (n=19) was examined for expression of CD16, CD62L, CD11b, CD11c, and CD54, and analyzed by flow cytometry. For oxidative burst and CD62L shedding analysis, CD16 and CD62L stained leukocytes were loaded with Dihydrorhodamine-123 (DHR-123) and stimulated with N-Formylmethionine-leucyl-phenylalanine (fMLF). Neutrophil subsets were characterized based on CD16 and CD62L expression. Marker surface expression was recorded on CD16+ neutrophils as median fluorescence intensity (MFI). Results: Neutrophil surface expression of CD62L was significantly reduced in COPD (median (IQR) MFI: 1156 (904, 1365)) compared with asthma (1865 (1157, 2408)) and healthy controls (2079 (1054, 2960)); p=0.028. COPD neutrophils also demonstrated a significant reduction in CD62L expression with and without fMLF stimulation. Asthma participants had a significantly increased proportion and number of CD62Lbright/CD16dim neutrophils (median: 5.4% and 0.14 × 109/L, respectively), in comparison with healthy (3.54% and 0.12 × 109/L, respectively); p<0.017. Conclusion: Reduced CD62L expression suggests blood neutrophils have undergone priming in COPD but not in asthma, which may be the result of systemic inflammation. The increased shedding of CD62L receptor by COPD blood neutrophils suggests a high sensitivity for activation.

Rationale: The macrolide antibiotic azithromycin reduces exacerbations in adults with persistent symptomatic asthma. However, owing to the pleotropic properties of macrolides, unintended bacteriological consequences such as augmented pathogen colonization or dissemination of antibiotic-resistant organisms can occur, calling into question the long-term safety of azithromycin maintenance therapy. Objectives: To assess the effects of azithromycin on the airway microbiota, pathogen abundance, and carriage of antibiotic resistance genes. Methods: 16S rRNA sequencing and quantitative PCR were performed to assess the effect of azithromycin on sputum microbiology from participants of the AMAZES (Asthma and Macrolides: The Azithromycin Efficacy and Safety) trial: a 48-week, double-blind, placebo-controlled trial of thrice-weekly 500 mg oral azithromycin in adults with persistent uncontrolled asthma. Pooled-template shotgun metagenomic sequencing, quantitative PCR, and isolate whole-genome sequencing were performed to assess antibiotic resistance. Measurements and Main Results: Paired sputum samples were available from 61 patients (n = 34 placebo, n = 27 azithromycin). Azithromycin did not affect bacterial load (P = 0.37) but did significantly decrease Faith¿s phylogenetic diversity (P = 0.026) and Haemophilus influenzae load (P, 0.0001). Azithromycin did not significantly affect levels of Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, or Moraxella catarrhalis. Of the 89 antibiotic resistance genes detected, five macrolide resistance genes and two tetracycline resistance genes were increased significantly. Conclusions: In patients with persistent uncontrolled asthma, azithromycin reduced airway H. influenzae load compared with placebo but did not change total bacterial load. Macrolide resistance increased, reflecting previous studies. These results highlight the need for studies assessing the efficacy of nonantibiotic macrolides as a long-term therapy for patients with persistent uncontrolled asthma.

Background Asthma pathophysiology and treatment responsiveness are predicted by inflammatory phenotype. However, the relationship between airway microbiology and asthma phenotype is poorly understood. Objective We aimed to characterize the airway microbiota in patients with symptomatic stable asthma and relate composition to airway inflammatory phenotype and other phenotypic characteristics. Methods The microbial composition of induced sputum specimens collected from adult patients screened for a multicenter randomized controlled trial was determined by using 16S rRNA gene sequencing. Inflammatory phenotypes were defined by sputum neutrophil and eosinophil cell proportions. Microbiota were defined by using a- and ß-diversity measures, and interphenotype differences were identified by using similarity of percentages, network analysis, and taxon fold change. Phenotypic predictors of airway microbiology were identified by using multivariate linear regression. Results Microbiota composition was determined in 167 participants and classified as eosinophilic (n = 84), neutrophilic (n = 14), paucigranulocytic (n = 60), or mixed neutrophilic-eosinophilic (n = 9) asthma phenotypes. Airway microbiology was significantly less diverse (P =.022) and more dissimilar (P =.005) in neutrophilic compared with eosinophilic participants. Sputum neutrophil proportions, but not eosinophil proportions, correlated significantly with these diversity measures (a-diversity: Spearman r = -0.374, P <.001; ß-diversity: r = 0.238, P =.002). Interphenotype differences were characterized by a greater frequency of pathogenic taxa at high relative abundance and reduced Streptococcus, Gemella, and Porphyromonas taxa relative abundance in patients with neutrophilic asthma. Multivariate regression confirmed that sputum neutrophil proportion was the strongest predictor of microbiota composition. Conclusions Neutrophilic asthma is associated with airway microbiology that is significantly different from that seen in patients with other inflammatory phenotypes, particularly eosinophilic asthma. Differences in microbiota composition might influence the response to antimicrobial and steroid therapies and the risk of lung infection.

Severe, steroid-resistant asthma is clinically and economically important since affected individuals do not respond to mainstay corticosteroid treatments for asthma. Patients with this disease experience more frequent exacerbations of asthma, are more likely to be hospitalized, and have a poorer quality of life. Effective therapies are urgently required, however, their development has been hampered by a lack of understanding of the pathological processes that underpin disease. A major obstacle to understanding the processes that drive severe, steroid-resistant asthma is that the several endotypes of the disease have been described that are characterized by different inflammatory and immunological phenotypes. This heterogeneity makes pinpointing processes that drive disease difficult in humans. Clinical studies strongly associate specific respiratory infections with severe, steroid-resistant asthma. In this review, we discuss key findings from our studies where we describe the development of representative experimental models to improve our understanding of the links between infection and severe, steroid-resistant forms of this disease. We also discuss their use in elucidating the mechanisms, and their potential for developing effective therapeutic strategies, for severe, steroid-resistant asthma. Finally, we highlight how the immune mechanisms and therapeutic targets we have identified may be applicable to obesity-or pollution-associated asthma.

Background Exacerbations of asthma cause a substantial global illness burden. Adults with uncontrolled persistent asthma despite maintenance treatment require additional therapy. Since macrolide antibiotics can be used to treat persistent asthma, we aimed to assess the efficacy and safety of oral azithromycin as add-on therapy in patients with uncontrolled persistent asthma on medium-to-high dose inhaled corticosteroids plus a long-acting bronchodilator. Methods We did a randomised, double-blind, placebo controlled parallel group trial to determine whether oral azithromycin decreases the frequency of asthma exacerbations in adults (=18 years) with symptomatic asthma despite current use of inhaled corticosteroid and long-acting bronchodilator, and who had no hearing impairment or abnormal prolongation of the corrected QT interval. Patients were randomly assigned (1:1) to receive azithromycin 500 mg or placebo three times per week for 48 weeks. Patients were centrally allocated using concealed random allocation from a computer-generated random numbers table with permuted blocks of 4 or 6 and stratification for centre and past smoking. Primary efficacy endpoints were the rate of total (severe and moderate) asthma exacerbations over 48 weeks and asthma quality of life. Data were analysed on an intention-to-treat basis. The trial is registered at the Australian and New Zealand Clinical Trials Registry (ANZCTR), number 12609000197235. Findings Between June 12, 2009, and Jan 31, 2015, 420 patients were randomly assigned (213 in the azithromycin group and 207 in the placebo group). Azithromycin reduced asthma exacerbations (1·07 per patient-year [95% CI 0·85¿1·29]) compared with placebo (1·86 per patient-year [1·54¿2·18]; incidence rate ratio [IRR] 0·59 [95% CI 0·47¿0·74]; p<0·0001). The proportion of patients experiencing at least one asthma exacerbation was reduced by azithromycin treatment (127 [61%] patients in the placebo group vs 94 [44%] patients in the azithromycin group, p<0·0001). Azithromycin significantly improved asthma-related quality of life (adjusted mean difference, 0·36 [95% CI 0·21¿0·52]; p=0·001). Diarrhoea was more common in azithromycin-treated patients (72 [34%] vs 39 [19%]; p=0·001). Interpretation Adults with persistent symptomatic asthma experience fewer asthma exacerbations and improved quality of life when treated with oral azithromycin for 48 weeks. Azithromycin might be a useful add-on therapy in persistent asthma. Funding National Health and Medical Research Council of Australia, John Hunter Hospital Charitable Trust.

Introduction Sputum colour is associated with neutrophilic inflammation in chronic bronchitis and chronic obstructive pulmonary disease (COPD). Neutrophilia and sputum expectoration is notable in asthma, but whether sputum colour is associated with and predicts the presence of neutrophilic inflammation in asthma is unknown. The objective of the study is to assess the ability of sputum colour in distinguishing asthma inflammatory phenotypes. Methods Induced sputum samples collected from 271 adults with stable asthma were retrospectively assessed. Sputum colour was determined using the BronkoTest sputum colour chart and correlated to differential cell counts and CXCL-8 concentration. Neutrophilic inflammation was defined as an age-corrected sputum neutrophil proportion (=61.6% for age 20¿40 years; =63.2% for age 40¿60 and =67.2% for age >60 years), whereas neutrophilic bronchitis (NB) was defined as high total cell count (=5.1×106 cells/mL) plus an increased age-corrected neutrophil proportion. The optimal cut-off for sputum colour to predict neutrophilic inflammation and NB was determined using receiver operator characteristic curve analysis. Results A sputum colour score of =3 represented and predicted neutrophilic inflammation with modest accuracy (area under the curve (AUC)=0.64; p<0.001, specificity=78.4%, sensitivity=49.2%). Participants with a sputum colour score of =3 had significantly (p<0.05) higher CXCL-8, total cells and neutrophil number and proportion. Sputum colour score was also positively correlated with these factors. Sputum colour score =3 predicted NB with reasonably good accuracy (AUC=0.79, p<0.001, specificity=79.3%, sensitivity=70.7%). Conclusions Visual gradation of sputum colour in asthma relates to high total cell count and neutrophilic inflammation. Assessment of sputum colour can identify adults with asthma who are likely to have NB without the need for sputum processing and differential cell count, which may facilitate asthma management.

Rationale: Severe, steroid-resistant asthma is the major unmet need in asthma therapy. Disease heterogeneity and poor understanding of pathogenic mechanisms hampers the identification of therapeutic targets. Excessive nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome and concomitant IL-1ß responses occur in chronic obstructive pulmonary disease, respiratory infections, and neutrophilic asthma. However, the direct contributions to pathogenesis, mechanisms involved, and potential for therapeutic targeting remain poorly understood, and are unknown in severe, steroid-resistant asthma. Objectives: To investigate the roles and therapeutic targeting of the NLRP3 inflammasome and IL-1ß in severe, steroid-resistant asthma. Methods: We developed mouse models of Chlamydia and Haemophilus respiratory infection-mediated, ovalbumin-induced severe, steroid-resistant allergic airway disease. These models share the hallmark features of human disease, including elevated airway neutrophils, and NLRP3 inflammasome and IL-1ß responses. The roles and potential for targeting of NLRP3 inflammasome, caspase-1, and IL-1ß responses in experimental severe, steroid-resistant asthma were examined using a highly selective NLRP3 inhibitor, MCC950; the specific caspase-1 inhibitor Ac-YVAD-cho; and neutralizing anti-IL-1ß antibody. Roles for IL-1ß-induced neutrophilic inflammation were examined using IL-1ß and anti-Ly6G. Measurements and Main Results: Chlamydia and Haemophilus infections increase NLRP3, caspase-1, IL-1ß responses that drive steroid-resistant neutrophilic inflammation and airway hyperresponsiveness. Neutrophilic airway inflammation, disease severity, and steroid resistance in human asthma correlate with NLRP3 and IL-1ß expression. Treatment with anti-IL-1ß, Ac- YVAD-cho, and MCC950 suppressed IL-1ß responses and the important steroid-resistant features of disease in mice, whereas IL-1ß administration recapitulated these features. Neutrophil depletion suppressed IL-1ß-induced steroid-resistant airway hyperresponsiveness. Conclusions: NLRP3 inflammasome responses drive experimental severe, steroid-resistant asthma and are potential therapeutic targets in this disease.

Background: COPD patients are often prescribed multiple medications for their respiratory disease and comorbidities. This can lead to complex medication regimens resulting in poor adherence, medication errors, and drug-drug interactions. The relationship between clinical outcomes and medication burden beyond medication count in COPD is largely unknown. Objectives: The aim of this study was to explore the relationships of medication burden in COPD with clinical outcomes, comorbidities, and multidimensional indices. Methods: In a cross-sectional study, COPD patients (n=222) were assessed for demographic information, comorbidities, medication use, and clinical outcomes. Complexity of medication regimens was quantified using the validated medication regimen complexity index (MRCI). Results: Participants (58.6% males) had a mean (SD) age of 69.1 (8.3) years with a postbronchodilator forced expiratory volume in 1 second % predicted of 56.5 (20.4) and a median of five comorbidities. The median (q1, q3) total MRCI score was 24 (18.5, 31). COPD-specific medication regimens were more complex than those of non-COPD medications (median MRCI: 14.5 versus 9, respectively; P<0.0001). Complex dosage formulations contributed the most to higher MRCI scores of COPD-specific medications while dosing frequency primarily drove the complexity associated with non-COPD medications. Participants in Global Initiative for Chronic Obstructive Lung Disease quadrant D had the highest median MRCI score for COPD medications (15.5) compared to those in quadrants A (13.5; P=0.0001) and B (12.5; P<0.0001). Increased complexity of COPD-specific treatments showed significant but weak correlations with lower lung function and 6-minute walk distance, higher St George¿s Respiratory Questionnaire and COPD assessment test scores, and higher number of prior year COPD exacerbations and hospitalizations. Comorbid cardiovascular, gastrointestinal, or metabolic diseases individually contributed to higher total MRCI scores and/or medication counts for all medications. Charlson Comorbidity Index and COPD-specific comorbidity test showed the highest degree of correlation with total MRCI score (¿=0.289 and ¿=0.326; P<0.0001, respectively). Conclusion: In COPD patients, complex medication regimens are associated with disease severity and specific class of comorbidities.

Background: Non-eosinophilic asthma (NEA) is a distinct, often corticosteroid-resistant inflammatory asthma phenotype. NK and NKT-like cells are effector lymphocytes that we have shown, like CD28null T cells, to be relatively resistant to steroids and major sources of pro-inflammatory/cytotoxic mediators. We hypothesized that these cells and mediators would be increased in peripheral blood in NEA. Methods: Adults with severe asthma and variable airflow obstruction, poorly controlled despite maintenance therapy with inhaled glucocorticosteroids and long-acting bronchodilators, were recruited. Blood was assessed in those with eosinophilic asthma (n = 12), NEA (n = 25) and healthy non-smoking controls (n = 30). We applied flow cytometry to measure T, CD28null, NK and NKT-like cells and their expression of granzyme B, perforin, and killer inhibitory/activating receptors CD94(Kp43), CD158b and CD107A. Intracellular pro-inflammatory cytokine production (IFN-¿ and TNF-a) was assessed in 18 controls and 10 patients with asthma/group. Results: In NEA, there was increased expression of granzyme B by CD8+ T cells vs. controls. There was increased expression of granzyme B and CD158 and decreased CD94 on NK cells, vs. healthy controls and those with eosinophilic asthma. IFN-¿ production by NK cells and TNF-a production by NKT-like cells in NEA were significantly increased vs. controls. In both eosinophilic and NEA phenotypes, there were significant increases in CD4+28null T cells (72% and 81% increases, respectively, vs. controls) and their expression of pro-inflammatory cytokines. Significant correlations were noted between blood CD4+28null T cells and neutrophil numbers in induced sputum, and between corticosteroid dose and blood NKT-like cells, and their production of granzyme B and TNF-a and NK IFN-¿. Conclusion and clinical relevance: In poorly controlled asthma, altered expression of cytotoxic/pro-inflammatory mediators can be seen on a variety of lymphocyte subsets in the peripheral blood; these changes are most apparent in NEA. Whether this pattern of expression is a marker of treatment responsiveness and future risk of exacerbations remains to be determined.

Mast cells are a resident inflammatory cell of the airways, involved in both the innate and adaptive immune response. The relationship between mast cells and inflammatory phenotypes and treatment response of asthma is not clear. Clinical characteristics of subjects with stable asthma (n=55), inflammatory cell counts and gene expression microarrays in induced sputum were analysed. Sputum mast cell subtypes were determined by molecular phenotyping based on expression of mast cell biomarkers (tryptase (TPSAB1), chymase (CMA1) and carboxypeptidase A3 (CPA3)). Effects of mast cell subtypes on steroid response were observed in a prospective cohort study (n=50). MCT (n=18) and MCT/CPA3 (mRNA expression of TPSAB1 and CPA3; n=29) subtypes were identified, as well as a group without mast cell gene expression (n=8). The MCT/CPA3 subtype had elevated exhaled nitric oxide fraction, sputum eosinophils, bronchial sensitivity and reactivity, and poorer asthma control. This was accompanied by upregulation of 13 genes. Multivariable logistic regression identified CPA3 (OR 1.21, p=0.004) rather than TPSAB1 (OR 0.92, p=0.502) as a determinant of eosinophilic asthma. The MCT/CPA3 subtype had a better clinical response and reduced signature gene expression with corticosteroid treatment. Sputum mast cell subtypes of asthma can be defined by a molecular phenotyping approach. The MCT/CPA3 subtype demonstrated increased bronchial sensitivity and reactivity, and signature gene expression, which was associated with airway eosinophilia and greater corticosteroid responsiveness.

Background and objective Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation and inflammation. Airway bacterial colonization is increased in COPD; however, the role of potentially pathogenic and non-pathogenic bacteria in the pathogenesis of disease is unclear. This study characterized the presence of bacteria in a well-characterized cohort of adults with COPD and healthy controls. Methods Adults with COPD (n = 70) and healthy controls (n = 51) underwent clinical assessment and sputum induction. Sputum was dispersed, and total and differential cell counts were performed. Bacteria were cultured, identified and enumerated. Supernatants were assessed for neutrophil elastase (NE) and IL-1ß. Common respiratory pathogens were also determined using real-time PCR. Results Participants with COPD had a typical neutrophilic inflammatory profile. The total load of bacteria was increased in COPD and was associated with poorer respiratory health status, as measured by the St George's Respiratory Questionnaire (Spearman's r = 0.336, P = 0.013). Significantly lower levels of culturable Bacillus species were identified compared with healthy controls. PCR analyses revealed increased rates of detection of potentially pathogenic bacteria with Haemophilus influenzae detection associated with higher sputum levels of NE and IL-1ß, while Streptococcus pneumoniae was more common in male ex-smokers with emphysema and a deficit in diffusion capacity. Conclusion Non-pathogenic and pathogenic bacteria were altered in the sputum of patients with COPD. These observations highlight the potential to identify treatment and management strategies that both target specific bacterial pathogens and restore the microbial balance, which may lead to reductions in inflammation and subsequent improvements in lung health.

Background and objective Neutrophilic inflammation has been implicated in non-eosinophilic asthma (NEA) in adults, but little is known about NEA in children/adolescents. We assessed clinical and inflammatory characteristics of NEA in adolescent asthma. Methods Airway inflammation, sputum endotoxin, airway hyper-reactivity, atopy and lung function were assessed in 77 adolescents with asthma and 68 without asthma (12-17 years). Asthma was identified on the basis of wheeze and asthma history. Results The proportion of NEA (sputum eosinophils <2.5%) was 54%. In this group, atopy, sputum neutrophil, eosinophil, eosinophil cationic protein (ECP), endotoxin, neutrophil elastase and IL-8 levels were not different from those without asthma. In contrast, eosinophilic asthma (EA) was associated with atopy and sputum ECP and IL-8. The majority of NEA had no evidence of inflammation; only 14% had neutrophilia (=61% neutrophils), compared with 11% of EA, and 15% of those without asthma. Small differences in FEV1 (NS) were found between EA and NEA, but symptom prevalence and severity was not different (63% of EA and 52% of NEA were classified moderate to severe). Conclusion NEA is common in adolescent asthma and has similar clinical characteristics as EA. Neutrophils do not appear to play a role in NEA in adolescents, and underlying mechanisms may not involve airway inflammation. Our study suggests that many adolescents with stable asthma do not appear to correspond to the conventional notion that asthma is 'an inflammatory disorder of the airways'. In the absence of overt inflammation, the mechanisms responsible for asthma symptoms in a large proportion of adolescent with asthma remain unknown.

BACKGROUND: Asthma is a heterogeneous chronic inflammatory disease in which host defense against respiratory viruses such as human rhinovirus (HRV) may be abnormal. This is a matter of some controversy, with some investigators reporting reduced type I interferon (IFN) synthesis and others suggesting that type I IFN synthesis is relatively normal in asthma. OBJECTIVE: The objective of this study was to examine the responsiveness of circulating mononuclear cells to HRV in a large cohort of participants with poorly controlled asthma and determine whether IFN-a and IFN-ß synthesis varies across different inflammatory phenotypes. METHODS: Eligible adults with asthma (n = 86) underwent clinical assessment, sputum induction, and blood sampling. Asthma inflammatory subtypes were defined by sputum cell count, and supernatant assessed for IL-1ß. Peripheral blood mononuclear cells (PBMCs) were exposed to HRV serotype 1ß, and IFN-a and IFN-ß release was measured by enzymelinked immunosorbent assay. RESULTS: Participants (mean age, 59 years; atopy, 76%) had suboptimal asthma control (mean asthma control questionnaire 6, 1.7). In those with neutrophilic asthma (n = 12), HRV1ß-stimulated PBMCs produced significantly less IFN-a than PBMCs from participants with eosinophilic (n = 35) and paucigranulocytic asthma (n = 35). Sputum neutrophil proportion and the dose of inhaled corticosteroids were independent predictors of reduced IFN-a production after HRV1ß exposure. CONCLUSIONS: Antiviral type I IFN production is impaired in those with neutrophilic airway inflammation and in those prescribed high doses of inhaled corticosteroids. Our study is an important step toward identifying those with poorly controlled asthma who might respond best to inhaled IFN therapy during exacerbations.

Viral respiratory infections trigger severe exacerbations of asthma, worsen disease symptoms, and impair lung function. To investigate the mechanisms underlying viral exacerbation, we established a mouse model of respiratory syncytial virus (RSV)-induced exacerbation after allergen sensitization and challenge. RSV infection of OVA-sensitized/challenged BALB/c mice resulted in significantly increased airway hyperresponsiveness (AHR) and macrophage and neutrophil lung infiltration. Exacerbation was accompanied by increased levels of inflammatory cytokines (including TNF-a, MCP-1, and keratinocyte-derived protein chemokine [KC]) compared with uninfected OVA-treated mice or OVA-treated mice exposed to UV-inactivated RSV. Dexamethasone treatment completely inhibited all features of allergic disease, including AHR and eosinophil infiltration, in uninfected OVAsensitized/challenged mice. Conversely, dexamethasone treatment following RSV-induced exacerbation only partially suppressed AHR and failed to dampen macrophage and neutrophil infiltration or inflammatory cytokine production (TNF-a, MCP-1, and KC). This mimics clinical observations in patients with exacerbations, which is associated with increased neutrophils and often poorly responds to corticosteroid therapy. Interestingly, we also observed increased TNF-a levels in sputum samples from patients with neutrophilic asthma. Although RSV-induced exacerbation was resistant to steroid treatment, inhibition of TNF-a and MCP-1 function or depletion of macrophages suppressed features of disease, including AHR and macrophage and neutrophil infiltration. Our findings highlight critical roles for macrophages and inflammatory cytokines (including TNF-a and MCP-1) in viral-induced exacerbation of asthma and suggest examination of these pathways as novel therapeutic approaches for disease management.

Background and objective Neutrophil extracellular traps (NETs) are web-like structures comprising DNA and antimicrobial proteins, expelled from neutrophils during NETosis. Persistence of NETs can be pro-inflammatory, yet their role in respiratory disease remains unclear. This study aimed to investigate the presence of NETs in sputum from patients with asthma and COPD, and the relationship of NETs with inflammatory phenotype and disease severity. Methods Induced sputum was collected from healthy controls, asthma and COPD patients. Extracellular DNA (eDNA) was quantified by PicoGreen. LL-37, a-defensins1-3, NE, IL-1ß and CXCL8 were quantified by ELISA. PAD4 and NLRP3 gene expression was performed using qPCR. NETs were imaged in sputum smears using immunofluorescence microscopy. Results Sputum eDNA and NET neutrophil antimicrobial proteins were significantly elevated in asthma and COPD compared with healthy controls. Levels of eDNA and NET components were significantly higher in neutrophilic versus non-neutrophilic asthma and COPD. NETs were clearly visualized in sputum smears. PAD4 mRNA was upregulated in neutrophilic COPD. The level of eDNA was higher in severe asthma. High eDNA levels were associated with heightened innate immune responses, including elevated CXCL8 and IL-1ß, and NLRP3 gene expression in both COPD and asthma. Antimicrobial proteins and eDNA were positively correlated with airway neutrophils, and negatively correlated with lung function and symptoms. Conclusion NETs are present in the airways of subjects with asthma and COPD. Accumulation of excessive NETs was associated with activation of innate immune responses contributing to disease pathogenesis in chronic airway disease.

Asthma is a chronic inflammatory disorder of the airways where bacteria may act as protagonists of chronic inflammation. Little is known about the relation of airway inflammation to the presence of specific bacterial taxa. We sought to describe the sputum microbiome in adults with poorly controlled asthma. DNA was extracted from induced sputum and microbial communities were profiled using 16S rRNA pyrosequencing. Bacterial species were characterised, and the relationship between microbial populations, asthma inflammatory subtypes and other covariates was explored. Real-time PCR was used to identify Tropheryma whipplei and Haemophilus influenzae in sputum. Adults with neutrophilic asthma had reduced bacterial diversity and species richness. Tropheryma was identified and confirmed with real-time PCR in 12 (40%) participants. Haemophilus occurred most often in a group of younger atopic males with an increased proportion of neutrophils. PCR confirmed the presence of H. influenzae in 35 (76%) participants with poorly controlled asthma. There are phenotype-specific alterations to the airway microbiome in asthma. Reduced bacterial diversity combined with a high prevalence of H. influenzae was observed in neutrophilic asthma, whereas eosinophilic asthma had abundant T. whipplei.

Background: Children with recurrent protracted bacterial bronchitis (PBB) and bronchiectasis share common features, and PBB is likely a forerunner to bronchiectasis. Both diseases are associated with neutrophilic inflammation and frequent isolation of potentially pathogenic microorganisms, including nontypeable Haemophilus influenzae (NTHi), from the lower airway. Defective alveolar macrophage phagocytosis of apoptotic bronchial epithelial cells (efferocytosis), as found in other chronic lung diseases, may also contribute to tissue damage and neutrophil persistence. Thus, in children with bronchiectasis or PBB and in control subjects, we quantified the phagocytosis of airway apoptotic cells and NTHi by alveolar macrophages and related the phagocytic capacity to clinical and airway inflammation. Methods: Children with bronchiectasis (n = 55) or PBB (n = 13) and control subjects (n = 13) were recruited. Alveolar macrophage phagocytosis, efferocytosis, and expression of phagocytic scavenger receptors were assessed by flow cytometry. Bronchoalveolar lavage fluid interleukin (IL) 1ß was measured by enzyme-linked immunosorbent assay. Results: For children with PBB or bronchiectasis, macrophage phagocytic capacity was significantly lower than for control subjects (P=.003 and P<.001 for efferocytosis and P=.041 and P = .004 for phagocytosis of NTHi; PBB and bronchiectasis, respectively); median phagocytosis of NTHi for the groups was as follows: bronchiectasis, 13.7% (interquartile range [IQR], 11%-16%); PBB, 16% (IQR, 11%-16%); control subjects, 19.0% (IQR, 13%-21%); and median efferocytosis for the groups was as follows: bronchiectasis, 14.1% (IQR, 10%-16%); PBB, 16.2% (IQR, 14%-17%); control subjects, 18.1% (IQR, 16%-21%). Mannose receptor expression was significantly reduced in the bronchiectasis group (P = .019), and IL-1ß increased in both bronchiectasis and PBB groups vs control subjects. Conclusions: A reduced alveolar macrophage phagocytic host response to apoptotic cells or NTHi may contribute to neutrophilic inflammation and NTHi colonization in both PBB and bronchiectasis. Whether this mechanism also contributes to the progression of PBB to bronchiectasis remains unknown.

Introduction: Sputum eosinophilia occurs in approximately one-third of stable chronic obstructive pulmonary disease (COPD) patients and can predict exacerbation risk and response to corticosteroid treatments. Sputum induction, however, requires expertise, may not always be successful, and does not provide point-of-care results. Easily applicable diagnostic markers that can predict sputum eosinophilia in stable COPD patients have the potential to progress COPD management. This study investigated the correlation and predictive relationship between peripheral blood and sputum eosinophils. It also examined the repeatability of blood eosinophil counts. Methods: Stable COPD patients (n=141) were classified as eosinophilic or noneosinophilic based on their sputum cell counts (=3%), and a cross-sectional analysis was conducted comparing their demographics, clinical characteristics, and blood cell counts. Receiver operating characteristic curve analysis was used to assess the predictive ability of blood eosinophils for sputum eosinophilia. Intraclass correlation coefficient was used to examine the repeatability of blood eosinophil counts. Results: Blood eosinophil counts were significantly higher in patients with sputum eosinophilia (n=45) compared to those without (0.3×109/L vs 0.15×109/L; P<0.0001). Blood eosinophils correlated with both the percentage (¿=0.535; P<0.0001) and number of sputum eosinophils (¿=0.473; P<0.0001). Absolute blood eosinophil count was predictive of sputum eosinophilia (area under the curve =0.76, 95% confidence interval [CI] =0.67¿0.84; P<0.0001). At a threshold of =0.3×109/L (specificity =76%, sensitivity =60%, and positive likelihood ratio =2.5), peripheral blood eosinophil counts enabled identification of the presence or absence of sputum eosinophilia in 71% of the cases. A threshold of =0.4×109/L had similar classifying ability but better specificity (91.7%) and higher positive likelihood ratio (3.7). In contrast, =0.2×109/L offered a better sensitivity (91.1%) for ruling out sputum eosinophilia. There was a good agreement between two measurements of blood eosinophil count over a median of 28 days (intraclass correlation coefficient =0.8; 95% CI =0.66¿0.88; P<0.0001). Conclusion: Peripheral blood eosinophil counts can help identify the presence or absence of sputum eosinophilia in stable COPD patients with a reasonable degree of accuracy.

BACKGROUND: Protracted bacterial bronchitis (PBB) is a common and treatable cause of chronic wet cough in children in which the mechanisms are not understood. Th is study investigates the IL-1 pathway and a neutrophil gene expression signature in PBB.

Background: The progression of obstructive airway diseases (OADs) including asthma, chronic obstructive pulmonary disease (COPD) and asthma-COPD overlap syndrome in older adults is not well understood. Objective: To examine the prognosis of OADs and to identify potential determinants for longitudinal changes in clinical outcomes. Methods: We consecutively recruited 99 older adults (>55 years) with OADs who underwent a multidimensional assessment at baseline and 4 years which involved spirometry, 6-min walk distance (6MWD), assessments of health status (Saint George's Respiratory Questionnaire, SGRQ), comorbidity, and serum and sputum biomarkers. All-cause mortality and respiratory hospitalisation during the follow-up period were recorded. Clinical outcomes were compared between basal and final visits, and changes in clinical outcomes were compared among asthma, COPD and asthma-COPD overlap groups. Associations between clinical parameters, biomarkers and prognosis were examined. Results: After a median follow-up of 4.2 years, outcome data were available for 75 (75.8%) patients. There were 16 (16.2%) deaths. The BODE index predicted all-cause mortality in older people with OADs. While spirometry, 6MWD and SGRQ deteriorated significantly over the 4 years, there was significant heterogeneity in the longitudinal changes in these clinical outcomes. Participants with COPD had a significant decline in FEV1 (p = 0.003), SGRQ (p = 0.030) and 6MWD [decline of 75.5 (93.4) m, p = 0.024]. The change in 6MWD was lower in the asthma-COPD overlap group. Airflow reversibility was associated with a reduced decline in 6MWD. Conclusion: COPD patients had a poor prognosis compared with asthma and asthma-COPD overlap patients. The BODE index is a useful prognostic indicator in older adults with OADs. Both airway disease diagnosis and BODE index warrant specific attention in clinical practice. © 2013 S. Karger AG, Basel.

Background: Asthma is a heterogeneous inflammatory disease and eosinophilic, noneosinophilic and neutrophilic forms are recognised. While clinically similar to eosinophilic asthma, patients with non-eosinophilic asthma have different responses to treatment and little is known about the triggers of symptoms and inflammation. Objective: This study sought to characterise asthma control, exacerbation frequency and potential triggers of non-eosinophilic and specifically neutrophilic asthma such as infection, gastroesophageal reflux disease, and rhinosinusitis. Methods: Adults with asthma (n=65; doctor's diagnosis plus demonstrated response to bronchodilator and/or airways hyperresponsiveness to hypertonic saline) were recruited from the Respiratory and Sleep Medicine Ambulatory Care Service at John Hunter Hospital, NSW, Australia. Questionnaires were administered to assess gastroesophageal reflux disease, rhinosinusitis and asthma control. A sputum induction was performed and sputum was processed for assessment of inflammatory cells, infection, and lipid laden macrophages (Oil Red O). Results: Participants with neutrophilic asthma (n=11, 23%) had a higher frequency of primary care doctor visits for asthma exacerbations and a high prevalence (>70%) of chest infections in the previous 12 months. There was also an increased prevalence of rhinosinusitis (64%) and increased symptoms of gastroesophageal reflux disease compared to those with eosinophilic asthma. Conclusions: The clinical pattern of neutrophilic asthma is different from paucigranulocytic and eosinophilic asthma with evidence of abnormal upper airways responses. Specific and targeted treatment of these airway problems may assist in the control and management of neutrophilic asthma.

Asthma is a heterogeneous inflammatory airways disorder where interleukin (IL)-1ß is thought to be a key mediator, especially in the neutrophilic subtype of asthma. The generation of active IL-1ß requires proteolytic cleavage typically mediated through the formation of a caspase-1-containing inflammasome. This study hypothesised that an IL-1ß endotype associated with the nucleotide-binding domain, leucine-rich repeat-containing family protein (NLRP)3/apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)/caspase-1 inflammasome is characteristic of patients with the neutrophilic subtype of asthma. Participants with asthma (n=85) and healthy controls (n=27) underwent clinical assessment, spirometry and sputum induction. Sputum was processed for differential cell count, gene expression and protein mediators. NLRP3 and caspase-1 expression was also determined by immunocytochemistry. Sputum macrophages were isolated (n=8) and gene expression of NLRP3 and IL-1ß determined. There was significantly elevated gene expression of NLRP3, caspase-1, caspase-4, caspase-5 and IL-1ß in participants with neutrophilic asthma. Protein levels of IL-1ß were significantly higher in those with neutrophilic asthma and correlated with sputum IL-8 levels. Sputum macrophages, as well as sputum neutrophils in neutrophilic asthma, expressed NLRP3 and caspase-1 protein. NLRP3 inflammasome is upregulated in neutrophilic asthma and may regulate the inflammation process observed in this asthma phenotype through production of IL-1ß. Copyright © ERS 2014.

Background: Severe asthma and chronic obstructive pulmonary disease (COPD) are chronic inflammatory airway diseases in which the mechanisms are not fully understood. A disintegrin and metalloproteinase domain 8 (ADAM8) is an enzyme expressed on most leucocytes and may be important for facilitating leucocyte migration in respiratory disease. Objective: To investigate ADAM8 mRNA and protein expression in asthma and COPD and its relationship between asthma severity and inflammatory phenotypes. Methods: Induced sputum was collected from 113 subjects with asthma (severe n = 31, uncontrolled n = 39 and controlled n = 35), 20 subjects with COPD and 21 healthy controls. Sputum ADAM8 mRNA expression was measured by qPCR, and soluble ADAM8 (sADAM8) protein was measured in the sputum supernatant by validated ELISA. Results: ADAM8 mRNA correlated with ADAM8 protein levels (r = 0.27, P < 0.01). ADAM8 mRNA (P = 0.004) and sADAM8 protein (P = 0.014) levels were significantly higher in both asthma and COPD compared with healthy controls. ADAM8 mRNA (P = 0.035) and sADAM8 protein (P = 0.002) levels were significantly higher in severe asthma compared with controlled asthma. Total inflammatory cell count (P < 0.01) and neutrophils (P < 0.01) were also elevated in severe asthmatic sputum. Although ADAM8 mRNA was significantly higher in eosinophilic and neutrophilic asthma (P < 0.001), sADAM8 did not differ between asthma inflammatory phenotypes. ADAM8 expression positively correlated with sputum total cell count and sputum neutrophils. Conclusions and Clinical Relevance: ADAM8 expression is increased in both severe asthma and COPD and associated with sputum total cell count and neutrophils. ADAM8 may facilitate neutrophil migration to the airways in severe asthma and COPD. © 2013 John Wiley & Sons Ltd.

Summary: Background: In asthma, the airway inflammatory phenotype influences clinical characteristics and treatment response. Although induced sputum is the gold standard test for phenotyping asthma, a more accessible method is needed for clinical practice. Objective: To investigate whether white blood cell counts and/or their derived ratios can predict sputum eosinophils or neutrophils in uncontrolled asthma. Methods: This cross-sectional study evaluated 164 treated but uncontrolled asthmatic patients with sputum induction and blood collection. Receiver-operating characteristic (ROC) curves were used to assess the relationship between blood and sputum parameters. Results: There was a significant positive relationship between blood eosinophil parameters and the percentage of sputum eosinophil count. A weak but significant correlation was found between sputum neutrophil percentage and blood neutrophil percentage (r = 0.219, P = 0.005). ROC curve analysis identified that blood eosinophil percentage count was the best predictor for eosinophilic asthma, with an area under the curve (AUC) of 0.907 (P < 0.001). The optimum cut-point for blood eosinophil percentage was 2.7%, and this yielded a sensitivity of 92.2% and a specificity of 75.8%. The absolute blood eosinophil count was also highly predictive with an AUC of 0.898 (P < 0.0001) at a blood eosinophil cut-off of 0.26 × 10⁹/L. The blood eosinophil/lymphocyte ratio (ELR) and eosinophil/neutrophil ratio (ENR) were increased in eosinophilic asthma, and the neutrophil/lymphocyte ratio (NLR) was increased in neutrophilic asthma. Neutrophilic asthma could also be detected by blood neutrophil percentages and NLR, but with less accuracy. Conclusions and Clinical Relevance: Blood eosinophil counts and derived ratios (ELR and ENR) can accurately predict eosinophilic asthma in patients with persistent uncontrolled asthma despite treatment. Blood neutrophil parameters are poor surrogates for the proportion of sputum neutrophils. Blood counts may be a useful aid in the monitoring of uncontrolled asthma. © 2014 John Wiley & Sons Ltd.

Background Airway inflammation is associated with asthma exacerbation risk, treatment response, and disease mechanisms. Objective This study aimed to identify and validate a sputum gene expression signature that discriminates asthma inflammatory phenotypes. Methods An asthma phenotype biomarker discovery study generated gene expression profiles from induced sputum of 47 asthmatic patients. A clinical validation study (n = 59 asthmatic patients) confirmed differential expression of key genes. A 6-gene signature was identified and evaluated for reproducibility (n = 30 asthmatic patients and n = 20 control subjects) and prediction of inhaled corticosteroid (ICS) response (n = 71 asthmatic patients). Receiver operating characteristic curves were calculated, and area under the curve (AUC) values were reported. Results From 277 differentially expressed genes between asthma inflammatory phenotypes, we identified 23 genes that showed highly significant differential expression in both the discovery and validation populations. A signature of 6 genes, including Charcot-Leydon crystal protein (CLC); carboxypeptidase A3 (CPA3); deoxyribonuclease I-like 3 (DNASE1L3); IL-1ß (IL1B); alkaline phosphatase, tissue-nonspecific isozyme (ALPL); and chemokine (C-X-C motif) receptor 2 (CXCR2), was reproducible and could significantly (P <.0001) discriminate eosinophilic asthma from other phenotypes, including patients with noneosinophilic asthma (AUC, 89.6%), paucigranulocytic asthma (AUC, 92.6%), or neutrophilic asthma (AUC, 91.4%) and healthy control subjects (AUC, 97.6%), as well as discriminating patients with neutrophilic asthma from those with paucigranulocytic asthma (AUC, 85.7%) and healthy control subjects (AUC, 90.8). The 6-gene signature predicted ICS response (>12% change in FEV1; AUC, 91.5%). ICS treatment reduced the expression of CLC, CPA3, and DNASE1L3 in patients with eosinophilic asthma. Conclusions A sputum gene expression signature of 6 biomarkers reproducibly and significantly discriminates inflammatory phenotypes of asthma and predicts ICS treatment response. This signature has the potential to become a useful diagnostic tool to assist in the clinical diagnosis and management of asthma. © 2013 American Academy of Allergy, Asthma & Immunology.

Purpose: The role of systemic inflammation on asthma-COPD overlap syndrome is unknown. This study aimed to examine systemic inflammation in asthma-COPD overlap syndrome, and to identify associations between clinical characteristics and inflammatory mediators in asthma-COPD overlap syndrome. Methods: In 108 adults older than 55 years comprising healthy controls (n=29), asthma (n=16), COPD (n=21) and asthma-COPD overlap syndrome (n=42), serum high sensitivity C-reactive protein and Interleukin 6 (IL-6) were assayed. Spirometry, induced sputum, quality of life, comorbidities and medications were assessed, and their associations with asthma-COPD overlap syndrome were analyzed using logistic regression. Associations between systemic inflammatory mediators and clinical characteristics were tested in multivariate linear regression models. Results: Patients with asthma-COPD overlap syndrome had significantly elevated IL-6 levels compared with healthy controls and asthmatics. Age, comorbidity index and IL-6 level were independently associated with asthma-COPD overlap syndrome. FEV1% predicted was inversely associated with IL-6 level, and cardiovascular disease was associated with an increased IL-6 level. Systemic markers were not associated with airway inflammation. Conclusions: Systemic inflammation is commonly present in asthma-COPD overlap syndrome, and asthma-COPD overlap syndrome resembled COPD in terms of systemic inflammation. IL-6 is a pivotal inflammatory mediator that may be involved in airflow obstruction and cardiovascular disease and may be an independent treatment target. © Copyright The Korean Academy of Asthma, Allergy and Clinical Immunology.

Background Asthma is a complex disease that involves both genetic factors and environmental exposures. Aberrant epigenetic modifications, such as DNA methylation, may be important in asthma development. Fetal exposure to maternal asthma during critical periods of in utero development may lead to epigenetic alterations that predispose infants to a greater risk of developing asthma themselves. We investigated alterations in the DNA methylation profile of peripheral blood from infants exposed to maternal asthma during pregnancy. Methods Peripheral blood was collected from 12-month-old infants born to women with (n = 25) and without (n = 15) doctor diagnosed asthma during pregnancy. Genomic DNA was extracted, bisulfite converted, and hybridized to Infinium Methylation 27 arrays (Illumina), containing over27,000 CpGs from 14,495 genes. CpG loci in only autosomal genes were classified as differentially methylated at the 99% level (P < 0.01, |DiffScore| > 22 and delta beta >0.06). Results There were 70 CpG loci, corresponding to 67 genes that were significantly differentially methylated. Twelve CpG loci (11 genes) showed greater than 10% comparative difference in DNA methylation, including hyper-methylated loci of FAM181A, MRI1, PIWIL1, CHFR, DEFA1, MRPL28, AURKA, and hypo-methylated loci of NALP1L5, MAP8KIP3, ACAT2, and PM20D1 in maternal asthma. Methylation of MAPK8IP3 was significantly negatively correlated with maternal blood eosinophils (r = -0.38; P = 0.022), maternal eNO (r = -0.44; P = 0.005), and maternal serum total IgE (r = -0.39, P = 0.015). Methylation of AURKA negatively correlated with maternal hemoglobin (r = -0.43; P = 0.008), infants height (r = -0.51; P < 0.001) and weight (r = -0.36; P = 0.021). Methylation of PM20D1 was lower in infants born to mothers with asthma on inhaled corticosteroid treatment. Methylation of PM20D1 was lower and MRI1 was higher in infants born to atopic mothers without asthma. Conclusions In an Australian study population, exposure to maternal asthma during pregnancy is associated with differential methylation profiles of infants' peripheral blood DNA, which may act as risk factors for future asthma development. © 2013 Wiley Periodicals, Inc.

Objectives: Outcome assessment is an important part of the management of airways disease, yet older adults may have difficulty with the burden of testing. This study evaluated the patient perception of tests used for the assessment of airways disease in older people. Data Source: Older adults (>55 years) with obstructive airway disease and healthy controls (N=56) underwent inhaler technique assessment, skin allergy testing, venepuncture, fractional exhaled nitric oxide (FENO) and gas diffusion measurement, exercise testing, sputum induction, and questionnaire assessment. They then completed an assessment burden questionnaire across five domains: difficulty, discomfort, pain, symptoms and test duration. Results: Test perception was generally favourable. Induced sputum had the greatest test burden perceived as being more difficult (mean 0.83, P=0.001), associated with more discomfort (mean 1.3, P<0.001), more painful (0.46, P=0.019), longer test duration (0.84, P<0.001) and worsening symptoms (0.55, P=0.001) than the questionnaires. FENO had a more favourable assessment but was assessed to be difficult to perform. Inhaler technique received the most favourable assessment. Conclusions: Older adults hold favourable perceptions to a range of tests that they might encounter in the course of their care for airway disease. The newer tests of sputum induction and FENO have some observed difficulties, in particular sputum induction. The results of this study can inform current practice by including details of the test and its associated adverse effects when conducting the test, as well as providing clear explanations of the utility of tests and how the results might aid in patient care. © 2013 John Wiley & Sons Ltd.

Background: COPD and asthma in older people are complex conditions associated with multiple clinical problems. The relative importance of these problems to both patients and physicians and the level of agreement between them is largely unknown. Methods: Older people with asthma and COPD underwent a multidimensional assessment to characterise the prevalence of clinical problems. Each individual's problems were then summarised and presented separately to the patient and physician to rate problem importance. Problems were scored using a 5-point Likert scale from unimportant to very important. Results: The highest-rated problems were dyspnoea, activity limitation and airway inflammation, and these areas had good patientphysician concordance. Poor concordance was found for inhaler technique adequacy, airflow obstruction and obesity. Good concordance was found for written action plans, but this was less important to both patients and physicians. Conclusions: In asthma and COPD, patients and their physicians agree about the importance of managing activity limitation, dyspnoea, and airway inflammation. Other areas of management had little concordance or were viewed as less important. Self-management skills were not rated as important by patients and this may hinder successful management. Eliciting problems and addressing their importance to treatment goals may improve care in COPD and asthma. © 2011 Primary Care Respiratory Society UK. All rights reserved.