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BACKGROUND: Mepolizumab is an anti-interleukin-5 monoclonal antibody treatment for severe eosinophilic asthma (SEA) that reduces asthma exacerbations. Residual airway inflammation on mepolizumab may lead to persistent exacerbations. Oral corticosteroids remain the main treatment for these residual exacerbations. OBJECTIVE: Our study aimed to explore the corticosteroid-responsiveness of airway inflammation after mepolizumab treatment to find potentially treatable inflammatory mechanisms beyond the IL-5 pathway. METHOD: The MAPLE trial was a multi-centre, randomized, double-blind, placebo-controlled, crossover study of 2 weeks of high-dose oral prednisolone treatment at stable state in 27 patients treated with mepolizumab for SEA. We analysed paired sputum (n=16) and plasma (n=25) samples from the MAPLE trial using high-throughput Olink® proteomics. We also analysed additional sputum proteins using ELISA. RESULTS: In patients receiving mepolizumab, prednisolone significantly downregulated sputum proteins related to type-2 inflammation and chemotaxis including IL-4, IL-5, IL-13, CCL24, CCL26, EDN, CCL17, CCL22, OX40 receptor, FCER2, and the ST2 receptor. Prednisolone also downregulated cell adhesion molecules, prostaglandin synthases, mast cell tryptases, MMP1, MMP12, and neuroimmune mediators. Neutrophilic pathways were upregulated. Type-2 proteins were also downregulated in plasma, combined with IL-12, IFN-γ, and IP-10. IL-10 and amphiregulin were upregulated. CONCLUSION: At stable state, prednisolone has broad anti-inflammatory effects on top of mepolizumab. These effects are heterogeneous and may be clinically relevant in residual exacerbations.
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BACKGROUND: In T2-mediated severe asthma, biologic therapies, such as mepolizumab, are increasingly used to control disease. Current biomarkers can indicate adequate suppression of T2 inflammation, but it is unclear whether they provide information about airway microbial composition. We investigated the relationships between current T2 biomarkers and microbial profiles, characteristics associated with a ProteobacteriaHIGH microbial profile and the effects of mepolizumab on airway ecology. METHODS: Microbiota sequencing was performed on sputum samples obtained at stable and exacerbation state from 140 subjects with severe asthma participating in two clinical trials. Inflammatory subgroups were compared on the basis of biomarkers, including FeNO and sputum and blood eosinophils. ProteobacteriaHIGH subjects were identified by Proteobacteria to Firmicutes ratio ≥0.485. Where paired sputum from stable visits was available, we compared microbial composition at baseline and following ≥12 weeks of mepolizumab. RESULTS: Microbial composition was not related to inflammatory subgroup based on sputum or blood eosinophils. FeNO ≥50 ppb when stable and at exacerbation indicated a group with less dispersed microbial profiles characterised by high alpha-diversity and low Proteobacteria. ProteobacteriaHIGH subjects were neutrophilic and had a longer time from asthma diagnosis than ProteobacteriaLOW subjects. In those studied, mepolizumab did not alter airway bacterial load or lead to increased Proteobacteria. CONCLUSION: High FeNO could indicate a subgroup of severe asthma less likely to benefit from antimicrobial strategies at exacerbation or in the context of poor control. Where FeNO is <50 ppb, biomarkers of microbial composition are required to identify those likely to respond to microbiome-directed strategies. We found no evidence that mepolizumab alters airway microbial composition.
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Asma , Humanos , Asma/diagnóstico , Asma/tratamento farmacológico , Asma/microbiologia , Eosinófilos , Escarro/microbiologia , Sistema Respiratório/microbiologia , BiomarcadoresRESUMO
To explore whether fractional exhaled nitric oxide (FeNO) non-suppression identifies corticosteroid resistance, we analysed inflammatory mediator changes during a FeNO suppression test with monitored high-intensity corticosteroid therapy. In linear mixed-effects models analysed over time, the 15 clinically distinct 'suppressors' (ie, ≥42% FeNO suppression) normalised Asthma Control Questionnaire scores (mean±SD, start to end of test: 2.8±1.4 to 1.4±0.9, p<0.0001) and sputum eosinophil counts (median (IQR), start to end of test: 29% (6%-41%) to 1% (1%-5%), p=0.0003) while significantly decreasing sputum prostaglandin D2 (254 (89-894) to 93 (49-209) pg/mL, p=0.004) and numerically decreasing other type-2 cytokine, chemokine and alarmin levels. In comparison, the 19 non-suppressors had persistent sputum eosinophilia (10% (1%-67%) despite high-intensity therapy) with raised end-test inflammatory mediator levels (1.9 (0.9-2.8)-fold greater than suppressors). FeNO non-suppression during monitored treatment implies biological corticosteroid resistance.
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Previous metagenomic studies in asthma have been limited by inadequate sequencing depth for species-level bacterial identification and by heterogeneity in clinical phenotyping. We hypothesize that chronic bacterial airways infection is a key "treatable trait" whose prevalence, clinical phenotype and reliable biomarkers need definition. In this study, we have applied a method for Oxford Nanopore sequencing for the unbiased metagenomic characterization of severe asthma. We optimized methods to compare performance of Illumina MiSeq, Nanopore sequencing, and RT-qPCR on total sputum DNA extracts against culture/MALDI-TOF for analysis of induced sputum samples from highly phenotyped severe asthma during clinical stability. In participants with severe asthma (n = 23) H. influenzae was commonly cultured (n = 8) and identified as the dominant bacterial species by metagenomic sequencing using an optimized method for Illumina MiSeq and Oxford Nanopore. Alongside superior operational characteristics, Oxford Nanopore achieved near complete genome coverage of H. influenzae and demonstrated a high level of agreement with Illumina MiSeq data. Clinically significant infection was confirmed with validated H. influenzae plasmid-based quantitative PCR assay. H. influenzae positive patients were found to have sputum neutrophilia and lower FeNO. In conclusion, using an optimized method of direct sequencing of induced sputum samples, H. influenzae was identified as a clinically relevant pathogen in severe asthma and was identified reliably using metagenomic sequencing. Application of these protocols in ongoing analysis of large patient cohorts will allow full characterization of this clinical phenotype. IMPORTANCE The human airways were once thought sterile in health. Now metagenomic techniques suggest bacteria may be present, but their role in asthma is not understood. Traditional culture lacks sensitivity and current sequencing techniques are limited by operational problems and limited ability to identify pathogens at species level. We optimized a new sequencing technique-Oxford Nanopore technologies (ONT)-for use on human sputum samples and compared it with existing methods. We found ONT was effective for rapidly analyzing samples and could identify bacteria at the species level. We used this to show Haemophilus influenzae was a dominant bacterium in the airways in people with severe asthma. The presence of Haemophilus was associated with a "neutrophilic" form of asthma - a subgroup for which we currently lack specific treatments. Therefore, this technique could be used to target chronic antibiotic therapy and in research to characterize the full breadth of bacteria in the airways.
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Asma , Infecções Bacterianas , Sequenciamento por Nanoporos , Bactérias/genética , Infecções Bacterianas/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Metagenômica/métodos , Sistema RespiratórioRESUMO
AIM: To update obstetric care providers about asthma management. SUMMARY: Asthma is the most frequent comorbid chronic illness in pregnancy. Convincing evidence shows that uncontrolled asthma magnifies the risk of maternal, fetal and neonate complications. Unfortunately, one in four women take no inhaler during pregnancy, and it is likely that decreased adherence, rather than changes in pathology, explains uncontrolled maternal asthma. Patient surveys reveal a need for information and reassurance. Although some molecules are preferred in pregnancy, there is currently no basis to withhold any asthma medication - old or new. Biomarkers such as blood eosinophils and fractional exhaled nitric oxide are an effective way to assess the risk of asthma attacks and the likelihood of responding to inhaled steroids. Furthermore, practice-changing trials in mild asthma show that switching reliever-only regimens to as-needed 'controller-and-reliever' therapy is effective. We suggest that applying these changes can alleviate women's concerns and improve outcomes.
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BACKGROUND: Clinical trials with mepolizumab, a humanised monoclonal antibody against interleukin-5, show a 50% reduction in severe asthma exacerbations in people with severe eosinophilic asthma. Exacerbations in patients treated with mepolizumab seem to be different to exacerbations in those given placebo, as patients treated with mepolizumab report fewer symptoms, have a lower sputum eosinophil count, and smaller fall in peak expiratory flow. We aimed to investigate the inflammatory phenotype and physiological characteristics of exacerbation events in patients with severe eosinophilic asthma who were treated with mepolizumab. METHODS: This multicentre, prospective, observational cohort study was carried out at four UK specialist severe asthma centres. Participants were aged 18-80 years, with severe eosinophilic asthma (Global Initiative for Asthma steps 4 and 5), and were eligible for mepolizumab therapy. All participants received mepolizumab 100 mg subcutaneously every 4 weeks, had a scheduled study visit when stable on mepolizumab (≥3 months on treatment), and measured daily peak flow and completed symptoms diaries throughout the course of the study. Participants attended their study centre for unscheduled exacerbation assessment when symptoms worsened outside of their normal daily variation and before commencing rescue treatment. If a participant was unable to attend their study centre for exacerbation or had initiated rescue treatment before the study visit, clinical details of the missed exacerbation were collected by clinical staff. In this exploratory study, the endpoint was 100 clinical assessments at exacerbation completed across all sites for participants on mepolizumab before initiation of rescue treatment. Characteristics of those who had exacerbations on mepolizumab were compared with those who did not, peak flow and symptoms diaries were compared for assessed versus missed exacerbations, and exacerbation phenotypes defined by sputum eosinophil cell count were compared. The utility of fractional exhaled nitric oxide (FeNO) and C-reactive protein in determining exacerbation phenotype on mepolizumab treatment were also assessed. This study is registered with ClinicalTrials.gov, NCT03324230. FINDINGS: Between Nov 30, 2017, and May 29, 2019, 145 participants were enrolled and treated with mepolizumab, five were excluded from the analysis. 172 exacerbations occurred, with 96 (56%) assessed before commencing rescue treatment. Compared with patients who did not exacerbate, patients who exacerbated had a higher exacerbation rate and more emergency department attendances in the year before commencing mepolizumab. The change in peak expiratory flow at nadir in the assessed exacerbation group was mean -40·5 L/min (SD 76·3) versus mean -37·0 L/min (93·0; p=0·84) in the missed exacerbation group, and there was no difference in reported symptom burden. When comparing exacerbations with a high sputum eosinophil count (≥2%; SEHIGH) with exacerbations with a low sputum eosinophil count (<2%; SELOW), the SEHIGH exacerbations were FeNO high (median difference 33 parts per billion [ppb; 95% CI 8 to 87]; p=0·0004), with lower FEV1 percent predicted (mean difference -15·9% [-27·0 to -4·8]; p=0·0075), lower FEV1 to forced vital capacity ratio (mean difference -10·3 [-17·0 to -3·6]; p=0·0043), and higher blood eosinophil counts (median difference 40 cells per µL [20 to 70]; p=0·0009). By contrast, SELOW exacerbations had higher C-reactive protein concentrations (median difference 12·7 mg/L [3·5 to 18·5]; p<0·0001), higher sputum neutrophil counts (median difference 52·7% [34·5 to 59·2]; p<0·0001), and were more likely to be treated with antibiotics (p=0·031). FeNO (≤20 or ≥50 ppb) was the most useful discriminator of inflammatory phenotype at exacerbation. The most common adverse event was hospital admission due to asthma exacerbation (17 [50%] of 34 events), none of the adverse events were study procedure related. INTERPRETATION: Exacerbations on mepolizumab are two distinct entities, which can largely be differentiated using FeNO: non-eosinophilic events are driven by infection with a low FeNO and high C-reactive protein concentration, whereas eosinophilic exacerbations are FeNO high. The results of the MEX study challenge the routine use of oral corticosteroids for the treatment of all asthma exacerbation events on mepolizumab, as well as the switching of biological therapies for treatment failure without profiling the inflammatory phenotype of ongoing asthma exacerbations. The results highlight clinically available tools to enable profiling of these residual exacerbations in patients treated with mepolizumab. FUNDING: UK Medical Research council.
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Antiasmáticos , Asma , Eosinofilia Pulmonar , Anticorpos Monoclonais Humanizados , Asma/induzido quimicamente , Asma/tratamento farmacológico , Eosinófilos , Humanos , Estudos Prospectivos , Eosinofilia Pulmonar/tratamento farmacológicoAssuntos
Antiasmáticos/efeitos adversos , Antiasmáticos/uso terapêutico , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/uso terapêutico , Asma/tratamento farmacológico , Eosinófilos/efeitos dos fármacos , Prednisolona/efeitos adversos , Idoso , Análise Química do Sangue , Feminino , Glucocorticoides/efeitos adversos , Glucocorticoides/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Prednisolona/uso terapêuticoAssuntos
Elastase de Leucócito/metabolismo , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imunoensaio , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Testes de Função Respiratória , Sensibilidade e Especificidade , Escarro/metabolismoRESUMO
This article was originally published under standard licence, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the paper have been modified accordingly.
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Human type-2 CD8+ T cells are a cell population with potentially important roles in allergic disease. We investigated this in the context of severe asthma with persistent airway eosinophilia-a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways of the same group of patients. In vitro PGD2 and LTE4 function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines, which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.
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Asma/tratamento farmacológico , Linfócitos T CD8-Positivos/efeitos dos fármacos , Lipídeos/farmacologia , Eosinofilia Pulmonar/tratamento farmacológico , Células A549 , Asma/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Quimiocinas/imunologia , Citocinas/imunologia , Humanos , Hipersensibilidade/tratamento farmacológico , Inflamação/tratamento farmacológico , Leucotrieno E4/imunologia , Contagem de Linfócitos/métodos , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Prostaglandina D2/imunologia , Eosinofilia Pulmonar/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologiaRESUMO
Near-patient testing (NPT) allows clinical decisions to be made in a rapid and convenient manner and is often cost effective. In COPD the peripheral blood eosinophil count has been demonstrated to have utility in providing prognostic information and predicting response to treatment during an acute exacerbation. For this potential to be achieved having a reliable NPT of blood eosinophil count would be extremely useful. Therefore, we investigated the use of the HemoCue® WBC Diff System and evaluated its sensitivity and specificity in healthy, asthmatic and COPD subjects. This method requires a simple skin prick of blood and was compared to standard venepuncture laboratory analysis. The HemoCue® WBC Diff System measured the peripheral blood eosinophil count in healthy, asthma and COPD subjects with very close correlation to the eosinophil count as measured by standard venepuncture. The correlations were unaffected by disease status. This method for the measurement of the peripheral blood eosinophil count has the potential to provide rapid near-patient results and thus influence the speed of management decisions in the treatment of airway diseases.