Integrated Species-Level Analysis of Metagenome and Inflammatory Mediators Reveals Dominant and Distinct Roles of  Haemophilus Influenzae and  Moraxella Catarrhalis in Severe Asthma

Abstract

Background: Persistent bacterial airways infection may drive immune pathology in ‘type-2 low’, neutrophilic asthma. The airway microbiome in asthma has not been characterised at species level by metagenomic sequencing, nor the relationships between specific species and mucosal immune responses defined.

Methods: We analysed the metagenome of sputum and nasal lavage using long-read Nanopore sequencing and qPCR in two cross-sectional Severe Asthma Cohorts, Wessex (n=66) and Oxford (n=30). We integrated species-level data with clinical parameters and 39 selected airway proteins using immunoassays and O-link proteomics.

Findings: Health and mild asthma were characterised by high sputum microbial diversity. By contrast in 23% (19/81) of participants with severe asthma the microbiome was dominated by a single respiratory pathogen, namely H. influenzae (n=10), M. catarrhalis (n=4), S. pneumoniae (n=4) and P. aeruginosa (n=1), with higher Proteobacteria:Firmicutes ratio. Neutrophilic asthma was associated with H. influenzae, M. catarrhalis, S. pneumoniae and T. whipplei; eosinophilic asthma with more M. catarrhalis, but lower abundance of H. influenzae, and S. pneumoniae. H. influenzae was associated with airway TNF and IL-10. M. catarrhalis associated with IL-1β, tryptase and YKL40. R. mucilaginosa associated negatively with FGF. Bayesian network analysis revealed close and distinct relationships of H. influenzae and M. catarrhalis with type-1 airway inflammation and clinical parameters. The microbiomes and cytokine milieu were highly distinct between upper and lower airways.

Interpretation: This species-level integrated analysis using Nanopore sequencing, reveals central, but distinct associations between H. influenzae and M. catarrhalis in airways inflammation in severe asthma.

Funding: Supported by the Medical Research Council (G0800649) and by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC). TSCH is supported by a Wellcome Trust Fellowship (211050/Z/18/z). MFJ was supported by a National Institute for Health Research Academic Clinical Fellowship Award and by the Nuffield Department of Medicine. Infrastructure support was funded by the National Institute for Health Research (NIHR) Southampton Respiratory Biomedical Research Unit and the NIHR through the Primary Care Research Network.

Declaration of Interest: TSCH has received grants from the Wellcome Trust, grants from The Guardians of the Beit Fellowship, and grants from the NIHR Oxford Biomedical Research Centre during the conduct of the study; and grants from Pfizer Inc., grants from University of Oxford, personal fees from Astra Zeneca, personal fees from TEVA, personal fees from Peer Voice outside the submitted work. MJ has received grants from the University of Oxford and NIHR Oxford Biomedical Research Centre. IDP has received speaker’s honoraria from Astra Zeneca, Boehringer Inglehiem, Aerocrine, Almirall, Novartis, Teva, Chiesi, Sanofi/Regeneron, Menarini, and GSK, and been on advisory panels for Genentech, Sanofi/Regeneron, Astra Zeneca, Boehringer Ingelheim, GSK, Novartis, Teva, Merck, Circassia, Chiesi, and Knopp, and received grants from the NIHR Oxford Biomedical Research Centre and Chiesi during the conduct of this study. CB and AA are employees of Astra Zeneca. PHH is an employee of GSK. AC has received funding from Airsonett and been on the advisory board for Sanofi Genzyme, Novartis, XIM and Exhalation Technology. EM, NDS, MT, GD, LCKL, PK, TLS, and TB declare they have no competing interests.

Ethical Approval: Oxford Severe Asthma Cohort conducted with NHS Research Ethics approval (08/H0406/189). Wessex Severe Asthma Cohort approved by Southampton and South-West Hampshire Research Ethics Committee A (09/H0502/37).

Keywords: Asthma, bacteria, microbiome, sputum, cytokine