Species-level, metagenomic and proteomic analysis of microbe-immune interactions in severe asthma.

BACKGROUND: The airway microbiome in severe asthma has not been characterised at species-level by metagenomic sequencing, nor have the relationships between specific species and mucosal immune responses in 'type-2 low', neutrophilic asthma been defined. We performed an integrated species-level metagenomic data with inflammatory mediators to characterise prevalence of dominant potentially pathogenic organisms and host immune responses. METHODS: Sputum and nasal lavage samples were analysed using long-read metagenomic sequencing with Nanopore and qPCR in two cross-sectional adult severe asthma cohorts, Wessex (n = 66) and Oxford (n = 30). We integrated species-level data with clinical parameters and 39 selected airway proteins measured by immunoassay and O-link. RESULTS: The sputum microbiome in health and mild asthma displayed comparable microbial diversity. By contrast, 23% (19/81) of severe asthma microbiomes were dominated by a single respiratory pathogen, namely H. influenzae (n = 10), M. catarrhalis (n = 4), S. pneumoniae (n = 4) and P. aeruginosa (n = 1). Neutrophilic asthma was associated with H. influenzae, M. catarrhalis, S. pneumoniae and T. whipplei with elevated type-1 cytokines and proteases; eosinophilic asthma with higher M. catarrhalis, but lower H. influenzae, and S. pneumoniae abundance. H. influenzae load correlated with Eosinophil Cationic Protein, elastase and IL-10. R. mucilaginosa associated positively with IL-6 and negatively with FGF. Bayesian network analysis also revealed close and distinct relationships of H. influenzae and M. catarrhalis with type-1 airway inflammation. The microbiomes and cytokine milieu were distinct between upper and lower airways. CONCLUSIONS: This species-level integrated analysis reveals central, but distinct associations between potentially pathogenic bacteria and airways inflammation in severe asthma.

MAIT cells and the microbiome.

Mucosal associated invariant T (MAIT) cells are innate-like T lymphocytes, strikingly enriched at mucosal surfaces and characterized by a semi-invariant αß T cell receptor (TCR) recognizing microbial derived intermediates of riboflavin synthesis presented by the MHC-Ib molecule MR1. At barrier sites MAIT cells occupy a prime position for interaction with commensal microorganisms, comprising the microbiota. The microbiota is a rich source of riboflavin derived antigens required in early life to promote intra-thymic MAIT cell development and sustain a life-long population of tissue resident cells. A symbiotic relationship is thought to be maintained in health whereby microbes promote maturation and homeostasis, and in turn MAIT cells can engage a TCR-dependent "tissue repair" program in the presence of commensal organisms conducive to sustaining barrier function and integrity of the microbial community. MAIT cell activation can be induced in a MR1-TCR dependent manner or through MR1-TCR independent mechanisms via pro-inflammatory cytokines interleukin (IL)-12/-15/-18 and type I interferon. MAIT cells provide immunity against bacterial, fungal and viral pathogens. However, MAIT cells may have deleterious effects through insufficient or exacerbated effector activity and have been implicated in autoimmune, inflammatory and allergic conditions in which microbial dysbiosis is a shared feature. In this review we summarize the current knowledge on the role of the microbiota in the development and maintenance of circulating and tissue resident MAIT cells. We also explore how microbial dysbiosis, alongside changes in intestinal permeability and imbalance between pro- and anti-inflammatory components of the immune response are together involved in the potential pathogenicity of MAIT cells. Whilst there have been significant improvements in our understanding of how the microbiota shapes MAIT cell function, human data are relatively lacking, and it remains unknown if MAIT cells can conversely influence the composition of the microbiota. We speculate whether, in a human population, differences in microbiomes might account for the heterogeneity observed in MAIT cell frequency across mucosal sites or between individuals, and response to therapies targeting T cells. Moreover, we speculate whether manipulation of the microbiota, or harnessing MAIT cell ligands within the gut or disease-specific sites could offer novel therapeutic strategies.

Identifying Bacterial Airways Infection in Stable Severe Asthma Using Oxford Nanopore Sequencing Technologies.

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.

Pellino-1 Regulates Immune Responses to Haemophilus influenzae in Models of Inflammatory Lung Disease.

Non-typeable Haemophilus influenzae (NTHi) is a frequent cause of lower respiratory tract infection in people with chronic obstructive pulmonary disease (COPD). Pellino proteins are a family of E3 ubiquitin ligases that are critical regulators of TLR signaling and inflammation. The aim of this study was to identify a role for Pellino-1 in airway defense against NTHi in the context of COPD. Pellino-1 is rapidly upregulated by LPS and NTHi in monocyte-derived macrophages (MDMs) isolated from individuals with COPD and healthy control subjects, in a TLR4 dependent manner. C57BL/6 Peli1-/- and wild-type (WT) mice were subjected to acute (single LPS challenge) or chronic (repeated LPS and elastase challenge) airway inflammation followed by NTHi infection. Both WT and Peli1-/- mice develop airway inflammation in acute and chronic airway inflammation models. Peli1-/- animals recruit significantly more neutrophils to the airway following NTHi infection which is associated with an increase in the neutrophil chemokine, KC, in bronchoalveolar lavage fluid as well as enhanced clearance of NTHi from the lung. These data suggest that therapeutic inhibition of Pellino-1 may augment immune responses in the airway and enhance bacterial clearance in individuals with COPD.