APPLICATION OF MODERN MOLECULAR MICROBIOLOGICAL TECHNIQUES TO IDENTIFY TREATABLE CHRONIC BACTERIAL AIRWAY INFECTION IN SEVERE ASTHMA

Background Haemophilus influenzae is emerging as the commonest pathogenic microorganism isolated from severe asthmatic airways; it is associated with sputum neutrophilia and may confer steroid resistance. Previous metagenomics studies in asthma were limited by lack of consistent clinical phenotyping and inadequate sequencing depth for species-level bacterial identification. We hypothesise that chronic bacterial infection constitutes a ‘treatable trait’ in non-eosinophilic severe asthma but its prevalence, clinical phenotype and reliable biomarkers need to be defined. Non-typeable Haemophilus influenzae strains (NTHi) can persist within the epithelium and cause episodic airways infections. Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes which protect against pulmonary bacterial infection, and respond to NTHi in the presence of professional antigen presenting cells (APCs) via MR1, a molecule expressed ubiquitously in many cell types including epithelial cells. It is not known whether NTHi-infected epithelial cells directly activate MAIT cells. Aims (1) Identify and characterise the sub-phenotype of severe asthmatics with chronic bacterial airways infection using modern molecular microbiological techniques (2) Determine whether MAIT cells directly respond to intra-epithelial infection with NTHi Methods Analysis of induced sputum samples from stable well-phenotyped severe asthmatics using culture, MALDI-TOF, RT-qPCR and metagenomic sequencing of total DNA extracts using the MiSeq platform. In vitro co-culture of MAIT cells with NTHi-infected bronchial epithelial cell lines (BEAS2B) or primary human airway epithelial cells. MAIT cell activation measured using intracellular cytokine staining. Results In a cohort of patients with severe asthma (n=23) H. influenzae was commonly cultured and subsequently identified as the dominant bacterial species by metagenomic sequencing (n=8, Median% Total bacterial reads=87.8%). Clinically significant infection was confirmed using a validated H. influenzae plasmid-based RT-qPCR assay. H. influenzae culture positive patients had sputum neutrophilia and lower FeNO. NTHi induces modest MAIT cell production of IFN-gamma which is partly MR1 dependent. Conclusions H. influenzae is a clinically-relevant pathogen in severe asthma that can be identified reliably using molecular microbiological methods. Ongoing analysis of a larger patient cohort will allow full characterisation of this clinical phenotype. MAIT cells are able to recognise airway epithelial cells infected with viable intracellular NTHi in the absence of APCs.