T3 Single cell analysis reveals mechanisms of azithromycin and regulation of mucosal immunity in severe asthma

Introduction Azithromycin reduces exacerbations in severe asthma,1 particularly if Haemophilus influenzae is present in the airways. Though this suggests antibacterial effects predominate, other antibiotics are not effective, implying other mechanisms including azithromycin’s additional antiviral and immunomodulatory functions may be important, whilst antimicrobial resistance is a significant concern. Our metagenomic studies showed airway dominance by specific potentially pathogenic organisms as a common ‘treatable trait’ in severe asthma.2 We sought to determine the azithromycin mechanisms of action to enable better targeting azithromycin therapy, or non-antibacterial macrolide compounds in airways disease. Objective Characterise changes in airway mucosal immunology and microbiology using bronchoscopy before and after therapeutic intervention with azithromycin. Methods Patients with severe asthma despite optimised inhaled therapy underwent detailed clinical phenotyping and bronchoscopy before and 3 months post-initiation of clinically-indicated azithromycin (250 mg, three times weekly). Endobronchial samples and nasal brushings underwent single cell immune-profiling (10X genomics). Bronchoalveolar lavage (BAL) and nasal samples were stored for metagenomic sequencing with Nanopore and immunoassays. Results Eleven patients (mean age±SD, 51.8±16.8 years; 63% male) with severe asthma and high symptom burden (mean ACQ-5±SD, 2.9±1.1) underwent pre-/post-azithromycin bronchoscopy. H. influenzae was isolated in 36% on BAL culture pre-azithromycin. No pathogenic organisms were isolated post-azithromycin therapy. Analyses performed on 55,962 airways cells (n=6) revealed significant downregulation of gene ontology categories related to innate immune defence responses and cytokine mediated signalling pathways, with upregulation of genes encoding extracellular matrix components (p adj<1x10-7) post-azithromycin therapy (table 1). This corresponded to reduced expression of CXCL10 and S100A8, with upregulation of CST1 and mast cell CPA3. CD8 T cells underwent the greatest transcriptional reprogramming, downregulating heat shock proteins and pathways implicated in T cell activation and cytokine responses. Conclusions In addition to known antimicrobial effects, single cell transcriptomics reveals azithromycin suppresses Th1 and neutrophilic airways inflammation, modulates T cell functions and upregulates corticosteroid responsiveness markers (CST1, CPA3). In ongoing work, transcriptomic data from the full study population (n=11) will be analysed and integrated with single cell T cell repertoires, comparing upper and lower airway responses, and integrated with airway metagenomics. References Gibson, et al. Lancet. 2017 Aug 12;390(10095):659–668. http://dx.doi.org/10.2139/ssrn.4473145