Integrative Proteomics and Phosphoproteomics of Asthmatic Airways Following RV Infection

Rhinovirus (RV) infection is associated with 60-80% of childhood asthma exacerbations in the emergency department. Global proteomics and phosphoproteomics were performed using human precision cut lung slice (PCLS) airways from deceased asthma and non-asthma donors with and without infection with RV in order to confirm previously identified dysregulated pathways and identify new areas of investigation. PCLS were prepared from 5 asthma and 4 non-asthma donor lungs for phosphoproteomic analyses. PCLS were infected with RV39 or treated with vehicle for 48 hours. Carbachol induced airway bronchoconstriction was measured pre-/post-infection. PCLS were collected and airways were microdissected for LC-MS/MS. Proteins and enriched phosphopeptides from each sample were labeled with TMT10plex isobaric tags and peptides were identified on an Orbitrap Eclipse Tribrid mass spectrometer. Asthma donors showed airway hyper-responsiveness to carbachol after RV infection (Control uninfected -0.6207, infected -0.4877; p=NS; Asthma uninfected -0.7113, infected 1.099; p<0.001). Global comparison between asthma and non-asthma airways identified 412 proteins and 1049 phosphopeptides differentially expressed using a p-value < 0.05 and an absolute fold change > 2 threshold. Comparison of asthma + RV to non-asthma + RV identified 381 significant proteins and 763 phosphopeptides. Pathologic pathways were identified with integrative bioinformatics and human protein-protein interactome network analyses. Dysregulated pathways in asthma infected compared to non-asthma infected involved inflammatory responses, TNF-a signaling, Interferon-g response, and IL-6/STAT3. Asthma donor airways were hyper-responsive after infection with RV, and this finding was associated with dysregulated pathways in the inflammatory response compared to similar non-asthma donors.