Systematic Analysis of Cell-Type Differences in the Epithelial Secretome Reveals Insights into the Pathogenesis of Respiratory Syncytial Virus–Induced Lower Respiratory Tract Infections

Abstract

Lower respiratory tract infections (LRTIs) from Respiratory Syncytial Virus (RSV) are due, in part, to secreted signals from lower airway cells that modify immune response and trigger airway remodeling. To understand this process, we applied an unbiased quantitative proteomics analysis of the RSV-induced epithelial secretory response in cells representative of the trachea (hBECs) vs small airway bronchiolar cells (hSAECs). A workflow was established using telomerase- immortalized human epithelial cells that revealed highly reproducible cell type-specific differences in both secreted proteins and nanoparticles (exosomes). Approximately one-third of secretome proteins are exosomal, with the remainder from lysosomal and vacuolar compartments. We applied this workflow to three independently derived primary human cultures from trachea (phBECs) vs bronchioles (phSAECs). 577 differentially expressed proteins from control supernatants and 966 differentially expressed proteins from RSV-infected cell supernatants were identified at a 1% false discovery rate (FDR). Fifteen proteins unique to RSV-infected phBECs were regulated by epithelial-specific ets homology factor (EHF). 106 proteins unique to RSV-infected hSAECs were regulated by the transcription factor NFκB. In this latter group, we validated the differential expression of Chemokine (C-C Motif) Ligand 20 (CCL20)/macrophage-inducible protein (MIP)3α, thymic stromal lymphopoietin (TSLP) and chemokine (CC) ligand 3-like 1(CCL3-L1) because of their roles in Th2 polarization. CCL20/MIP3α was the most active mucin-inducing factor in the RSV-infected hSAEC secretome, and was differentially expressed in smaller airways in a mouse model of RSV infection. These studies provide insights into the complexity of innate responses, and regional differences in epithelial secretome participating in RSV LRTI-induced airway remodeling.

BRD4 Couples NF-κB/RelA with Airway Inflammation and the IRF-RIG-I Amplification Loop in Respiratory Syncytial Virus Infection

“…2G). We also examined the presence of RSV virions in cellular supernatants using selective SRM-MS assays developed by us previously (29). All RSV proteins were detected in the supernatant in patterns identical to that shown for RSV N protein, where cells treated with JQ1 had slightly higher levels of RSV proteins (Fig.…”

“…hSAECs are immortalized with human telomerase and CDK4 expression; these maintain a stable epithelial morphology, express differentiated cytokeratin (CK) isoforms, form pseudostratified columnar epithelial structures, undergo cell cycle arrest, and transition into mesenchymal cells like primary bronchiolar cells (26)(27)(28). Moreover, hSAECs have similar genomic signatures and exhibit similar RSV-induced innate responses as those of primary SAECs (22,27,29). Upon RSV adsorption, the initiation of RSV transcription triggers the innate response.…”

Selective Antagonists of the Bronchiolar Epithelial NF-κB-Bromodomain-Containing Protein 4 Pathway in Viral-Induced Airway Inflammation

“…This model used telomerase-immortalized human small airway epithelial cells (hSAECs), differentiated epithelial cells that exhibit similar innate responses as primary SAECs whose stable phenotype enables genomic manipulation. hSAECs permit study of signalling pathways relevant to those in vivo (Tian et al, 2017; Zhao et al, 2017). To establish stable RelA-deficient hSAECs, we used CRISPR/Cas9 genome editing to introduce a frameshift mutation in Exon 4 of RelA (Figure 1A), resulting in a complete absence of the 65 kDa RelA by Western immunoblot (Figure 1B).…”

“…Once explanation may be provided by our systems-level proteomics studies that have shown cell-type differences in innate cytokine production in response to RSV infection. Here, we observed that, although a significant amount of proteins were shared between tracheal and bronchiolar cells, the bronchiolar cells selectively secreted TSLP, CCL20 and IL6, proteins that promote Th2 and mucin expression important in the pathogenesis of RSV-induced lower respiratory tract infections (Zhao et al, 2017). These subtle differences in chemokine expression patterns by different epithelial cell types are therefore immunologically significant.…”

“…Airway epithelial cells dynamically respond to viral patterns in a cell-type specific pattern by synthesizing and secreting over 400 proteins including CXC and CC-type chemokines, type I and III IFNs, and IFN stimulated genes (ISGs) (Zhao et al, 2017). Similarly, resident alveolar macrophages synthesize cytokine mediators that play roles in initial anti-viral defense and inflammation (Kolli et al, 2014).…”