Revealing the impact of Pseudomonas aeruginosa quorum sensing molecule 2'-aminoacetophenone on human bronchial-airway epithelium and pulmonary endothelium using a human-airway-on-a-chip
Pseudomonas aeruginosa (PA) causes severe respiratory infections utilizing multiple virulence functions. Our previous findings on PA quorum sensing (QS)-regulated small molecule, 2'-aminoacetophenone (2-AA), secreted in host infected tissues, revealed its effect on immune and metabolic functions favoring a long-term presence of PA in host tissues. However, studies on 2-AA's effect on bronchial-airway epithelium and pulmonary endothelium remain elusive. To evaluate 2AA's spatiotemporal changes in the human airway, considering endothelial cells as the first point of contact when the route of lung infection is through the bloodstream, we utilized the microfluidic bronchial-airway-on-a-chip lined by polarized human bronchial-airway epithelium and pulmonary endothelium. Using this platform, we performed RNA-sequencing to analyse responses of 2-AA treated primary human pulmonary microvascular endothelium (HPMEC) and adjacent primary normal human bronchial epithelial (NHBE) cells from healthy female donors and potential cross-talk between these cells. Analyses unveiled specific signaling and biosynthetic pathways to be differentially regulated by 2-AA in epithelial cells, including HIF-1 and pyrimidine signaling, glycosaminoglycan, and glycosphingolipid biosynthesis, while in endothelial cells were fatty acid metabolism, phosphatidylinositol and estrogen receptor signaling, and the proinflammatory signaling pathways.  As part of the significant overlap in gene regulation and signaling pathways in both cell types impacted by 2-AA were genes implicated in immune response and cellular functionality. Moreover, we found cellular responses differentially regulated in each cell type, affecting barrier permeability, cholesterol metabolism, and oxidative phosphorylation.  These 2-AA-mediated distinct responses may result from the cross-talk between these cells. Murine in-vivo and additional in vitro cell culture studies confirmed cholesterol accumulation and its effect on lung physiology by 2-AA. Results also revealed specific biomarkers associated with cystic fibrosis and idiopathic pulmonary fibrosis to be modulated by 2-AA in both cell types, with the cystic fibrosis transmembrane regulator expression to be affected only in endothelial cells. The 2-AA-mediated effects on epithelial and endothelial cells within a microphysiological dynamic environment that mimics the human lung airway enhance our understanding of the influence of this QS signaling molecule in HPMEC  and NHBE cells. This study provides novel insights into their functions and potential interactions, paving the way for innovative therapeutic strategies to combat PA lung infections. Overall design: Using the microfluidic human airway-on-a-chip, RNA seq profiling was performed of untreated and 2-AA (20 µM) treated HPMEC (human pulmonary microvascular endothelial cells) and NHBE (normal human bronchial epithelial) cells with continuous flow of