Revealing the impact of Pseudomonas aeruginosa quorum sensing molecule 2'aminoacetophenone on the human bronchial-airway epithelium and pulmonary endothelium using a human airway-on-a-chip

Shifu Aggarwal^1,2Arijit Chakraborty^1,2,3Vijay K. Singh¹Stephen Lory²Katia Karalis⁴Laurence Rahme^1,2,3*

• ¹Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States
• ²Department of Microbiology, Harvard Medical School, Boston, United States
• ³Shriners Children's Boston, Boston, Massachusetts, United States
• ⁴Regeneron Pharmaceuticals, New York, United States

Pseudomonas aeruginosa (PA) causes severe respiratory infections utilizing multiple virulence functions. Our earlier work demonstrated that the Pseudomonas aeruginosa quorum-sensing molecule 2′-aminoacetophenone (2-AA) reprograms host immune and metabolic pathways, thereby facilitating the pathogen's long-term persistence.However, its specific impact on the bronchial airway epithelium and pulmonary endothelium remains unknown. To evaluate the spatiotemporal changes in 2-AA within the human airway, considering endothelial cells as the primary point of contact when the route of lung infection is hematogenous, we utilized the airway-on-a-chip platform lined by polarized human bronchial airway epithelium and pulmonary endothelium.Using this platform, we performed RNA sequencing to analyse the responses of 2-AAtreated primary human pulmonary microvascular endothelial cells (HPMEC) and adjacent primary normal human bronchial epithelial cells (NHBE) from healthy female donors, as well as potential cross-talk between these cells. Analyses revealed that 2-AA differentially regulates specific signaling and biosynthesis pathways in epithelial cells, including HIF-1 and pyrimidine signaling, as well as glycosaminoglycan and glycosphingolipid biosynthesis. On the other hand, in endothelial cells, fatty acid metabolism, phosphatidylinositol signaling, estrogen receptor signaling, and inflammatory signaling pathways have been identified. Significant overlap was found in both cell types in response to 2-AA in genes implicated in immune response and cellular functions. In contrast, we found that genes related to barrier permeability, cholesterol metabolism, and oxidative phosphorylation were differentially regulated in response to 2-AA exposure in the studied cell types. Murine in-vivo and additional in vitro cell culture studies confirmed cholesterol accumulation in epithelial cells. Results also revealed that specific biomarkers associated with cystic fibrosis and idiopathic pulmonary fibrosis were modulated by 2-AA in both cell types, with the expression of cystic fibrosis transmembrane regulator being affected only in endothelial cells.The effects of 2-AA on epithelial and endothelial primary cells studied within a dynamic microphysiological environment that mimics the human lung airway deepen our understanding of this quorum sensing (QS) signaling molecule. This study offers novel insights into the functions and interactions of 2-AA, paving the way for innovative, cellspecific therapeutic strategies to combat Pseudomonas aeruginosa lung infections.