Protein phosphatase 2A mediates influenza-induced inhibition of lung alveolar defense

/ABSTRACT Influenza A virus (IAV) infects over 30 million people annually throughout the United States. Those with severe IAV infection often develop secondary bacterial coinfection by Staphylococcus aureus (SA), leading to acute lung injury (ALI) with high mortality. Though antiviral and antibiotic therapies are available, increasing rates of resistance among IAV and SA has rendered these therapies increasingly ineffective. Thus, new understanding of how IAV promotes secondary SA infection is needed to develop novel treatment approaches. Since the critical site of IAV-SA coinfection is lung alveoli, this F31 project focuses on studies that use alveolar epithelial cells and intact alveoli of live lungs to define the molecular mechanisms by which IAV inhibits alveolar defense to promote SA coinfection. Reports indicate that IAV induces both dephosphorylation and ubiquitination of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ion channel that drives alveolar wall liquid (AWL) secretion, a major alveolar lung defense. The hypothesis of this F31 project is that protein phosphatase 2A (PP2A) dephosphorylates CFTR to cause CFTR ubiquitination in IAV-exposed alveolar epithelial cells, leading to loss of AWL secretion and alveolar SA retention. Aim 1 seeks to define the role of PP2A in IAV- induced CFTR dephosphorylation and ubiquitination using murine lung epithelial-12 cells and cultured primary mouse and human alveolar type 2 (AT2) cells isolated from mouse and human lungs by flow cytometry. PP2A will be inhibited by pharmacologic and genetic approaches. The effect of PP2A inhibition on CFTR dephosphorylation and ubiquitination will be determined by immunoblot or mass spectrometry, which will also provide an opportunity to define the specific amino acid sites that are dephosphorylated and ubiquitinated in IAV infection. Aim 2 seeks to determine the effect of alveolar cell type-specific PP2A deletion on IAV-induced AWL inhibition and alveolar SA retention using transgenic mice that harbor inducible PP2A deletion in alveolar type 1 (AT1) or AT2 cells. Confocal imaging of live, intact lung alveoli will be used to define the role of PP2A in IAV- induced inhibition of AWL secretion and SA retention. Proof-of-concept experiments using wild-type mice will support the transgenic mouse experiments. The long-term goals of this F31 project are to gain new insights into IAV-SA pathogenesis in lung alveoli and, if the preliminary findings bear out, to identify PP2A as a novel therapeutic target. Importantly, this project will provide outstanding training and career development toward the establishment of an independent research career. Specific training goals include the generation of a new first- author publication, acquisition of critical technical and scientific skills, and preparation for high-quality post- doctoral research fellowship training. Project Number: 1F31HL179970-01 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: Stephanie Tang | Institution: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI, NEW YORK, NY | Award Amount: $49,538 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F10A-R (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F31HL17997001