PB1-F2 mediates hypoxemia in influenza - Alveolar type II (ATII) cells are the primary site for influenza A virus (IAV) replication in the distal lung and central players in the pathogenesis of IAV-induced ARDS. ATII cells also regulate alveolar lining fluid depth by alveolar fluid clearance (AFC). Prior studies show infection with H1N1 IAV A/WSN/33 (WSN) causes ARDS in wild-type (WT) C57BL/6J mice by 2 days post-inoculation (dpi). WSN-induced ARDS in WT mice is accompanied by a significant reduction in AFC rate, decreased ATII cell mitochondrial (mt) oxidative phosphorylation (OXPHOS), and a switch to aerobic glycolysis as a primary means of ATP synthesis. Post-infection treatment with the liponucleotide cytidine 5’-diphosphocholine (CDP-choline) improves gas exchange, restores normal AFC, and reduces pulmonary inflammation, without altering WSN replication or improving surfactant function. CDP-choline also prevents WSN-induced ATII cell mt depolarization and restores OXPHOS but does not prevent the glycolytic shift. Pilot studies using inducible, ATII cell-specific Transcription Factor A, Mitochondrial (TFAM)-KO mice show that the beneficial effects of CDP-choline treatment on WSN-induced hypoxemia and AFC impairment are ATII cell mt-dependent, but its anti-inflammatory effects are not. This implies that induction of hypoxemia by WSN is directly related to its effects on ATII cell OXPHOS and results from impaired AFC rather than pulmonary inflammation or surfactant dysfunction. In addition, infection of mice with H1N1 IAV strains that lack functional PB1-F2 had no detrimental effects on oxygenation in vivo or ATII cell OXPHOS ex vivo, showing WSN effects on both are PB1-F2-dependent. Hence, it is hypothesized that the PB1-F2 protein of IAV interacts with MAVS to depolarize mt which inhibits ATII cell OXPHOS, thereby reducing mt ATP generation. This causes an energy crisis in ATII cells, which impairs AFC and results in hypoxemia that promotes progression to ARDS. This hypothesis will be tested in 2 independent but complementary Specific Aims. In Aim 1, WT mice will be infected with PB1-F2-expressing and PB1-F2-negative H1N1 IAV strains to define their effects on oxygenation, AFC rate, lung mechanics, pulmonary edema, and pulmonary inflammation in vivo, and on ATII cell mt function ex vivo. MAVS-KO and WT/MAVS-KO chimeric mice will then be infected with WSN to show IAV effects on the above parameters are non-myeloid MAVS-dependent. Proximity ligation assays will be used to confirm the interaction between MAVS and PB1-F2 in ATII cells. Aim 2 will use human precision-cut lung slices to validate mouse data and to define the effects of IAV and its PB1-F2 protein on the human ATII cell secretome, energetics, gene expression, and ultrastructure. Proposed studies will use innovative experimental tools to provide additional evidence (and a potential mechanism) for the finding that PB1-F2 is an important virulence determinant of IAVs and may show that new IAV strains should be assessed for their ability to inhibit OXPHOS. They will also demonstrate a causal link between AFC impairment (secondary to PB1-F2/MAVS-mediated ATII cell mt dysfunction) and development of hypoxemia in influenza, which has broader relevance to other forms of ARDS.
