View Abstract

A3843 - Acutely Lethal Influenza Infection Alters Murine Alveolar Epithelial Cell Energy Production
Author Block: L. M. Doolittle1, D. Guttridge2, I. C. Davis1; 1Veterinary Biosciences, The Ohio State University, Columbus, OH, United States, 2Cancer Biology and Genetics, The Ohio State University, Columbus, OH, United States.
RATIONALE: Alveolar type II (ATII) epithelial cells promote normal lung function and facilitate respiration via a number of energetically expensive processes unique to this cell type, including pulmonary surfactant synthesis and secretion and vectorial ion transport. ATII cells are also the primary site of influenza A virus (IAV) replication within the distal lung. Mitochondrial membranes are predominantly composed of phospholipids. Correct membrane structure is crucial for mitochondrial function, including ATP production by the electron transport chain (ETC). We showed that IAV infection reduces the levels of major mitochondrial membrane phospholipids in ATII cells. We therefore hypothesized that IAV infection would alter ATII cell energy production by compromising ATII cell mitochondrial membrane integrity.
METHODS: C57BL/6 mice were inoculated intranasally with 10,000 p.f.u./mouse influenza A/WSN/33 (H1N1), which induces ARDS in mice by 6 days post-inoculation (d.p.i.). Controls were mock-infected with virus diluent. ATII cells were isolated by a standard lung digestion protocol at 6 d.p.i. Mitochondrial respiration rates were measured in live ATII cells using a Seahorse XFe Analyzer with the Mito Stress Test kit. ATII cell protein expression was quantified by Western blotting, and gene expression was measured by qRT-PCR. Mitochondrial membrane potential and transition pore permeability were measured by flow cytometry, using DiLC1(5) and calcein dyes.
RESULTS: At 6 d.p.i., ATII cells from infected mice had reduced mitochondrial membrane potential. However, mitochondrial transition pore permeability did not change between groups. Relative to mock-infected mice, mitochondrial respiration rates were decreased in ATII cells at 6 d.p.i. Additionally, inhibition of ATP synthase by oligomycin indicated the rate of mitochondrial ATP production was reduced in ATII cells at 6 d.p.i. ATII cells from infected mice showed altered gene expression of several ETC enzymes compared to controls, including subunits of ATPase (Complex V) and NADH dehydrogenase (Complex I). Protein expression of several ETC complexes was also reduced at 6 d.p.i.
CONCLUSIONS: Composition of the ETC, which is embedded in the inner mitochondrial membrane, is altered in ATII cells following IAV infection. Reduced mitochondrial respiration and ATP production may be the result of changes in mitochondrial membrane integrity and ETC assembly. Reduced membrane potential indicates reduced ETC function in ATII cells. Similar mitochondrial transition pore permeability in both groups suggests these changes in mitochondrial function are not due to increased ATII cell apoptosis at 6 d.p.i. Changes in mitochondrial energy production in ATII cells may impact ATII cell processes which help maintain normal lung function.