Pathogen Res 175: 110C119, 2013. antigen podoplanin (PODO). ATII cells had been EpCAM+ and 2 also,3-connected sialosaccharide+. Infections with influenza A/WSN/33 pathogen caused severe hypoxemia and pulmonary edema. This was accompanied by loss of whole lung GFP fluorescence, reduced ATII cell yields, increased ATII cell apoptosis, reduced SP-C gene and protein expression in Saccharin 1-methylimidazole ATII cell lysates, and increased PODO gene and protein levels. Flow cytometry indicated that infection decreased GFP+/PODO? cells and increased GFP?/PODO+ and GFP?/PODO? cells. Very few GFP+/PODO+ cells were detectable. Finally, infection resulted in a significant decline in EpCAM expression by PODO+ cells, but had limited effects on 2,3-linked sialosaccharides. Our findings indicate that influenza infection results in a progressive differentiation of ATII cells into ATI-like cells, possibly via an SP-C?/PODO? intermediate, to replace dying or dead ATI Saccharin 1-methylimidazole cells. However, impaired SP-C synthesis is likely to contribute significantly to reduced lung compliance in infected mice. and were approved by The Ohio State University Institutional Animal Care and Use Committee. Ethical considerations precluded performance of survival studies, and every effort was made to minimize animal suffering. Preparation of viral inoculum. All studies used egg-grown mouse-adapted influenza A/WSN/33 (H1N1) virus. Absence of contamination with was confirmed by PCR (Charles River Research Animal Diagnostic Services, Wilmington, MA). Absence of endotoxin contamination was confirmed by a standard amebocyte assay (Lonza, Basel, Switzerland). Mouse inoculation. Pathogen-free, 8- to 12-wk-old SP-CGFP mice of either gender were anesthetized by intraperitoneal injection of ketamine (8.7 mg/kg)/xylazine (1.3 mg/kg), individually marked, and then inoculated Rabbit Polyclonal to TSC2 (phospho-Tyr1571) intranasally with 10,000 plaque-forming units (pfu)/mouse of H1N1 virus in 50 l PBS/0.1% BSA, as in our previous studies. In our hands, this inoculum induces hypoxemia and lung dysfunction in wild-type mice by 2 days postinfection (dpi), and results in 100% mortality by 8 dpi (median time to death: 7 days), but does not infect the brain (2, 3, 62). Conscious mice were weighed every other day following infection, and carotid arterial O2 saturation was recorded by pulse oximetry, as in our previous studies (1, 2). Data for each experimental group were derived from at least three independent infections. Lung wet-to-dry weight ratio. Lung wet-to-dry weight ratio was measured as previously described (2). Briefly, mice were killed by intraperitoneal injection of ketamine (87 mg/kg)/xylazine (13 mg/kg) and then exsanguinated. Both lungs were removed, weighed, and dried in an oven at 55C for 3 days. After being dried, the lungs were weighed again. Wet-to-dry weight ratio was then calculated as an index of intrapulmonary fluid accumulation, without correction for blood content. Measurement of viral titers. Viral titers were determined from serial dilutions of lung homogenates by a fluorescent-focus assay in NY3 fibroblasts (derived from STAT1?/? mice), as previously described (24). Whole organ imaging. Immediately before imaging, mice were killed as above. Both lungs were removed by careful dissection, and total lung GFP fluorescence was detected Saccharin 1-methylimidazole using the In Vivo Imaging System (IVIS)-200 bioluminescent imaging system (Perkin Elmer, Waltham, MA). ATII cell isolation. ATII cells were isolated from SP-CGFP mice by a standard lung digestion protocol (12). Briefly, mice were killed, the pulmonary artery was cannulated, and the lungs were perfused with normal saline in situ to flush out blood. The trachea was cannulated, and 2 ml dispase II (5 U/ml in PBS; Becton-Dickinson, San Jose, CA) were injected in the lungs followed by 0.3 ml warmed low-melting-point agarose (1% in PBS) to prevent the isolation of Clara cells and upper airway epithelial cells. The lungs were cooled on ice, dissected free, rinsed with saline, and placed in 5 ml dispase to digest at room temperature for 60 min with gentle rocking. Pancreatic DNase (0.01% in DMEM; Sigma-Aldrich) was added for the final 5 min of incubation. Lung tissue was teased apart, and the resulting cell suspension was filtered sequentially through 100-, 40-, and 21-m sterile nylon meshes. Leukocytes were removed by panning with rat polyclonal anti-murine CD45 and anti-murine CD16/CD32 antibodies (both Becton-Dickinson) for 2 h at 37C. Nonadherent cells were collected, pelleted by centrifugation, resuspended in normal saline, and counted using a hemocytometer. Purity of isolated ATII cell preparations was determined by visualization of lamellar bodies in modified Papanicolaou-stained cytospins. Western blot. ATII cell preparations were homogenized in Cell Lysis buffer 9803.