Methods

Mice. PDGFRa-GFP (7), SPC-CreERT2 (8), K5-CreERT2 (5), ROSA-mTmG (9), ROSA-Tomato (8) and Rosa-fGFP alleles have been previously described. To lineage trace AEC2, 8-week-old mice heterozygous for both SPC-CreERT2 and a ROSA reporter were given tamoxifen (0.25 mg/g body weight) dissolved in corn oil via intraperitoneal injection every other day for three doses. A chase period of 30 days was used between tamoxifen injection and subsequent influenza infection. To lineage trace cells expressing Krt5, adult mice heterozygous for both K5-CreERT2 and ROSA-mTmG were given a single intraperitoneal dose of tamoxifen (0.123 mg/g body weight) 10 days after infection with PR8, when Krt5 1 cells are abundant in the distal mouse lung. All studies were performed according to protocols approved by the University of California, San Francisco Institutional Animal Care and Use Committee.

Influenza infections. Mice were anesthetized with isoflurane and infected intranasally with D-PBS (vehicle), 3 3 105 focus forming units (FFU) of influenza A/H3N2 A/Aichi/02/68 3 A/Puerto Rico/8/34 (X31) or 200–280 FFU of influenza A/H1N1/Puerto Rico/8/34 (PR8) dissolved in 30 ml D-PBS at 8–12 weeks of age. BLEOMYCIN. Six-to 8-week-old female C57BL/6 mice were intratracheally instilled with saline or 2.0–2.5 units/kg bleomycin (Sigma-Aldrich). Mice were killed on Day 17. The lungs were lavaged, followed by snap freezing in liquid nitrogen for protein extraction. ASSESSMENT OF LUNG INJURY. C57BL/6 mice were given high-dose ketamine and xylazine and were subsequently killed by bilateral thoracotomy. Blood hemoglobin concentration was assessed, and lung homogenate and homogenate supernatant were collected, weighed, and desiccated for wet-to-dry ratio and excess lung water measurements, as described (10).