IAV infection and animal experiments

Wild type C57BL/6J mice were obtained from Jackson Laboratory (Bar Harbor, ME). Wild type C57BL/6J mice at 6–8 weeks old, sex- and age-matched Runx3 KO and littermate control mice on C57BL/6 genetic background were anesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (8.5 mg/kg). These mice were then administered via intranasal instillation of 35 μl sterile saline or 35 μl saline containing 1 to 1.5 LD₅₀ (~ 330-500 plaque-forming units (pfu)) H1N1 PR/8/34 (Charles River, Wilmington, MA). Viral dilution from stock with saline and animal infection were performed in a class II biosafety cabinet, and the infected mice were housed in a designated BSL2 room. Personnel conducting the experiments were rigorously trained and protected by use of PPE (gloves, masks, gowns and shoe covers). Working surfaces were decontaminated after experiments with 70% ethanol, and hand-washing with an alcohol gel was done after experiments according to the BSL2 research safety and work practices manual. Mice were observed daily for signs of distress by monitoring general appearance, respiratory difficulties, body weight loss and animal survival. Mice which lost more than 30% of their initial body weight were humanely euthanized by CO₂ inhalation followed by cervical dislocation. On days 3, 6, 9, and 12 post-infection (pi), the infected Runx3 KO and control mice were anesthetized with injectable anesthesia and then sacrificed by exsanguination to harvest tissues. For the collection of bronchoalveloar lavage fluids (BALFs), a small incision of trachea was made to insert a 21G cannula which was then firmly fixed. The lungs were lavaged with 0.75 ml saline six times to collect BALFs. The first two BALFs were combined for cytokine ELISA assays, and lung immune cells from six BALFs were combined for flow cytometry analysis for each mouse. After that, the lungs were perfused and harvested for experiments. In some cases, the lungs were directly perfused without collecting BALFs. For IAV infected wild type mice, animals were euthanized and lungs were perfused and harvested on designated time points. Whole lungs were fixed in 10% formalin for at least 48 h and formalin-preserved lungs were processed and embedded in paraffin via standard procedures.