Influenza Infection and Immune Cell Modulation

All mice were breed and housed in a pathogen-free environment and used at 7–14 weeks of age for all experiments. NS1-GFP virus was a generous gift from the Adolfo Garcia-Sastre laboratory. Influenza A viruses PR8 (H1N1) and NS1-GFP [27 (link)] were grown in the allantoic cavity of day 10 chicken embryos as described previously [26 (link)]. Mice were infected with 250 EID₅₀ units of PR8 (0.1LD₅₀), or 10⁵ EID₅₀ NS1-GFP [26 (link)]. All infectious doses were administered i.n. in 50μL of serum-free Dulbecco’s Modified Eagle Medium (Invitrogen) following ketamine and xylazine anesthesia. For i.n. transfer of cells, 500,000 AlvMΦs were given in 50uL of serum-free Dulbecco’s Modified Eagle Medium (Invitrogen) following ketamine and xylazine anesthesia. Irradiation and bone marrow transplantation mice were irradiated with 9.5 Gy and, within 24hours, i.v. injected with RBC-lysed bone marrow cells (1–3 × 10⁶) [26 (link)]. For AlvMΦ depletion CD11c-DTr+ and CD11c-DTr- BALB/C littermates were given 40ng of DTx i.n. following ketamine and xylazine anesthesia. Acivicin was diluted in serum-free Dulbecco’s Modified Eagle Medium (Invitrogen) and 2.5mg/kg was given i.n. in 50uL following ketamine and xylazine anesthesia. 10mg/kg of Zafirlukast was given daily on days 0–3 PI by i.p. injection in 1mL of saline with 1%DMSO and 2% hydroxypropyl-β- cyclodextrin (HPCD).