BAL supernatants (25 μl) were used to determine neutrophil myeloperoxidase (MPO) using a mouse-specific ELISA kit (R&D Systems, Minneapolis, MN) according to manufacturer instructions. Secreted MUC5AC protein was also determined in BAL supernatants following the published method 29 (link). Briefly, an aliquot of BAL supernatant (20 μl) was loaded in each well of an ELISA plate containing a goat polyclonal anti-MUC5AC capture antibody (1:40 dilution of sc-16903; Santa Cruz Biotechnology Inc., Dallas, TX) in pH 9.5 bicarbonate-carbonate coating buffer (BD OptEIA Reagent; BD Biosciences, San Diego, CA). The plate was incubated at 48°C until the reaction was dry (>5 hr). The wells were washed and blocked overnight with an assay diluent containing 10% fetal bovine serum (BD Opt EIA) at 4°C. The samples were then incubated with a biotinylated monoclonal anti-MUC5AC detection antibody (1:100 of Clone 45M1, Thermo Scientific, Waltham, MA) for 1.5 hr at 37°C. Following incubation with a peroxidase-conjugated secondary antibody (1:2500, goat anti-mouse-IgG-HRP), color change was developed by adding the TMB substrate solution (BD Biosciences). Optical density was measured at 450 nm after the stop buffer was added.