Mice were sensitized intranasally for 3 days with carrier-free recombinant mouse (rm)-IL-33 (0.5 µg per mouse in 50 µL, BioLegend) to induce AHR or with PBS, as the control. In some experiments, mice were challenged for 4 consecutive days with 100 μg of Alternaria alternata extracts (Greer Laboratories). To block glycolysis in vivo, mice were injected intraperitonially on 2 consecutive days with 500 mg kg−1 of 2-Deoxyglucose (2-DG) (Sigma-Aldrich) or with PBS (vehicle). For the neutralization models, Rag2−/− mice received an intraperitoneal injection of PD-1 blocking antibody (500 μg per mouse, clone 29 F.1A12; BioXcell) or a rat IgG2aκ isotype control 1 day before rm-IL-33 or Alternaria intranasal administration.
On day 4, lung function was evaluated by direct measurement of lung resistance and dynamic compliance (cDyn) in restrained, tracheostomized, and mechanically ventilated mice using the FinePointe RC system (Buxco Research Systems) under general anesthesia. Mice were sequentially challenged with aerosolized PBS (baseline), followed by increasing doses of methacholine ranging from 5 to 40 mg mL−1. Maximum lung resistance and minimum compliance values were recorded during a 3-min period after each methacholine challenge. AHR data were analyzed by repeated measurements of a general linear model.
On day 4, lung function was evaluated by direct measurement of lung resistance and dynamic compliance (cDyn) in restrained, tracheostomized, and mechanically ventilated mice using the FinePointe RC system (Buxco Research Systems) under general anesthesia. Mice were sequentially challenged with aerosolized PBS (baseline), followed by increasing doses of methacholine ranging from 5 to 40 mg mL−1. Maximum lung resistance and minimum compliance values were recorded during a 3-min period after each methacholine challenge. AHR data were analyzed by repeated measurements of a general linear model.