All mice used for experiments in this study were on a C57BL/6 background. Transgenic mice that express a FLAG-tagged form of activated human IKK-β containing S177E and S188E mutations under the control of a tetracycline operator minimal CMV promoter and a tetracycline-controlled transcription silencer under the control of the Clara cell 10 kDa (CC10) promoter have been previously described (FLAG-IKK-β mice) (13). To activate NF-κB in the airway epithelium, hemizygous FLAG-IKK-β male mice were crossed with homozygous female mice expressing reverse tetracycline transactivator (rtTA) under control of the Club cell–specific protein promoter (CCSP-rtTA mice). Approximately half of the pups from these matings expressed all 3 transgenes and activated NF-κB in the airway epithelium after treatment with Dox. These mice were designated IKTA mice, and the remaining pups in each litter that expressed only the CCSP-rtTA transgene were used as littermate controls. For transgene activation in neonatal lungs, lactating dams were administered 100 mg/L of Dox (Sigma-Aldrich) in drinking water from P3 to P5 (saccular-stage transgene activation) or from P10 to P12 (alveolar-stage transgene activation). In some experiments, IKTA mice deficient in IL-1R1 were also used. IL-1R1–/– mice (B6.129S7-Ilr1 tm1Imx /J, stock 003245) were purchased from The Jackson Laboratory. FLAG-IKK-β and CC10-rtTA mice were separately backcrossed to IL-1R1–/– mice to generate FLAG-IKK-β and CCSP-rtTA mice deficient in IL-1R1 respectively. IL-1R1–/– FLAG-IKK-β and IL1R1–/– CCSP-rtTA mice were then crossed to generate IKTA mice (and littermate controls) deficient in IL-1R1.

To block neutrophil influx, neonatal IKTA mice (and littermate controls) were i.p. administered a neutralizing anti-Ly6G antibody (20 μg of antibody per pup; Bio X Cell, clone 1A8, BP0075-1) or an IgG1a isotype control antibody (20 μg of antibody per pup; Bio X Cell, BP0089) on P2 prior to Dox administration from P3 to P5. Lungs were then harvested on P5 for additional studies.

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