In all instances recombinant proteins, extracts, and LPS were given in endotoxin-free PBS (Sigma-Aldrich) or PBS as a control.
rmIL-25 (2 μg/dose, made in-house, Janssen Pharmaceuticals), rmIL-33 (0.5 μg/dose, Peprotech) or rmEar11 (2 μg/dose) were given on 2 or 3 consecutive days (as indicated) and tissues harvested 24 h after the final dose. rmEar11 (2 μg/dose) or CXCL1 (0.5 μg/ml, R and D Systems; <100 EU/mg protein) were given as a single dose and tissues were harvested at 12 h post injection. rmEar11 and CXCL1 were boiled for 60 min to denature the protein.
RWP (100–200 μg/dose, RWP, Ambrosia artemisiifolia, short form, Greer Laboratories) was given intranasally on 5 consecutive days. All tissues were harvested 24 h after the final dose. Alternatively, three doses RWP were administered intranasally over 3 days and lung CD45+CD11c+F4/80+SiglecF+ alveolar macrophages purified 24 h after the final dose. Macrophage gene expression was analysed by qPCR.
LPS (1–2 μg/dose, Ultrapure LPS-EB from Escherichia coli 0111: B4, InvivoGen) was given intranasally on three consecutive days. Analyses were performed 24 h after the final dose. In some experiments, lung CD45+CD11c+F4/80+SiglecF+ alveolar macrophages were purified and gene expression analysed by qPCR.
rmIL-25 (2 μg/dose, made in-house, Janssen Pharmaceuticals), rmIL-33 (0.5 μg/dose, Peprotech) or rmEar11 (2 μg/dose) were given on 2 or 3 consecutive days (as indicated) and tissues harvested 24 h after the final dose. rmEar11 (2 μg/dose) or CXCL1 (0.5 μg/ml, R and D Systems; <100 EU/mg protein) were given as a single dose and tissues were harvested at 12 h post injection. rmEar11 and CXCL1 were boiled for 60 min to denature the protein.
RWP (100–200 μg/dose, RWP, Ambrosia artemisiifolia, short form, Greer Laboratories) was given intranasally on 5 consecutive days. All tissues were harvested 24 h after the final dose. Alternatively, three doses RWP were administered intranasally over 3 days and lung CD45+CD11c+F4/80+SiglecF+ alveolar macrophages purified 24 h after the final dose. Macrophage gene expression was analysed by qPCR.
LPS (1–2 μg/dose, Ultrapure LPS-EB from Escherichia coli 0111: B4, InvivoGen) was given intranasally on three consecutive days. Analyses were performed 24 h after the final dose. In some experiments, lung CD45+CD11c+F4/80+SiglecF+ alveolar macrophages were purified and gene expression analysed by qPCR.