Allergen-induced inflammation was localized to the lungs of C57BL/6J mice (n = 3 per group) via a series of 6 allergen challenges. (a) Time course of representative PAS staining, taken 6 hrs post indicated allergen challenge. Upper row scale bar, 400 µm; lower row, 100 µm. (b) Time course of pulmonary IL-4, IL-5, IL-13, IFN-γ and IL-17a mRNA expression 6 hours post indicated allergen challenge. (*P < 8.44E⁻⁰⁵, #P = 0.0008, +P = 0.001). (c) Time course of IL-25, IL17RB, IL-33, IL-17b, and IL17d mRNA expression 6 hours post indicated allergen challenge. ST2 and IL-22 transcripts were not detectable. (*P < 0.007). (d and e) IL-17RB⁺ lung subsets from naïve and allergen sensitized C57BL/6J mice (n = 5 per group) were assessed by flow cytometry for IL-4 and IL-13 production. (f) Representative flow plots of intracellular cytokine staining in IL-17RB⁺ CD11b⁺ Gr-1^mid cells from naïve and allergen sensitized C57BL/6J mice. Gray: n-1 staining, black: naïve IL-17RB⁺ CD11b⁺ Gr-1^mid, red: allergen challenged IL-17RB⁺ CD11b⁺Gr-1^mid (g) Pulmonary IL-17RB^−/− CD11b⁺ Gr-1^mid populations are significantly increased following allergen sensitization. (*P = 0.0026). Results are representative of two independent experiments. (h) Morphology of myeloid cells isolated from the lungs of allergen-sensitized mice. Cells were sorted as CD11b⁺ Gr-1^mid FcγR⁺ IL-17RB⁺ CD4⁻ CD8⁻ B220⁻ IL-7Rα⁻ Sca1⁻ c-kit⁻ and stained with H+E. Scale bar 50 µm. All data are presented as mean ± s.e.m.

(a) PAS staining of lungs from WT and Il17rb^−/− mice following chronic allergen sensitization. Scale bar 200 µm. (b) Lungs from allergic mice (n = 5 animals per group) were harvested 24 h post final allergen challenge and whole lung homogenates were analyzed for cytokine production by bioplex. (*P = 0.001, #P = 0.048, +P < 0.05 versus WT allergen). (c) Cytokine production from draining lymph nodes cells of allergic mice (n = 5 animals per group). Bars represent the mean ± s.e.m. from triplicate wells. (N.D. = not detected, *P = 2.46E⁻⁰⁵, #P = 8.79E⁻⁰⁸, +P = 4.00E⁻⁰⁶ versus WT allergen). (d) Flow cytometric analysis of lungs from allergic WT and Il17rb^−/− mice (n = 5 animals per group), (*P < 0.03). (e) Intracellular cytokine staining for IL-4 and IL-13 producing cells in allergic WT and Il17rb^−/− mice (n = 4 animals per group). Results are gated on CD11b⁺ Gr-1^mid cells. Bars represent the mean ± s.e.m. for each group (*P < 0.05, #P < 0.0009). Data are representative of two independent experiments.

4 get mice (n = 4 animals per group) were IT dosed with vehicle or 0.5 µg IL-25 for 4 days, and the inflammatory response was investigated 24 h post final IT. (a) Histograms of lung tissue from 4get mice treated with vehicle or IL-25, gated on total lung, CD11b⁺, T2M, CD4⁺ Lymphocytes, and Lin⁻ Sca⁺ c-kit⁺ cells respectively. (b) GFP⁺ and CD11b⁺ Gr-1^mid populations in the lung were assessed by flow cytometry, (*P < 0.026). (c) Pulmonary IL-17RB⁺ CD11b⁺ GFP/IL-4⁺ cell numbers following IL-25 administration. Data are representative of two independent experiments. (d) Pulmonary IL-13⁺ populations following IL-25 treatment (n = 5 animals per group, *P = 0.038). (e, f, and g) QPCR analysis of IL-4, IL-5, and IL-13 transcripts in T2M cells. Cells were isolated from C57BL/6J mice dosed with 0.5 µg IL-25 for 4 days (n = 5 animals per group), and plated in triplicate. T2M cells were isolated using MACS magnetic bead enrichment followed by FACS. mRNA was isolated from naïve C57BL/6J mice, CD11b depleted lung from IL-25 treated mice, and T2M cells isolated from IL-25 treated mice. All data are presented as mean ± s.e.m.

(a) Characterization of pulmonary T2M cells by cell surface receptor expression. 4get mice were challenged (as previously described) with intratracheal IL-25 to induce recruitment of T2M cells to the lung. T2M cells were identified by gating on CD11b⁺ Gr1^mid IL-17RB⁺ GFP/IL-4⁺ cells, and expression of various surface markers was then assessed. Gray shaded area: isotype, red line; T2M cells. Data are representative of 2 independent experiments. (b) Hierarchical clustering and heat map generated from microarray analysis of pulmonary T2M cells compared to Eosinophils, Neutrophils, and Macrophages. Colors illustrate fold changes among 1,880 probes for which T2M cells exhibited a minimum 3 fold difference in expression from 2 or more cell types, and an average expression value of at least 2⁶, normalized to average T2M expression levels. (c) Venn diagram illustrating differences in locus expression between T2M cells and other myeloid populations. 366 probes were differentially expression between T2M cells and all cell types analyzed, 1,880 probes differed between T2M cells and at least 2 of the comparison populations.

(a) Representative PAS staining indicates IL-25-induced mucus production in 4get mice (n = 5 per group) is not altered by dexamethasone administration. Scale bar 400 µm; inset 100 µm. (b) Airway hyperreactivity (*P < 0.01 versus methacholine treated vehicle). (c) QPCR analysis of whole lung following dexamethasone treatment. (d) Representative flow plots of GFP/IL-4⁺ CD11b⁺ Gr-1^mid pulmonary populations. (e) Total numbers of pulmonary GFP/IL-4⁺ cells. Bars represent the mean ± s.e.m. of 4 mice per group. *P < 0.01 versus vehicle alone. Data are representative of two independent experiments. (f) Representative histology from recipients of T2M transfer stained with PAS, 24 hours post final IL-25 administration. Scale bar 400 µm; inset 100 µm. T2M cells were isolated by MACS enrichment and FACS, and 2.0×10⁵ cells were instilled into the airways of Il17rb^−/− mice. Mice received 4 total treatments, consisting of 0.5 µg IL-25, T2M cells alone, or IL-25 + T2M cells. (g) QPCR expression of IL-13 following T2M transfer (*P = 0.017, #P = 0.042). (h) QPCR expression for the mucus specific gene muc5ac (*P < 0.026). Bars represent the mean ± s.e.m. of 4 mice per group. Results are representative of 3 independent experiments.

Granulocytes were isolated from peripheral blood samples donated by asthmatic (n = 9) or non-asthmatic (n = 8) volunteers and analyzed by flow cytometry. (a) Representative dot plots of granulocytes stained for IL-17RB, normalized to 400k events. (b) Percent total IL-17RB⁺ granulocytes isolated from peripheral blood of volunteer donors. Bars represent the mean ± s.e.m. for each group, (*P = 0.0031). (c) Representative histograms of IL-17RB⁺ granulocytes from an asthmatic donor indicate the majority of IL-17RB⁺ granulocytes are CD11b⁺ CD16⁺ CD177⁺. Cells were gated on total IL-17RB⁺ cells and assessed for surface marker expression. (d) Percent total CD11b⁺ CD16⁺ CD177⁺ IL-17RB⁺ granulocytes from volunteer donors. Bars represent the mean ± s.e.m. for each group, (*P = 0.023). (e) Percent total CD11b⁺ CD16⁺ CD177⁺ IL-17RB⁺ cells from whole blood of volunteer asthmatic donors, cultured in vitro for 2 hours ± 50 ng mL⁻¹ IL-25. (f) Representative intracellular cytokine staining for IL-4 and IL-13 from whole blood from an asthmatic volunteer, cultured for 2 hours with RPMI 1640.