Airway responsiveness (a) **P<0.01, ***P<0.001 (one-way ANOVA), cytokine production in HDM-restimulated lung cells (b), frequency of pulmonary CD4⁺IL-17A⁺ cells (c), IL-17A MFI (d), frequency of pulmonary CD4⁺IL-17F⁺ cells (e), and IL-17F MFI (f) were measured in HDM-challenged A/J and C3H/HeJ mice. *P<0.05, **P<0.01, ***P<0.001 comparing A/J vs. C3H/HeJ (one-way ANOVA), and †P<0.05, comparing PBS vs. HDM (one-way ANOVA). Results are representative of three independent experiments (mean ± s.e.m. of 8–12 mice per group).

A/J mice were treated with HDM or PBS and isotype or IL-17A blocking mAbs and airway function (a) (*P<0.05, ***P<0.001), BAL cellularity (b) (#P<0.05 versus HDM + Iso, one-way ANOVA) and mucus staining using periodic acid-Schiff (PAS) (c) were assessed. Supernatants from medium (open bars) and HDM-restimulated (closed bars) lung cell cultures were assayed for production of (d) IL-4, (e) IL-5, and (f) IL-13 by ELISA, (†P<0.05, ††P<0.01 [one-way ANOVA]). Airway reactivity was measured in C3H/HeJ mice treated with PBS, HDM or HDM + 15 µg rIL-17A, 3 days after the last allergen challenge (g), ***P<0.001 (one-way ANOVA). Results are from two experiments [mean ± s.e.m. of 6–8 mice per group (a–f)] or representative of one experiment [mean ± s.e.m. of 4–8 mice per group (g)].

A/J mice were treated with rmIL-13, rmIL-17A, or a combination of both and airway responsiveness was assessed (a) *P<0.05, **P<0.01 (one-way ANOVA). Lung expression of Il4ra1 (b), Il13ra1 (c), Il13ra2 (d), Tff2, (e), Arg1 (f), C3 (g), and Cebpb (h) were measured by real-time PCR, †P<0.05, ††P<0.01, †††P<0.001, (one-way ANOVA). Results are representative of two independent experiments (mean ± s.e.m. of 8 mice per groups).

Flow cytometric analysis of phosphoErk1/2 in IL-17A treated lung cells from naïve A/J and C3H/HeJ mice (a). Effect of IL-17A on expression of MHC class II and costimulatory molecules (CD80, CD86 and B7-DC) on BMDCs from naïve A/J and C3H/HeJ mice as analyzed by flow cytometry (shown as fold increase over media-treated cells) (b). Real-time PCR analysis of the IL-17A-responsive genes, Cepbd (c), Cebpb (d), and Cxcl1 (e) and CXCL1 secretion (f) from media- or IL-17A-stimulated A/J and C3H/HeJ lung fibroblasts *P<0.05, **P<0.01 comparing A/J vs C3H/HeJ (two-tailed Student’s t-test). Il17ra mRNA in untreated A/J and C3H/HeJ BMDC or lung fibroblasts determined by real-time PCR (g). Results represent one to two separate experiments (mean ± s.e.m. of triplicate wells).

C5-sufficient and C5-deficient strains were treated with HDM as described in Methods. Frequency of lung T_H17 cells and IL-17A MFI were analyzed by flow cytometry and serum C5a was measured by ELISA (shown as fold change over PBS-treated mice) (a, b). BALB/c mice were treated with C5aR or IL-17A blocking antibodies as described in the Methods section. Airway responses (c), and the frequency of CD4⁺IL17A⁺ cells was determined by flow cytometry (d) *P<0.05; **P<0.01 (one-way ANOVA). BMDCs from BALB/c control or C5ar-deficient mice were treated with media or HDM (100 µg/ml) and supernatants were analyzed for IL-23 (e), IL-6 (f), and IL-1β (g) production. Supernatants from HDM-treated BMDCs from A/J and C3H/HeJ mice were assayed for IL-23 (h), IL-6 (i) and IL-1β (j) production by ELISA **P<0.01; ***P<0.001 (Student’s t-test). Results are representative of one [4 mice per strain - (a,b); 4–6 mice per group - (c,d)] or two to four independent experiments [mean ± s.e.m. of 4 individual samples, (e–j)].

BMDCs from wild-type (BALB/c) and C3ar KO mice were treated with media or HDM and supernatants were analyzed for IL-23 (a), IL-6 (b), and IL-1β (c) production, ***P<0.001 (two-tailed Student’s t-test). BALB/c, and C3ar KO mice were exposed to HDM as described in the Methods section to assess airway function (d), and the frequency of lung T_H17 cells (CD4⁺IL-17A⁺) cells (e) and IL-17A MFI (f) by flow cytometry ††P<0.01, †††P<0.001 (two-tailed Student’s t-test). A/J and C3H/HeJ mice were treated with a single exposure of PBS or HDM, and sacrificed 72h afterwards, lungs were harvested for RNA extraction, and gene expression of C3 (g) and C3ar (h). ##P<0.01, ###P<0.001 (two-tailed Student’s t-test). Results are representative of three independent experiments [mean ± s.e.m. of four individual samples (a–c)], one experiment with five to six mice per group (d–e) or three separate experiments [mean ± s.e.m. of eight mice per group (g,h)].

Primary mouse tracheal epithelial cells from A/J and C3H/HeJ mice were cultured in media or in 100 ng/ml mIL-17A and C3 message was determined by real-time PCR (a), **P<0.01 (two-tailed Student's t-test). A/J mice were treated HDM or PBS and isotype or IL-17A blocking mAbs as described in Methods to quantify C3a in BAL by ELISA (b), †P<0.05 (two-tailed Student's t-test). C3a concentrations in the supernatants of BMDC (C57BL/6) treated with media or 100 ng/ml IL-17A (c), #P<0.05 (two-tailed Student's t-test) Concentrations of C3a in the BAL of A/J mice receiving rIL-17A (5 ug) intratracheally (d). Results are representative of one to two independent experiments (mean ± s.e.m. of three to eight individual samples). *P<0.05, **P<0.01, (two-tailed Student’s t-test).

IL-10 production in HDM-treated BMDCs (BALB/c) in the presence of medium, rC3a, or rC5a (a), *P<0.05, ***P<0.001 (one way ANOVA). IL-23 production by HDM-treated BMDCs (BALB/c) with or without rC5a in the presence of IgG1, anti-IL-10 or rIL-10 (b). IL-23 (c) and IL-10 (d) production by BMDCs (BALB/c) pre-treated (1 h) with DMSO, SP600125, AS602868, or rapamycin, then with HDM (100 µg/ml) in the presence or absence of rC5a. Phospho c-Jun and total c-Jun in media- or C5a-treated RAW 264.7 cells (e). AP-1 activity in RAW 264.7 cells were treated as indicated (SP = SP600125) (f). AP-1 activity in RAW 264.7 cells stimulated as indicated (g). Il23a promoter activity in RAW 264.7 cells co-transfected with p-cDNA3.1, or vectors containing c-Jun or c-Fos cDNAs (h). Jun and Fos mRNA was measured in A/J and C3H/HeJ BMDC (i). Results are representative of two to three independent experiments (mean ± s.e.m. from 4–8 individual samples).