IL-17-PE (clone TC11-18H10.1) with antibodies from Biolegend, used at 0.2 mg/16106 cells according to Biolegend protocols using Biolegend buffers. Prior to staining for cytokines, CD4-APC stained cells were activated with TPA (12-O-Tetradecanoylphor- bol 13-acetate, 25 ng/ml) and ionomycin (250 ng/ml) in the presence of the Golgi inhibitor, monensin, for 4 hrs at 37uC in 5% CO2. Cells were analyzed by flow cytometry using MACSQuant (Miltenyi) cytometer. Foxp3, IFN-c, IL-4 and IL-17 positive cells were measured within the CD4+ gate. Neutralization of IL-17 and ELISA IL-17 was neutralized by intravenous injections of anti-IL-17 mAb (specific for IL-17A and not reactive with IL-1F, rat clone 50104, endotoxin-free from R&D Systems) 100 mg at sensitization and 50 mg on days 0, 3, 6, 9, 12, and 15 of oxazolone treatment. Control mice were similarly treated with isotype control rat IgG2ak mAb (clone 16-4321, endotoxin-free from eBioscience). The amount of CXCL-1 in ears was measured by ELISA as previously described [25], after each ear was homogenized with Polytron in 0.5 ml of PBS (without Ca/Mg) with 1 mM EDTA, 1 mM PMSF, 1:100 dilution of protease inhibitors (Sigma P1860), and 0.5% Triton X-100, followed by sonication and centrifuga- tion. Supporting Information Figure S1 Pglyrp32/2 and Pglyrp42/2 mice have in- creased Th17 cells in the affected skin in the oxazolone atopic dermatitis model. Expression of a panel of marker genes characteristic of various inflammatory cell types in the ears of mice after sensitization and 10 applications of oxazolone to the ears every other day measured by qRT-PCR (day 20) is shown. For WT mice (top panel), the ratio of the amount of mRNA in oxazolone-treated to untreated mice for each gene (fold induction by oxazolone) is shown; for Pglyrp2/2 mice, the results are the ratios of fold induction of each gene by oxazolone in Pglyrp2/2 mice to fold induction of each gene by oxazolone in WT mice (which represents the fold difference in the response to oxazolone in Pglyrp2/2 versus WT mice). The results are means 6 SEM of 3 arrays from 4–5 mice/group and are shown as heat maps in Figure 6A in the main article. (PDF) Figure S2 In the oxazolone contact dermatitis model in Pglyrp12/2 and Pglyrp22/2 mice most cell types are decreased in the affected skin, but Treg cells and B cells are increased. Expression of a panel of marker genes characteristic of various inflammatory cell types in the ears of mice after sensitization and 6 hrs after single application of oxazolone to the ears measured by qRT-PCR is shown. For WT mice (top panel), the ratio of the amount of mRNA in oxazolone- treated to untreated mice for each gene (fold induction by oxazolone) is shown; for Pglyrp2/2 mice, the results are the ratios of fold induction of each gene by oxazolone in Pglyrp2/2 mice to fold induction of each gene by oxazolone in WT mice (which represents the fold difference in the response to oxazolone in Pglyrp2/2 versus WT mice). The results are means 6 SEM of 3 arrays from 4–5 mice/group and are shown as heat maps in Figure 6B in the main article. (PDF) Figure S3 Multiple inflammatory and immune genes are induced at an early stage of oxazolone model of atopic dermatitis. Expression of a panel of cytokines, Pglyrp3/4 Promote Treg and Inhibit Th17 Responses chemokines, and other marker genes characteristic of Th1, Th2, Th17, Treg, NK, and other cell types in the ears of mice after sensitization and 7 applications of oxazolone to the ears every other day measured by qRT-PCR (day 13). For WT mice (top panel), the ratio of the amount of mRNA in oxazolone-treated to untreated mice for each gene (fold induction by oxazolone) is shown; for Pglyrp32/2 or Pglyrp42/2 mice, the results are the ratios of fold induction of each gene by oxazolone in Pglyrp2/2 mice to fold induction of each gene by oxazolone in WT mice (which represents the fold difference in the response to oxazolone in Pglyrp2/2 versus WT mice). The results are means 6 SEM of 3 arrays from 4–5 mice/group and are shown as heat maps in Figure 7A in the main article. (PDF) Figure S4 Th17 gene expression profile is preferentially induced in the oxazolone model of atopic dermatitis in Pglyrp32/2 and Pglyrp42/2 mice. Expression of a panel of cytokines, chemokines, and other marker genes characteristic of Th1, Th2, Th17, Treg, NK, and other cell types in the ears of mice after sensitization and 10 applications of oxazolone to the ears every other day shows higher induction of several Th17 marker genes in Pglyrp32/2 and Pglyrp42/2 compared to WT mice measured by qRT-PCR. For WT mice (top panel), the ratio of the amount of mRNA in oxazolone-treated to untreated mice for each gene (fold induction by oxazolone) is shown; for Pglyrp2/2 mice, the results are the ratios of fold induction of each gene by oxazolone in Pglyrp2/2 mice to fold induction of each gene by oxazolone in WT mice (which represents the fold difference in the response to oxazolone in Pglyrp2/2 versus WT mice). The results are means 6 SEM of 3–4 arrays from 4–5 mice/group and are shown as heat maps in Figure 7A in the main article. (PDF) Figure S5 Expression of most immune genes is reduced in the oxazolone model of contact dermatitis in Pglyrp2/2 mice. Expression of a panel of cytokines, chemokines, and other marker genes characteristic of Th1, Th2, Th17, Treg, NK, and other cell types in the ears of mice after sensitization and a single application of oxazolone to the ears shows lower induction of immune marker genes in Pglyrp2/2 mice compared to WT mice measured by qRT-PCR. For WT mice (top panel), the ratio of the amount of mRNA in oxazolone-treated to untreated mice for each gene (fold induction by oxazolone) is shown; for Pglyrp2/2 mice, the results are the ratios of fold induction of each gene by oxazolone in Pglyrp2/2 mice to fold induction of each gene by oxazolone in WT mice (which represents the fold difference in the response to oxazolone in Pglyrp2/2 versus WT mice, negative numbers show lower gene induction in Pglyrp2/2 than in WT mice). The results are means 6 SEM of 3–4 arrays from 4–5 mice/group and are shown as heat maps in Figure 7B in the main article. (PDF) Acknowledgments We are grateful to Patrick Bankston for help in interpreting histology slides, and Robert Rukavina, Julie Cook, Panida Girddonfag, and Tiffany Caluag for maintaining and breeding our mice. Author Contributions Conceived and designed the experiments: SYP DG CHK RD. Performed the experiments: SYP DG RD. Analyzed the data: SYP DG CHK RD. Wrote the paper: RD. PLoS ONE | www.plosone.org 15 September 2011 | Volume 6 | Issue 9 | e24961