Pglyrp3/4 Promote Treg and Inhibit Th17 Responses Figure 6. Pglyrp32/2 and Pglyrp42/2 mice have increased Th17 cells in the affected skin in the atopic dermatitis model, whereas Pglyrp12/2 and Pglyrp22/2 mice have most cell types decreased (except B cells and Treg cells) in the contact dermatitis model. Expression of a panel of marker genes characteristic of various inflammatory cell types in the ears of mice after (A) sensitization and 10 applications of oxazolone to the ears every other day, or (B) after sensitization and 6 hrs after a single application of oxazolone to the ears, measured by qRT-PCR is shown. For WT mice (left panels in A and B), 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 (right panels in A and B), 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 of 3 arrays from 4–5 mice/group in heat map format. The means 6 SEM bar graphs for these results are shown in Figures S1 and S2. doi:10.1371/journal.pone.0024961.g006 Th cell types were significantly higher in Pglyrp32/2 mice compared to WT mice (Figure 8B and C). These results indicate initial (day 13) preferential activation of Th2 cells in the affected ears and draining lymph nodes in Pglyrp32/2 mice compared to WT mice, consistent with B-cell- dependence of atopic dermatitis model. However, continued treatment with oxazolone (20 days) showed a switch to preferential infiltration of the affected ears with Th17 cells in Pglyrp32/2 mice compared to WT mice (Figure 8), consistent with our mRNA gene expression data (Figures 6 and 7 and Figures S1, S3, and S4). IL-17 is required for enhanced response to oxazolone in Pglyrp32/2 mice To further study the role of IL-17 (Th17 cytokine) in high sensitivity of Pglyrp32/2 mice to oxazolone-induced atopic dermatitis, we determined the protein levels of an IL-17-induced chemokine, CXCL-1, in the ears of WT and Pglyrp32/2 mice. CXCL-1 was undetectable (,7 pg/ear) in the ears of untreated mice, and after sensitization and 20 days of skin treatment with oxazolone, the amount of CXCL-1 increased to .350 pg/ear in Pglyrp32/2 mice, the level that was significantly higher than in WT mice (Figure 9A). To determine whether IL-17 is required for the high sensitivity of Pglyrp32/2 mice to atopic dermatitis, we compared the severity of ear inflammation in oxazolone-treated Pglyrp32/2 mice in which IL-17 activity was inhibited with neutralizing anti-IL-17 mAb. In vivo neutralization of IL-17 activity in Pglyrp32/2 mice in the oxazolone-induced atopic dermatitis significantly reduced ear inflammation, compared to mice treated with an isotype control IgG (Figure 9B). These results demonstrate that IL-17 is required PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24961