Figure 9. IL-17 is required for enhanced response to oxazolone in Pglyrp32/2 mice. (A) The level of IL-17-induced chemokine, CXCL-1, is higher in the ears of Pglyrp32/2 mice than WT mice after sensitization and application of oxazolone for 20 days. (B) Ear swelling in Pglyrp32/2 mice sensitized and treated 7 times with oxazolone every other day and also treated with neutralizing anti-IL-17 mAbs is lower than in Pglyrp32/2 mice similarly treated with oxazolone and isotype control IgG. Means 6 SEM; N = 6 mice/group; significance of differences between Pglyrp32/2 and WT mice (A) or IgG control and anti-IL-17 mAbs-treated mice (B): *, P,0.05; **, P,0.005. doi:10.1371/journal.pone.0024961.g009 had significantly lower numbers of CD4+FoxP3+ Treg cells in the affected skin compared to WT mice measured by flow cytometry (Figure 8B–D). These results suggest impaired recruitment and/or maintenance of Treg cells in the inflamed skin in Pglyrp32/2 and Pglyrp42/2 mice. To further investigate whether Pglyrp32/2 mice have less efficient generation of induced Treg cells in lymphoid tissues in general or less efficient recruitment and/or maintenance of these cells in the inflamed skin, we compared the numbers of Treg cells in the draining cervical lymph nodes and in the spleen of WT and Pglyrp32/2 mice treated with oxazolone. Oxazolone-treated Pglyrp32/2 mice on day 13 had lower numbers of Treg cells than WT mice in cervical lymph nodes and spleen (Figure 8B). However, at the peak of inflammation (day 20) Pglyrp32/2 mice had similar numbers of Treg cells in the draining lymph nodes and spleen compared to WT mice (Figure 8C). These results indicate that Pglyrp32/2 mice can eventually generate sufficient numbers of induced Treg cells in lymphoid organs and suggest a possible reduced migration and retention of Treg cells in the affected skin. There could be at least two reasons for this less efficient recruitment of Treg cells to the skin in Pglyrp32/2 mice: insufficient production of Treg-attracting chemokines in the skin, and/or insufficient expression of receptors for these chemokines in Treg cells in Pglyrp32/2 mice. Our results show lower expression of mRNA for Treg-attracting chemokines, CCL-27 (both on days 13 and 20) and CCL-17 (on day 20), in the ears of oxazolone- treated Pglyrp32/2 mice compared to WT mice (Figure 7A and Figures S3 and S5), indicating insufficient production of Treg- attracting chemokines in the skin in Pglyrp32/2 mice. To investigate the second of the above-mentioned possibilities, we determined whether Treg cells in the draining cervical lymph nodes in Pglyrp32/2 mice had sufficient expression of receptors for Treg-attracting chemokines (Ccr4, Ccr8, and Ccr10). The expression of mRNA for Ccr4, Ccr8, and Ccr10 in the draining cervical lymph nodes in oxazolone-treated Pglyrp32/2 mice and WT mice was similar (Figure 8E). These results support the Pglyrp3/4 Promote Treg and Inhibit Th17 Responses conclusion that Treg cells in the draining lymph nodes in oxazolone-treated Pglyrp32/2 mice have sufficient expression of receptors for Treg-attracting chemokines, but that these Treg cells are not recruited to the inflamed skin, likely because of the insufficient production of Treg-attracting chemokines in the skin (Figure 7A and Figures S3 and S4). Our results thus indicate that Pglyrp3 promotes efficient population of the skin with Treg cells in oxazolone-induced atopic dermatitis. Induction of Treg cells in Pglyrp32/2 mice reduces Th17 cells and sensitivity to atopic dermatitis To further investigate the role of Treg cells in high sensitivity of Pglyrp32/2 mice to atopic dermatitis, we induced generation of Treg cells by application of vitamin D to the skin (which is known to induce Treg cells [30,31]) together with the sensitizing allergen, oxazolone. Vitamin D applied to the ears of Pglyrp32/2 mice together with oxazolone significantly reduced ear swelling compared to Pglyrp32/2 mice similarly treated with oxazolone alone (Figure 10A). Vitamin D applied to the ears (together with oxazolone) also significantly increased the percentages of Treg cells both in the ears and cervical lymph nodes (as expected [30,31]), and, moreover, it significantly reduced the percentages of Th17 cells in the ears compared to the ears treated with oxazolone alone, measured on day 20 by flow cytometry (Figure 10B–D). Thus, increasing the numbers of Treg cells in the affected skin in Pglyrp32/2 mice to the numbers found in WT mice could revert the inflammatory phenotype of Pglyrp32/2 mice to the less inflammatory phenotype characteristic of WT mice. These results further demonstrate the critical role of Treg cells in preventing high levels of Th17 cells in the skin and excessive inflammation in the oxazolone model of atopic dermatitis. In summary, our results indicate that in WT mice Pglyrp3 and Pglyrp4 promote efficient population of the skin with Treg cells in the experimental model of atopic dermatitis. Discussion Skin diseases such as atopic dermatitis and contact dermatitis involve complex interactions of many cell types. Atopic dermatitis is thought to have Th2 bias [2,26], but recent findings also show involvement of Th17 cells [32,33]. The initially identified in vivo role of Th17 cells was promoting some autoimmune diseases and recruitment of PMNs to the sites of inflammation [34–37]. However, Th17 cells have many other functions – they play a role in inflammatory bowel diseases, skin diseases, asthma, graft rejection, atherosclerosis, periodontal disease, and arthritis [32,33,38–40]. We extend these findings by showing that Th17 cells exacerbate skin inflammation in experimental model of atopic dermatitis in a PGRP-dependent manner. We demonstrate here that Pglyrp32/2 and Pglyrp42/2 mice develop more severe oxazolone-induced atopic dermatitis than WT mice. By contrast, Pglyrp12/2 mice develop less severe oxazolone-induced atopic dermatitis and also less severe contact dermatitis than WT mice. Thus, individual PGRPs play distinct roles in these two models of skin diseases: in WT mice Pglyrp3 and Pglyrp4 protect mice from the development of experimental atopic dermatitis, whereas Pglyrp1 enhances the development of both atopic and contact dermatitis and Pglyrp2 has less effect on both disease models. The common mechanism underlying these protective effects of PGRPs is decreased recruitment and activity of Treg cells and enhanced production and activation of Th17 cells in the affected skin in Pglyrp32/2 and Pglyrp42/2 mice, which results in more severe inflammation and keratinocyte proliferation. Thus, in WT PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e24961