Pglyrp42/2 mice have decreased numbers of Treg cells and increased numbers of Th17 cells in the inflamed skin, compared to WT mice. Allergens and proinflammatory stimuli (such as oxazolone) in WT mice initially induce vigorous cytokine and chemokine production. However, upon chronic exposure, WT mice recruit and maintain large numbers of Treg cells in the inflamed skin and are able to limit the proinflammatory response by reducing the number of proinflammatory genes that are activated and reducing the level of their activation. By contrast, Pglyrp32/2 and Pglyrp42/2 mice have fewer Treg cells and higher numbers of Th17 cells in the affected skin and are unable to limit inflammatory responses. This T cell imbalance in Treg/Th17 cells in Pglyrp32/2 and Pglyrp42/2 mice could come from reduced recruitment of Treg cells and increased recruitment of Th17 cells to the affected skin, and/or from enhanced local differentiation of T cells into Th17 cells (including conversion of Treg cells into Th17 cells) under the influence of locally produced chemokines and cytokines. T cell populations are dynamic and have considerable plasticity based on local cytokine milieu, as, for example, Treg cells can differentiate into Th17 under the influence of locally-produced proinflammatory cytokines [45,46]. The enhanced recruitment and differentiation of Th17 cells is supported by higher production of Th17 cell promoting cytokines (IL-17, IL-22, IL-23) in the inflamed skin in Pglyrp32/2 and Pglyrp42/2 mice. Decreased recruitment of Treg cells to the inflamed skin is supported by the presence of adequate numbers of Treg cells and adequate expression of receptors for Treg-attracting chemokines on these Treg cells in the draining lymph nodes and spleen in oxazolone-treated Pglyrp32/2 mice, but lower numbers in the skin than in WT mice. These results suggest efficient generation of induced Treg cells in lymphoid organs but defective recruitment to the inflamed skin. This mechanism is further supported by decreased production of Treg cell-attracting chemokines (CCL1, CCL17, CCL27) in the skin of Pglyrp32/2 and Pglyrp42/2 mice. Thus both increased recruitment and generation of Th17 cells and decreased recruitment and retention of Treg cells in the skin are likely responsible for increased inflammation in Pglyrp32/2 and Pglyrp42/2 mice. Pglyrp3 and Pglyrp4 are primarily expressed in keratinocytes and other epithelial cells, but not in immune cells and stimulation of immune cells does not induce expression of Pglyrp3 and Pglyrp4 [7,8,18,20,21]. Our results extend these findings by showing expression of Pglyrp3 and Pglyrp4 in differentiated keratinocytes in untreated and oxazolone-treated skin, and no expression of Pglyrp3 and Pglyrp4 in the inflammatory cells infiltrating the skin and in the draining lymph nodes, which primarily contain resting and activated lymphocytes and antigen-presenting cells that migrated from the inflamed skin. Thus, Pglyrp3 and Pglyrp4 most likely exert their anti-inflammatory effect in the skin through their expression in keratinocytes. Keratinocytes are an important local source of chemokines and cytokines, and activation of keratinocytes by proinflammatory stimuli also leads to increased expression of Pglyrp3 and Pglyrp4 in the skin, which correlates with the ability of WT mice to reduce chronic inflammation in the skin. By contrast, increased Pglyrp1 expression in the inflamed skin likely comes from the influx of PMNs, because PMNs express high amounts of Pglyrp1 in their granules and, unlike other PGRPs, Pglyrp1 expression is not increased by proinflammatory stimuli in epithelial cells, including keratinocytes [7,8,14,15]. Thus, the effects of PGRPs in the inflamed skin are likely exerted through a change in the local production of chemokines and cytokines in the skin, which modulates the recruitment and activity of these Treg and Th17 cells. In Pglyrp32/2 and Pglyrp42/2 mice reduced numbers of Treg cells allow dominating expansion of Th17 cells, Pglyrp3/4 Promote Treg and Inhibit Th17 Responses which can increase inflammatory responses in the atopic dermatitis model, but may reduce Th1-mediated response in the contact dermatitis model by shifting T cell differentiation into Th17 cells instead of Th1 cells. Thus in WT mice, compared to Pglyrp32/2 and Pglyrp42/2 mice, the immune balance is shifted towards Th1 cells, which are protective against microbial infections (rather than Th17 cells) and towards Treg cells that control detrimental inflammation induced by proinflammatory chemicals and allergens. Our results suggest that defects in Pglyrp3 and Pglyrp4 genes could be predisposing to atopic dermatitis through the aforementioned shifts in immune homeostasis. Materials and Methods Ethics statement All experiments on mice were performed according to the guidelines and approved by the Indiana University School of Medicine–Northwest Institutional Animal Care and Use Com- mittee (approval number IUSM-NW-16). Mice We generated Pglyrp12/2, Pglyrp22/2, Pglyrp32/2, and Pglyrp42/2 mice as described previously [15,22,25]. We generated Pglyrp12/2Pglyrp22/2, Pglyrp12/2Pglyrp32/2, and Pglyrp22/2 Pglyrp32/2 double knockout mice and Pglyrp12/2Pglyrp22/2 Pglyrp32/2 and Pglyrp12/2Pglyrp22/2Pglyrp42/2 triple knockout mice by breeding single and double knockout mice (all on BALB/c background) and screening for homozygous deletion of each Pglyrp gene by PCR analysis of genomic DNA as previously described [15,22,25]. The lack of expression of the Pglyrp genes was confirmed by qRT-PCR in mRNA from the ears. Double and triple homozygous Pglyrp knockout mice were viable and fertile, bred normally, and yielded the expected male:female ratios and similar litter size as the wild type and heterozygous mice. They had similar weight as the WT and single Pglyrp knockout mice and developed normally with no obvious defects. Their major internal organs had normal macroscopic appearance, and normal histological appearance on hematoxylin/eosin-stained sections. All mice used in experiments were 8–10 week-old and on BALB/c background. The original colony founder WT BALB/c breeder mice were obtained from Harlan-Sprague-Dawley. All knockout mice were backcrossed to the same WT BALB/c mice from our breeding colony, and all WT and knockout mice were bred and kept under conventional pathogen-free conditions in the same room in our facility to minimize the influence of differences in the environment. For each experiment, mice from several different cages and breeder pairs were used. The BALB/c background of Pglyrp-deficient mice and their negative status for all common viral and bacterial pathogens and parasites were confirmed as previously described [22]. Oxazolone atopic dermatitis and contact dermatitis models To induce atopic dermatitis female mice were first sensitized with 10 ml of 5% oxazolone (in 80% acetone, 20% olive oil) applied to the abdomen (after removing hair with Nair cream); 6 days later applications of 30 ml of 0.1% oxazolone (in 80% acetone, 20% olive oil) to each ear (15 ml to each side) were started (day 0) and continued every other day through day 18 [26]. In some experiments to induce Treg cells [30,31] 3 mM vitamin D (1a,25-dihydroxyvitamin D3 from Sigma) was added to the oxazolone solution and used for the initial sensitization and applications to the ears. Ear thickness was measured each time PLoS ONE | www.plosone.org 13 September 2011 | Volume 6 | Issue 9 | e24961