colitis [22] and to modulate the development of experimental arthritis [25]. Here we tested this hypothesis using PGRP-deficient mice and mouse models of chemically-induced atopic dermatitis and contact dermatitis. Our results show that Pglyrp32/2 and Pglyrp42/2 mice (but not Pglyrp22/2 mice) are more sensitive to the development of experimental atopic dermatitis than wild type (WT) mice. The common mechanism underlying this increased sensitivity of Pglyrp32/2 and Pglyrp42/2 mice is reduced recruitment of Treg cells to the skin and enhanced production and activation Th17 cells in Pglyrp32/2 and Pglyrp42/2 mice, which results in more severe inflammation and keratinocyte proliferation. By contrast, Pglyrp12/2 mice are less sensitive than WT mice to both experimental atopic dermatitis and contact dermatitis. Thus, Pglyrp3 and Pglyrp4 limit over-activation of Th17 cells by promoting accumulation of Treg cells at the site of chronic inflammation, which protects the skin from exaggerated inflam- matory response to cell activators and allergens. By contrast, Pglyrp1 has an opposite pro-inflammatory effect in the skin. Results Pglyrp32/2 and Pglyrp42/2 mice have enhanced inflammatory response in the oxazolone model of atopic dermatitis Repeated epicutaneous sensitization with oxazolone is an established mouse model of atopic dermatitis [3,26]. Initial sensitization of mice with oxazolone through abdominal skin, followed by 6-day rest and application of oxazolone to the ears every other day for 20 days induced in WT BALB/c mice progressive moderate inflammation manifested by some redness and swelling (Figure 1A). Similar application of oxazolone to Pglyrp32/2 or Pglyrp42/2 mice induced significantly enhanced inflammation, manifested first by increased redness and significantly increased swelling, accompanied by scaling (Figure 1A). This enhanced response was unique for Pglyrp32/2 and Pglyrp42/2 mice, because it was not observed in Pglyrp12/2, Pglyrp22/2, and Pglyrp12/2 Pglyrp22/2 mice, and was dominant, because it was still observed in Pglyrp12/2Pglyrp32/2 and Pglyrp22/2Pglyrp32/2 double-knock- out mice and in Pglyrp12/2Pglyrp22/2Pglyrp32/2 and Pglyrp12/2 Pglyrp22/2Pglyrp42/2 triple-knockout mice (Figure 1A). Deletion of Pglyrp1 had the opposite effect – Pglyrp12/2 single knockout mice showed reduced ear swelling on days 16 through 20 of oxazolone application (Figure 1A). These results indicate that in WT mice both Pglyrp3 and Pglyrp4 have a protective effect against severe atopic dermatitis-like inflammation, whereas Pglyrp1 has an enhancing proinflammatory effect and Pglyrp2 has little effect on the response to oxazolone. To determine the pathologic basis of higher sensitivity of Pglyrp32/2 and Pglyrp42/2 mice to oxazolone, we compared the histology of oxazolone-induced skin lesions in WT and Pglyrp- deficient mice. Ears in untreated WT mice have one- to two-cell thick epidermis and few-cell thick subepidermal layer with blood vessels, sebaceous glands, hair follicles, muscle bundles, and central fat and connective tissue layer, with total thickness of approximately 200 mm. Histology of all untreated Pglyrp-deficient mice was similar to WT mice (Figure 2). Sensitization and 10 oxazolone applications to the ears (every other day) induced strong inflammatory response that was very severe in Pglyrp32/2 mice and Pglyrp42/2 mice. Cross-sections of the oxazolone-treated ears revealed severe acanthosis (thickening of the epidermis due to proliferation of keratinocytes), parakeratosis (retention of kerati- nocytes’ nuclei in stratum corneum), and marked thickening of the sub-epidermal layer with spongiosis (intercellular edema) and Pglyrp3/4 Promote Treg and Inhibit Th17 Responses dense cellular infiltrates (composed primarily of mononuclear cells and some polymorphonuclear cells) that were all highly prominent in Pglyrp32/2 mice and Pglyrp42/2 mice (Figure 2A) and in all other double and triple knockout mice deficient in Pglyrp3 (not shown). All these changes are highly characteristic of atopic dermatitis lesions. These mice did not develop rete pegs (downward papillary projections of epidermis), which are charac- teristic of psoriasis, but not atopic dermatitis. WT mice (Figure 2A), Pglyrp12/2 mice, Pglyrp22/2 mice, and Pglyrp12/2Pglyrp22/2 mice (not shown) all showed much less severe acanthosis, parakeratosis, edema, and cell infiltrations, and less thickening of the subepidermal layer. Inflammatory response to oxazolone was accompanied by a marked increase in the concentration of serum IgE, consistent with oxazolone inflammation being a model of atopic dermatitis (which is type I hypersensitivity associated with increased IgE production). The serum IgE levels in Pglyrp32/2 mice and Pglyrp42/2 mice, as well as Pglyrp12/2Pglyrp32/2 and Pglyrp22/2Pglyrp32/2 double- knockout mice and in Pglyrp12/2Pglyrp22/2Pglyrp32/2 and Pglyrp12/2Pglyrp22/2Pglyrp42/2 triple-knockout mice were all significantly higher than in WT and in Pglyrp12/2, Pglyrp22/2, and Pglyrp12/2Pglyrp22/2 mice (Figure 3). Pglyrp2 also played a minor role, because Pglyrp22/2Pglyrp32/2 double-knockout mice and Pglyrp12/2Pglyrp22/2Pglyrp32/2 and Pglyrp12/2Pglyrp22/2 Pglyrp42/2 triple-knockout mice had higher serum IgE levels than Pglyrp32/2 and Pglyrp42/2 single-knockout mice. These results demonstrate that deletion of Pglyrp3 or Pglyrp4 highly predisposes mice to atopic dermatitis-like lesions in response to oxazolone, and thus in WT mice Pglyrp3 or Pglyrp4 protect the skin from excessive inflammation in the oxazolone model of atopic dermatitis. Pglyrp-deficient mice have reduced response in the oxazolone model of contact dermatitis Single epicutaneous sensitization with oxazolone followed by a single epicutaneous challenge in a different area is an established mouse model of allergic contact dermatitis (contact hypersensitiv- ity) due to type IV hypersensitivity [27,28]. Sensitization of mice with oxazolone through abdominal skin, followed by 6-day rest and a single application of oxazolone to the ears induced on the next day a strong inflammatory response in WT BALB/c mice, manifested by redness and swelling (Figure 1B). Similar application of oxazolone to Pglyrp-deficient mice induced significantly lower inflammation, manifested by significantly reduced ear swelling compared to WT mice, especially in Pglyrp12/2 mice, and to some extent in Pglyrp22/2 and Pglyrp42/2 mice. The reduced responsiveness was dominant, because it was still observed in Pglyrp12/2Pglyrp22/2 (most affected), Pglyrp12/2Pglyrp32/2, and Pglyrp22/2Pglyrp32/2 double-knockout mice and in Pglyrp12/2 Pglyrp22/2Pglyrp32/2 triple-knockout mice (Figure 1B). To determine the pathologic basis of this lower response to oxazolone in the contact dermatitis model in Pglyrp-deficient mice, we compared the ear histology in oxazolone-treated WT and Pglyrp-deficient mice. Sensitization and single oxazolone application to the ears induced strong inflammatory response in WT mice manifested by marked spongiosis of the sub- epidermal layer with cellular infiltrates of epidermal and sub- epidermal layers with mononuclear and polymorphonuclear cells (but no acanthosis, parakeratosis, rete pegs, or scaling, which histologically differentiates contact dermatitis from atopic dermatitis and psoriasis) (Figure 2B). Pglyrp12/2 and Pglyrp12/2Pglyrp22/2 mice still had cellular infiltrates, but had substantially reduced swelling, compared to WT mice, mostly due to reduced edema (Figure 2B). These results indicate that in PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24961