Pglyrp3/4 Promote Treg and Inhibit Th17 Responses inflammatory response in WT mice with marked spongiosis of the sub-epidermal layer (Sp) and cellular infiltrates of epidermal and sub-epidermal layers, composed of mononuclear and polymorphonuclear cells; 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. H&E stained cross-sections; bar = 200 mm for all panels, except high magnification insets (the magnified areas are shown by rectangles). doi:10.1371/journal.pone.0024961.g002 treatment with oxazolone induced increased Pglyrp1 mRNA expression in the ears in all strains of mice (except Pglyrp12/2 mice, in which Pglyrp1 gene is deleted) that was significantly higher than in untreated mice (Figure 4). The expression of Pglyrp1 was significantly higher in all Pglyrp-deficient mice2 than in WT mice early (day 13) in the atopic dermatitis model, which correlates with their higher inflammatory response. This increase in Pglyrp1 mRNA in the ears is likely due to increased infiltration with PMNs, because PMNs highly express Pglyrp1 and expression of Pglyrp1 is not inducible in any of the cell types previously studied, including lymphocytes, monocytes, macrophages, and keratino- cytes [8,14,15]. The expression of Pglyrp2 was only modestly increased early in the atopic dermatitis model and returned to the untreated level later in this model, and was not significantly changed at any time point in the contact dermatitis model, consistent with the little effect of Pglyrp2 in both atopic and contact dermatitis models (Figure 1). Pglyrp1 and Pglyrp2 were also constitutively expressed in the cervical lymph nodes at a similar level as in the ears, but following oxazolone treatment their expression in the cervical lymph nodes did not significantly change (data not shown), despite extensive stimulation and expansion of immune cells in the draining lymph nodes, which increased in diameter from ,0.5 mm in untreated mice to .3 mm after 20 days of oxazolone treatment. Pglyrp3 has high constitutive expression in untreated skin [8,21]. Pglyrp3 mRNA expression initially increased and later decreased in the atopic dermatitis model, and decreased in the contact dermatitis model. Pglyrp4 has lower constitutive expres- sion in untreated skin than Pglyrp3. Expression of Pglyrp4 mRNA was highly increased in the atopic dermatitis model and was the highest in Pglyrp22/2Pglyrp32/2 and Pglyrp32/2 mice, suggesting a possibility of compensatory expression of Pglyrp4 in mice deficient in Pglyrp3 or changes in the regulation of transcription or stability Figure 3. Pglyrp32/2 and Pglyrp42/2 mice have higher serum IgE levels than WT mice in the oxazolone model of atopic dermatitis. Sensitization followed by 10 applications of oxazolone to the ears every other day induced higher level of serum IgE in Pglyrp32/2 and Pglyrp42/2 mice than in WT, Pglyrp12/2 or Pglyrp22/2 mice; means 6 SEM of 8–20 mice/group; *, P,0.002; **, P,0.0001, Pglyrp2/2 versus WT. doi:10.1371/journal.pone.0024961.g003 of Pglyrp4 mRNA, since Pglyrp3 and Pglyrp4 genes are tightly linked in the psoriasis sensitivity locus on chromosome 3 in mice and their expression is correlated with the expression of keratinocytes differentiation genes [21]. To further investigate which cells express Pglyrp3 and Pglyrp4 in the untreated and oxazolone-treated skin, we analyzed the expression of Pglyrp3 and Pglyrp4 proteins by immunohistochemistry. Pglyrp3 and Pglyrp4 were expressed in epidermal keratinocytes in untreated ears, and in oxazolone-treated ears they were primarily expressed in the upper layers of differentiated keratinocytes (Figure 5). Pglyrp3 and Pglyrp4 expression was not induced in other cell types, such as infiltrating inflammatory cells in oxazolone-treated ears. More- over, Pglyrp3 and Pglyrp4 were not expressed in the cervical lymph nodes in untreated mice and their expression there was not induced following oxazolone treatment (data not shown). These results indicate that Pglyrp3 and Pglyrp4 are primarily expressed in keratinocytes, but not in immune or inflammatory cells, and thus these results raise the possibility that Pglyrp3 and Pglyrp4 exert their protective effects in the oxazolone-induced atopic dermatitis by modulating the function of keratinocytes, which are known to produce many pro-inflammatory cytokines and chemokines. Pglyrp32/2 and Pglyrp42/2 mice have increased Th17 cells and Th17 responses in the skin To determine the cellular basis for the differences in the inflammatory responses in Pglyrp-deficient mice, we next deter- mined the types of inflammatory cells in the ears in both models of skin inflammation, and then we determined which cell types significantly differed in Pglyrp-deficient mice compared to WT mice. This was first accomplished by measuring the amounts of mRNA for several marker genes characteristic of various immune and inflammatory cell types in the untreated and the affected ears. To determine which marker genes (and thus cell types) are increased or decreased in Pglyrp-deficient mice compared to WT mice, we calculated how many times higher or lower they were induced in Pglyrp-deficient mice than in WT mice (fold induction in Pglyrp-deficient mice/fold induction in WT mice). Prolonged treatment with oxazolone in the atopic dermatitis model induced increases in mRNA of several cell types in WT mice, and especially mature B cells, CD8+ T cells, monocytes, PMNs, and mast cells, as well as changes associated with de- differentiation, proliferation, and activation of keratinocytes, which are all expected changes consistent with the atopic dermatitis model (Figure 6A and Figure S1). Atopic dermatitis- sensitive mice (Pglyrp32/2, Pglyrp42/2, and Pglyrp22/2Pglyrp32/2 mice) had increased expression of genes characteristic of B cells and T cells. Also increased was Rorct mRNA, which is preferentially expressed in Th17 cells [29] (Figure 6A and Figure S1). Short-term treatment with oxazolone in the contact dermatitis model in WT mice induced increases primarily in monocytes and PMNs at 6 hrs after challenge (Figure 6B and Figure S2) and later in T cells (18 hrs, not shown). Contact dermatitis-resistant mice (Pglyrp12/2, Pglyrp22/2, and Pglyrp12/2Pglyrp22/2 mice) had initially (6 hrs after challenge) increased B cells and Treg cells and decreased other cell-types, compared to WT mice (Figure 6B and Figure S2). Later (18 hrs after challenge) the cell markers in PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24961