Pglyrp3/4 Promote Treg and Inhibit Th17 Responses Figure 4. Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4 expression is increased in the affected skin in the atopic dermatitis and contact dermatitis models of inflammation. The amounts of each PGRP mRNA in WT mice and in the indicated Pglyrp-deficient mice sensitized and treated with oxazolone every other day (atopic dermatitis model) or sensitized and treated with oxazolone at 0 hrs only (contact dermatitis model) were measured by qRT-PCR. The results are means of 3–4 mice 6 SEM; *, P,0.04; **, P,0.001, treated versus untreated; ¤, P,0.04; ¤¤, P,0.001, Pglyrp2/2 versus WT. doi:10.1371/journal.pone.0024961.g004 Pglyrp22/2 and Pglyrp12/2Pglyrp22/2 mice were similar (most cells) or increased (monocytes), compared to WT mice (not shown), consistent with comparable cell infiltrates seen in all mice on tissue sections on day 1 (Figure 2B). To further define the cell types responsible for differential sensitivity of Pglyrp-deficient mice to atopic dermatitis and contact dermatitis, we measured the expression of an extended panel of cytokines, chemokines, and other marker genes characteristic of Th1, Th2, Th17, Treg, NK, and other cell types to determine which of these genes were differentially induced in the affected skin in Pglyrp-deficient mice, compared to WT mice. We included these cell types, because in addition to Th1 and Th2 cells, Th17 cells and other cell types may also be involved in these skin diseases. Prolonged epicutaneous sensitization with oxazolone (20 days), which induced atopic dermatitis-like skin inflammation, was accompanied in WT mice by high activation (more than 15-fold) of 14 out of 44 studied genes in the affected skin. These genes were Ifng (Th1), Il4 and Il10 (Th2), Cxcl2, Cxcl5, and Il21 (Th17), Cxcl9 and Cxcl10 (NK cells), and Ccl3, Ccl4, Ccr5, Fasl, Il1b, and Il6 (several cell types) (Figure 7A and Figures S3 and S4). Atopic dermatitis-sensitive mice initially (day 13) had several genes activated higher than in WT mice, characteristic of several cell types, including Th2 and Th17 (Figure 7A and Figure S3). Later (day 20), atopic dermatitis-sensitive mice (Pglyrp32/2, Pglyrp42/2, and Pglyrp22/2Pglyrp32/2 mice) had four genes characteristic of Th17 cells (Cxcl1, Cxcl5, IL17a, IL22) and one gene characteristic of several cell types (Ccl2) activated more than three-fold higher in Pglyrp-deficient than in WT mice (Figure 7A and Figure S4). A single oxazolone challenge in sensitized WT mice (in the contact dermatitis model) also induced many genes characteristic of several cell types, and the early (6 hrs) activation of these genes in Pglyrp-deficient mice was mostly reduced, compared to WT mice (Figure 7B and Figure S5). These results are consistent with lower clinical responses of Pglyrp-deficient mice to a single oxazolone challenge in the contact dermatitis model (Figures 1B and 2B). The above results indicate that the atopic dermatitis-sensitive Pglyrp-deficient mice have increased activity of Th17 cells in the affected skin, compared to WT mice. To further investigate the role Th17 cells (and other Th cell types) in increased sensitivity of Pglyrp-deficient mice in atopic dermatitis model, we used flow cytometry to directly measure Th cell types in the ears, draining lymph nodes, and the spleen. Untreated ears in WT and Pglyrp2/2 mice had ,400 CD4+ cells/ear, whereas after sensitization and 20 days of oxazolone treatment the numbers of CD4+ cells/ear increased .50 times to ,18,000–19,000/ear in WT and Pglyrp32/2 mice (Figure 8A). Regarding Th cell subpopulations, oxazolone treatment for 13 days induced significantly higher numbers of Th2 cells (CD4+IL- 4+) in the affected ears in Pglyrp32/2 mice compared to WT mice, whereas oxazolone treatment for 20 days induced significantly PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24961