before oxazolone application with Digimatic Micrometer (Mitu- toyo, Japan) under constant pressure at the lowest setting. Ear swelling was determined by subtracting the untreated ear thickness. The significance of differences in ear swelling was determined using t-test. To induce contact dermatitis female mice were first sensitized with 50 ml of 2% oxazolone (Sigma, in 80% acetone, 20% olive oil) applied to the abdomen (after removing hair with Nair cream) and 5 ml applied to each paw. The contact dermatitis reaction was then elicited 6 days later with a single application of 20 ml of 1% oxazolone (in 80% acetone, 20% olive oil) to each ear (10 ml to each side) [27,28]. Ear thickness was measured as described above before and 24 and 48 hrs after oxazolone application, and ear swelling was determined by subtracting the untreated ear thickness. The significance of differences in ear swelling was determined using t-test. Histology and immunohistochemistry For histological analysis ears were fixed in Bouin’s fixative, postfixed in 70% ethanol, and embedded in paraffin, and 5 mm cross-sections were stained with hematoxylin/eosin, and eval- uated microscopically. For immunohistochemistry, antibodies to mouse Pglyrp3 and Pglyrp4 were obtained by immunizing rabbits with peptides corresponding to the following amino acids: CLVPQHSEIPKKA for Pglyrp3 (exon 5), and CWENPQTDQV- SEG for Pglyrp4 (exon 2), coupled to KLH, followed by affinity purification on SulfoLink gel (Pierce) with corresponding peptides linked through the N-terminal Cys, elution with Tris-glycine buffer, pH 2.5, and dialysis against PBS, pH 7.2. A rabbit IgG antibody to a different peptide, which did not react with mouse Pglyrp3 and Pglyrp4, prepared and purified by the same method, was used as a negative control. Paraffin 5 mm cross-sections of were stained by the immunoperoxidase method as previously described [8], including standard deparaffinization, re-hydration, quenching of endogenous peroxidase by 30 min incubation in 0.3% H2O2, and incubation with 0.5 mg/ml of anti-Pglyrp3 or Pglyrp4 antibodies or control IgG overnight, followed by biotinylated second Ab and Vectastain Elite ABC kit (Vector) with DAB as a substrate (which generates brown reaction product) and counterstaining with hematoxylin (blue). RNA and quantitative real-time reverse transcription PCR (qRT-PCR) RNA was isolated from either the entire untreated or treated ears or lymph nodes using the TRIZOL method (InVitrogen), followed by digestion with RNase-free DNase (Qiagen) and purification on RNeasy spin columns using RNeasy Minikit (Qiagen). Quantitative reverse transcription real-time PCR (qRT- PCR) was used to quantify the amounts of mRNA in the ears or lymph nodes using custom RT2 Profiler PCR Arrays designed by us and manufactured by Qiagen/SA Biosciences, as previously described [22,25]. The arrays typically included 30 to 44 assay genes, 5 housekeeping genes and reverse transcription efficiency and DNA contamination controls. All primer sets were from Qiagen/SA Biosciences, except the following primers designed by us: Pglyrp1, exons 1 and 2 primers, GTGGTGATCTCACACA- CAGC and GTGTGGTCACCCTTGATGTT; Pglyrp2, exons 3 and 4 primers, ACCAGGATGTGCGCAAGTGGGAT and AGTGACCCAGTGTAGTTGCCCA; and Pglyrp4, exons 4 and 5 primers, CGACCAGGGCTACAAGAA and CCAGG- CAGTCTTCACTTTTC. cDNA was synthesized from 2 mg of RNA using RT2 PCR Array First Strand Kit (Qiagen/SA Biosciences) and the arrays were performed according to the manufacturer instructions using Qiagen/SA Biosciences Master Pglyrp3/4 Promote Treg and Inhibit Th17 Responses Mix. The lists of genes are provided in the figures. The experiments were performed on RNA pooled from 4–5 mice/ group and repeated 3 times usually with another set of 4–5 mice/ group (usually total of 8–10 mice per treatment). For each gene, DCt was calculated using the same threshold (0.2) for all genes and Ct#35 considered as no expression, followed by normalization to 5 housekeeping genes (Hsp90ab1, Gusb, Hprt1, Gapdh, and Actb) included in each array, followed by calculation of DDCt for each gene from two arrays: DDCt = DCt12DCt2, where DCt1 is the oxazolone treated mice and DCT2 is the untreated mice, using the program provided by Qiagen/SA Biosciences. This calculation gives the fold increase in expression of each gene in the treated mice versus untreated mice per mg RNA. The genomic DNA contamination controls, reverse transcription controls, and positive PCR controls were included in each array and were all passed. Additional control to assure amplification from RNA, but not from possible contaminating DNA included parallel reaction sets from which reverse transcrip- tase was omitted, and which showed no amplification. To compare baseline gene expression in untreated mice, DCT1 was from untreated PGRP-deficient mice and DCT2 was from untreated WT mice. The results were reported as mean fold increases after oxazolone treatment (treated/untreated) for WT mice, or ratios of fold increases in Pglyrp-deficient to WT mice, calculated as follows: [(Pglyrp2/2 treated)/(Pglyrp2/2 untreated)]/[(WT treat- ed)/(WT untreated)] and presented as heat maps or bar graphs. The latter fold differences (ratios) of .1 or ,1 reflect higher or lower expression levels of the genes (respectively) in Pglyrp-deficient than in WT mice. Heat maps were generated using Java TreeView after converting ,1 ratios to negative fold difference using the formula: (21)/ratio. In some bar graph figures, ,1 ratios were also converted to negative fold difference using the formula: (21)/ ratio. The significance of differences in gene activation between groups of mice was determined using the two-sample one-tailed t- test, and typically the differences of .2 fold were significant at P,0.05. Expression of mRNA for PGRPs and some chemokine receptors was similarly measured by qRT-PCR using Qiagen/ SA Biosciences First Strand Kit (with random primers) and amplification for 40 cycles with Qiagen/SA Biosciences SYBR Green Master Mix, and calculated using comparative cycle threshold method with 5 housekeeping genes (Hsp90ab1, Gusb, Hprt1, Gapdh, and Actb) as controls. Isolation of cells and flow cytometry Mouse ears were placed in RPMI-1640 with 3 mg/ml of Dispase II (Roche), separated into dorsal and ventral halves and scored on the dermal side with a scalpel. The tissue was digested for 8 hrs at 37uC in 5% CO2. Dermis was then separated from the epidermis and epidermis was further digested with 0.25% trypsin in RPMI-1640 for 10 min at 37uC. Cells were washed twice with RPMI-1640 with 5% fetal bovine serum (FBS) and incubated for 20 hrs in the same medium at 37uC in 5% CO2. Cells were then strained through a 40 mm filter and resuspended at 2.06107 cells/ ml in RPMI-1640 with 5% FBS. Single cells from cervical lymph nodes and spleen were obtained by passing the tissue through a 40 mm filter, red blood cells were removed from the spleen cells with a lysis buffer (Biolegend), and cells were suspended at 2.06107 cells/ml in RPMI-1640 with 5% FBS. 16106 cells were stained with CD4-APC (clone RM4-5, Biolegend) antibody for 20 min at 4uC. CD4-stained cells were then stained for Foxp3-PE (clone FJK-16s, eBioscience) or for cytokines IFN-c-PE (clone XMG1.2), IL4-PE (clone 11B11) and PLoS ONE | www.plosone.org 14 September 2011 | Volume 6 | Issue 9 | e24961