S of immunization (Figure 3a and b). At the dose of

S of immunization (Figure 3a and b). At the dose of 2 that is commonly used in this FITC assay, there was no significant difference in the degree of migration between EP3KO and B6 mice in accordance with our previous report [10]. Intriguingly, however, the number of migrated FITC+ cells was apparently increased in EP3KO mice when the concentration of FITC was 0.5 (Figure 3a and b). We measured the PGE2 levels in FITC-applied skin. We collected the skin samples using punch biopsy (8 mm in diameter) 24 hours after 0.5 FITC application. Skin sections were homogenized in PBS 10457188 and PGE2 levels in the supernatant were measured with a PGE2 EIA kit (Cayman Chemical). PGE2 levels with or without FITC applications were comparablebetween B6 and EP3KO mice (Table S1). These data suggest that the difference of migrated DCs between B6 and EP3KO mice depends not on the expression level of PGE2 in the skin, but on the EP3 signaling after binding of PGE2 to EP3 receptor. This indicates that EP3 attenuates the migration of cutaneous DCs only when the antigen is applied at a suboptimal dose. We Title Loaded From File further examined the number of migrated FITC+ cutaneous DCs by dividing into Memory Th1 repertoire.Persisting bim2/2 SMARTA “Memory” Cells are Functionally DefectiveThe Langerin+ DCs (including LCs and Langerin+ dermal DCs) and Langerin- dermal DCs, and found that both subsets were increased in EP3KO mice (Figure 3c). Therefore, EP3 signaling restrains the migration of cutaneous DCs when the external stimuli are subtle.EP3 Signaling Regulates the Cutaneous DC FunctionsFigure 3. Enhanced cutaneous DC migration in EP3KO mice. (a) The number of FITC+ MHC class II+ DCs in the draining lymph nodes from EP3KO and WT mice 96 hours after application of 0.5 or 2 FITC (n=4). (b) Representative flow cytometry of lymph node cells. (c) The numbers of FITC+ LCs (FITC+ Langerin+ cells) or dermal DCs (FITC+ Langerin- cells) in the draining lymph nodes of the mice after application of 0.5 FITC. White and black columns indicate B6 and EP3KO mice, respectively (n=4). Each data represents the mean + SD. *p<0.05.doi: 10.1371/journal.pone.0069599.gSuboptimally primed CHS response was inhibited by EPWe examined whether the restriction on cutaneous DC functions by EP3 is also involved in the CHS response that is a murine allergic contact dermatitis model. CHS is a cascade of sequential events that starts with the sensitization phase and is terminated by the priming of hapten-specific T cells. Reexposure of the hapten initiates the second phase of CHS, which is called the elicitation phase. It is well known that cutaneous DCs play a pivotal role in the sensitization phase by migrating into the draining lymph nodes to activate T cells. By sensitizing abdomens with 50 of 0.5 2,4-dinitro-1fluorobenzene (DNFB) five days before, we could elicit earswelling in both B6 and EP3KO mice after 24 hours of challenge with 20 of 0.3 DNFB. Importantly, sensitization with 0.05 DNFB was suboptimal for normal B6 mice and did not elicit ear swelling after the challenge. However, the same sensitization did elicit ear swelling in EP3KO mice at a comparable level to that of optimally sensitized mice (Figure 4a). Histological analysis confirmed significant inflammation in the ears of 0.05 DNFB-sensitized EP3KO mice (Figure 4b and Table S2). Accordingly, there was approximately twice the amount of IFN- transcripts in the draining lymph nodes of 0.05 DNFB-sensitized and challenged EP3KO mice compared to that of B6 mice (Figure 4c). Moreover, careful observation revealed that EP3KO mice exhibi.S of immunization (Figure 3a and b). At the dose of 2 that is commonly used in this FITC assay, there was no significant difference in the degree of migration between EP3KO and B6 mice in accordance with our previous report [10]. Intriguingly, however, the number of migrated FITC+ cells was apparently increased in EP3KO mice when the concentration of FITC was 0.5 (Figure 3a and b). We measured the PGE2 levels in FITC-applied skin. We collected the skin samples using punch biopsy (8 mm in diameter) 24 hours after 0.5 FITC application. Skin sections were homogenized in PBS 10457188 and PGE2 levels in the supernatant were measured with a PGE2 EIA kit (Cayman Chemical). PGE2 levels with or without FITC applications were comparablebetween B6 and EP3KO mice (Table S1). These data suggest that the difference of migrated DCs between B6 and EP3KO mice depends not on the expression level of PGE2 in the skin, but on the EP3 signaling after binding of PGE2 to EP3 receptor. This indicates that EP3 attenuates the migration of cutaneous DCs only when the antigen is applied at a suboptimal dose. We further examined the number of migrated FITC+ cutaneous DCs by dividing into Langerin+ DCs (including LCs and Langerin+ dermal DCs) and Langerin- dermal DCs, and found that both subsets were increased in EP3KO mice (Figure 3c). Therefore, EP3 signaling restrains the migration of cutaneous DCs when the external stimuli are subtle.EP3 Signaling Regulates the Cutaneous DC FunctionsFigure 3. Enhanced cutaneous DC migration in EP3KO mice. (a) The number of FITC+ MHC class II+ DCs in the draining lymph nodes from EP3KO and WT mice 96 hours after application of 0.5 or 2 FITC (n=4). (b) Representative flow cytometry of lymph node cells. (c) The numbers of FITC+ LCs (FITC+ Langerin+ cells) or dermal DCs (FITC+ Langerin- cells) in the draining lymph nodes of the mice after application of 0.5 FITC. White and black columns indicate B6 and EP3KO mice, respectively (n=4). Each data represents the mean + SD. *p<0.05.doi: 10.1371/journal.pone.0069599.gSuboptimally primed CHS response was inhibited by EPWe examined whether the restriction on cutaneous DC functions by EP3 is also involved in the CHS response that is a murine allergic contact dermatitis model. CHS is a cascade of sequential events that starts with the sensitization phase and is terminated by the priming of hapten-specific T cells. Reexposure of the hapten initiates the second phase of CHS, which is called the elicitation phase. It is well known that cutaneous DCs play a pivotal role in the sensitization phase by migrating into the draining lymph nodes to activate T cells. By sensitizing abdomens with 50 of 0.5 2,4-dinitro-1fluorobenzene (DNFB) five days before, we could elicit earswelling in both B6 and EP3KO mice after 24 hours of challenge with 20 of 0.3 DNFB. Importantly, sensitization with 0.05 DNFB was suboptimal for normal B6 mice and did not elicit ear swelling after the challenge. However, the same sensitization did elicit ear swelling in EP3KO mice at a comparable level to that of optimally sensitized mice (Figure 4a). Histological analysis confirmed significant inflammation in the ears of 0.05 DNFB-sensitized EP3KO mice (Figure 4b and Table S2). Accordingly, there was approximately twice the amount of IFN- transcripts in the draining lymph nodes of 0.05 DNFB-sensitized and challenged EP3KO mice compared to that of B6 mice (Figure 4c). Moreover, careful observation revealed that EP3KO mice exhibi.