We further analyzed the transcriptional mechanisms underlyin

We further analyzed the transcriptional mechanisms underlying the synergistic action of IL-23 and PGE2 and found that this action is mediated by not only STAT3 but also CREB1 and NF-κB. Involvement of CREB1 is analogous to that in the PGE2-EP2/EP4–mediated Il12rb2 induction during TH1 cell differentiation and might be consistent with the findings by Hernandez et al showing that the CREB1/CRTC2 pathway regulates expression of IL-17A and IL-17F and that TH17 differentiation is defective in CRTC2 mutant mice. IL-23R and IL-12Rβ2 make a pair with the same molecule, IL-12Rβ1, to form IL-23R and IL-12R, respectively. It is interesting that the same pathway regulates expression of these 2 genes. We have also used T wehi from p105 NF-κB1–deficient mice and CTP-NBD and unraveled the involvement of NF-κB in the IL-23/cAMP–induced Il23r expression in TH17 cells. Consistent with these findings, we previously found that PGE2, through EP2 or EP4, activates NF-κB1 containing NF-κB in various types of cells, including macrophages and dendritic cells, and induces expression of inflammation-related genes, including COX2, which then produces PGE2 and amplifies this process.47, 54, 64 Thus our present findings further extend the importance of this COX2–PGE2–EP2/EP4–NF-κB loop to generation of TH17 cell pathogenicity. On the other hand, Boniface et al suggested that PGE2-induced enhancement of Il23R expression in human TH17 cells was mediated by the IL-1β–IL-1 receptor pathway. This is also a possibility in mice because upregulated expression of Il1r1 and Il1b was detected in clusters 1U and 4U by using our microarray analysis (Fig 4, B, left). However, we assume that this mechanism is not critical in our experiment because addition of anti–IL-1β antibody to the medium did not reduce Il23r induction (see Fig E7 in this article\'s Online Repository at www.jacionline.org). In addition to Il23r, our microarray analysis has revealed that stimulation of EP2/EP4 signaling together with IL-23 facilitates expression of a variety of pathogenic TH17 signature genes (ie, Il17a, Il17f, Il18r1, and Tgfb3). Interestingly, PGE2-EP2/EP4 signaling also upregulated expression of various genes related to chemotaxis and migration, such as S1pr1, Ccr2, Cxcl3, Cx3cr1, Cxcr4, Sema4f, Sell, Sema3c, and Sema6a (Fig 4, B, left). These results suggest that PGE2-EP2/EP4 signaling can contribute to migration, infiltration, and accumulation of TH17 cells into the inflammatory lesion. On the other hand, the addition of db-cAMP downregulated expression of Il10, Il2, Il4, and Il9, which are known as suppressive factors for TH17 cells. Although some of these results, such as IL-17A, are consistent with the previous findings in human TH17 cells, our study did not detect induction of IFN-γ and T-bet in cultured TH17 cells, which might reflect the stages of TH17 cells examined in each study.20, 24, 65 It should also be noted that our analysis was carried out on the whole CD4+ T-cell population pretreated with IL-6 and TGF-β1 and stimulated with each stimulus, in which IL-17A+ cells comprised about 10% of cells. Therefore single-cell RNA sequencing analysis is desired to establish gene expression signatures specific to TH17 cells matured with each stimulus. Nonetheless, the most important point in our study was that the EP2/EP4 signaling in TH17 cells identified here is critical in eliciting their pathogenicity in vivo in immune inflammation. We tested this issue in an IL-23–induced mouse psoriasis model. Intriguingly, not only the systemic inhibition of EP2/EP4 signaling with the EP4 antagonist in EP2 KO mice but also selective loss of EP2 and EP4 in T cells almost completely suppressed inflammation induced by IL-23. This was accompanied by suppression of accumulation of IL-17A+ and IL-17A+IFN-γ+ T cells and suppression of expression of Il17a, Ifng, and Il23r genes in the lesion. These results suggest that PGE2-EP2/EP4 signaling functions is critical to generation of pathogenic TH17 cells induced by IL-23 in situ. Of those TH17 cells, antigen-specific TH17 cells were shown to be involved specifically in the pathogenesis of mouse models of autoimmune inflammation, including experimental autoimmune encephalomyelitis, type 1 diabetes, and psoriasis. Quite recently, it was also reported that mPGES1 is involved in generation of antigen-specific TH17 cells by regulating PGE2 production in a T-cell autocrine and paracrine manner. Our present findings combined with these findings suggest that PGE2 plays an important role in psoriasis through regulation of antigen-specific pathogenic TH17 cells.