Prostaglandin E₂ stimulates adaptive IL-22 production and promotes allergic contact dermatitis

Abstract

Background

Atopic dermatitis (AD) and allergic contact dermatitis (ACD) are both forms of eczema and are common inflammatory skin diseases with a central role of Th22-derived IL-22 in their pathogenesis. Although prostaglandin E₂ (PGE₂) is known to promote inflammation, little is known about its role in processes related to AD and ACD development, including IL-22 up-regulation.

Objectives

To investigate whether PGE₂ has a role in IL-22 induction and development of ACD, which has increased prevalence in patients with AD.

Methods

T-cell cultures and in vivo sensitization of mice with haptens were used to assess the role of PGE₂ in production of IL-22. The involvement of PGE₂ receptors and their downstream signals were also examined. The effects of PGE₂ were evaluated using the oxazolone (OXA)-induced ACD mouse model. The relationship of PGE₂ and IL-22 signaling pathways in skin inflammation were also investigated using genomic profiling in human lesional AD skin.

Results

PGE₂ induces IL-22 from T cells through its receptors EP2 and EP4 and involves cyclic adenosine monophosphate (cAMP) signaling. Selective deletion of EP4 in T-cells prevents hapten-induced IL-22 production in vivo, and inhibition of PGE₂ synthesis limits atopic-like skin inflammation in the OXA-induced ACD model. Moreover, both PGE₂ and IL-22 pathway genes were coordinately up-regulated in human AD lesional skin, but were below significant detection levels after corticosteroid or ultraviolet band B (UVB) treatments.

Conclusions

Our results define a crucial role for PGE₂ in promoting ACD by facilitating IL-22 production from T-cells.

Introduction

Atopic dermatitis (AD) and allergic contact dermatitis (ACD) are both forms of eczema and are common chronic inflammatory skin diseases. The prevalence of AD is up to 3-7% in adults and up to 25% among children^1,2 and the prevalence of ACD is up to 7% among the general population³. ACD and AD potentially share common cellular mechanisms and there is an increased rate of ACD in patients with AD³. Histologic features of affected eczematous skin include epidermal hyperplasia and spongiosis, and infiltration of immune cells (T-cells, dendritic cells/DCs, eosinophils, etc) in the dermis^1,3,4. Barrier dysfunction is also recognized as important for development of AD^5–7. In response to antigen and hapten sensitization, keratinocytes are activated to produce pro-inflammatory cytokines. Concurrently skin resident innate immune cells like dendritic cells capture antigens and migrate to secondary lymphocytes, where they present antigens to T lymphocytes for their activation and differentiation into effector T cells, resulting in ACD initiation when the antigen or hapten is repeatedly applied to the skin³. Both type 1 and 2 helper T (Th1 and Th2) cells, characterized by production of IFN-γ or IL-4/IL-13, respectively, has been thought to contribute to ACD pathogenesis depending on the antigens⁸. For example, while the majority of haptens (e.g. 1-Fluoro-2,4-dinitrobenzene) induce a Th1-dominant response, some others like fluorescein isothiocyanate induce Th2-dominant responses⁹. While single hapten elicitation induces delayed-type hypersensititivity, as a mouse model of human contact dermatitis¹⁰, repeated hapten challenge results in a chronic late-phase and immediate-type hypersensitivity response, which are clinically relevant to the acute phase of AD in humans^11–13. However, recent studies have shown that IL-17 and IL-22, produced by Th17 and other activated T cells including Th22 cells, are also critical in mediating the initiation and progression of ACD and AD^14–20. In patients with ACD to nickel, there were upregulated levels of IL-22 in both serum and inflamed skin^19,20. Furthermore, IL-22 levels in serum from patients with AD are higher than those from healthy individuals^21,22, and IL-22⁺ T cells infiltrate into lesional AD skin²³, where IL-22 induces epidermal hyperplasia and inhibits epidermal differentiation^24,25. Importantly, a neutralizing anti-IL-22 antibody (NCT01941537) or targeting components of the IL-23/Th17 pathway, are currently being tested in clinical trials^4,26. To refine future therapies, there is a need to better understand the mechanisms that drive IL-22 production in response to cutaneous antigen stimulation.

Prostanoids, including prostaglandin D2 (PGD₂), PGE₂, PGF_2α, PGI₂ and thromboxane A₂, are bioactive lipid mediators that are generated from arachidonic acid by cyclooxygenases (COX) and then respective PG synthases. PGs have various roles in inflammatory skin diseases through regulating functions of immune cells, including Th1/Th17 T-cells, T regulatory cells, mast cells and DCs²⁷. PGE₂ is synthesized by microsomal prostaglandin E synthases (mPGES1 and mPGES2) and has essential roles in modulating various inflammatory responses by binding to PG receptors (EP1-4) on cell surfaces. Many cutaneous cells, including keratinocytes, mast cells, eosinophils, fibroblasts, DCs and lymphocytes, express PGs and PG receptors²⁸. Increased PGE₂ expression in biologically active amounts has been reported in both lesional and non-lesional skin from AD patients²⁹. Blockade of PG production by a COX2 inhibitor was reported to enhance eosinophil infiltration and elevate IL-4 expression in lesions of an OVA-sensitized mouse model³⁰. In contrast, PGE₂ was also suggested to induce AD by favoring a Th2 immune milieu and directly enhancing B-cell production of IgE^31,32. Furthermore, we and others have recently reported that PGE₂ through EP2 and EP4 receptors augmented IL-17 and IL-22 productions, and blockade of PGE₂ signaling during T-cell differentiation limited acute contact hypersensitivity^33–35. These findings suggest that PGE₂ may have both suppressive and provocative roles in the development of ACD and AD²⁸. However, it is unclear how PGE₂ regulates IL-22 production and chronic, atopic skin inflammation.

Here we report that PGE₂ promotes IL-22 production from Th17 and Th22 T-cells through its receptors EP2 and EP4. This effect is mediated by cAMP-protein kinase A (PKA) signaling and induction of aryl hydrocarbon receptor (AHR), a transcription factor critical for both adaptive and innate IL-22 expression³⁶. T-cell specific EP4 deficiency diminishes hapten-induced IL-22-expressing T cells in skin-draining lymph nodes (LNs). Accordingly, inhibition of PGE₂ production limits skin inflammation in an animal model for ACD induced by repeated OXA challenges in mice. Furthermore, genes related to PGE₂ signaling are over-expressed in human atopic lesional skin, and positively correlate with expression of IL-22 pathway genes. This relationship between expression of PGE₂ and IL-22-related genes is no longer evident after successful corticosteroid or UVB treatments. These findings suggest that PGE₂ facilitates ACD through promoting adaptive IL-22 signaling.

Methods

Mice

Wild-type C57BL/6 mice were purchased from Harlan UK. Lck-Cre mice were crossed to lox-flanked Ptger4 (EP4-floxed) mice³⁷ to generate mice with selective EP4 deficiency in T cells (EP4cKO) as previously reported^34,35. All mice were bred and maintained under specific pathogen-free conditions in accredited animal facilities in the University of Edinburgh and Kyoto University. Mice were aged >7 weeks old at the beginning of use and sex-matched. All experiments were conducted in accordance with the UK Scientific Procedures Act of 1986 and had local institutional ethical approval.

Reagents

Antibodies to mouse CD3 (clone 145-2C11), CD28 (clone 37.51), CD45 (clone 30-F11), CD4 (clone L3T4), CD8 (clone 53-6.7), IL-17A (clone eBio17B7) and IL-22 (clone IL22J0P) were from eBioscience or Biolegend. Mouse CD4 microbeads were from Miltenyi Biotec. Recombinant human TGF-β1 and recombinant mouse IL-6 and IL-23 were purchased from Biolegend. PGE₂, 17-phenyl trinor PGE₂ (EP1/3 agonist), Butaprost (EP2 agonist), CAY10598 or L-902,688 (EP4 agonist), PF-04418948 (EP2 antagonist), and L-161,982 (EP4 antagonist) were from Cayman. Db-cAMP, PKA Inhibitor 14-22 (PKI), CH-223191, oxalozone, indomethacin, phorbol myristate acetate (PMA), Ionomycin were from Sigma or Calbiochem.

Oxazolone-induced ACD model

The OXA-induced mouse ACD model was induced as reported¹¹. Briefly, mice were sensitized with 3% OXA in EtOH on shaved abdominal skin and after 5 days were repeatedly challenged with 0.6% OXA in EtOH on one ear once every two days for a total of 5 challenges. The opposing ear was challenged with vehicle (pure EtOH) to serve as a control. Mice were culled at 6 h after the last challenge and the ears and ear-draining LNs collected for further analysis. Ear samples were fixed with 10% neutral buffered formalin solution (Sigma), embedded in paraffin wax, and 5μm sections used for staining with hematoxylin and eosin (H&E). In some experiments, EP4cKO or control mice were sensitized with 0.5% dinitrofluorobenzene (DNFB), and 5 days later skin-draining LNs were collected for further analysis.

T-cell isolation and culture

Mouse CD4⁺ T-cells were isolated from spleens using autoMACS (Miltenyi). Cells were cultured in complete RPMI1640 medium containing 10% FBS and stimulated with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) antibodies plus various cytokines and other compounds as indicated in figures. For certain experiments, Th17 cells were differentiated from CD4⁺ T-cells by TGF-β1 and IL-6 for 3-4 d. IL-22 levels in supernatants were measured using mouse IL-22 enzyme-linked immunosorbent assay (ELISA) Ready-SET-Go! kits (eBioscience).

Surface and intracellular staining

For surface staining, cells were stained on ice for 30 min with anti-CD45, anti-CD3e, anti-CD4 and anti-CD8 Abs. For intracellular staining of IL-22, cells were stimulated with PMA and ionomycin for 4-5h in the presence of GolgiPlug (BD Bioscience). Cells were then harvested and fixed by BD Cytofix/Cytoperm Fixation Buffer (BD Bioscience) for 30min and then stained with anti-human/mouse IL-22 (clone IL22JOP, eBioscience) or anti-human IL-22 in BD Perm/Wash Buffer for 1h. Flow cytometry was performed on a BD LSRFortessa (BD Bioscience) and analyzed by FlowJo software (Tree Star).

Real-time PCR

RNA purification from T-cells was performed using the Rneasy Mini Kit (Qiagen). cDNA was obtained by reverse transcription using High-capacity cDNA Reverse Transcription Kits (ABI). Samples were analyzed by real-time PCR with SYBR Premix Ex Taq II (Tli RNase H Plus) kit (Takara) or GoTaq qPCR Master Mix (Promega) on the Applied Biosystem 7900HT Fast machine. The following primers were used. Gapdh forward, 5’-TGAACGGGAAGCTCACTGG-3’; Gapdh reverse, 5’-TCCACCACCCTGTTGCTGTA-3’. Il22 forward, 5'-CATGCAGGAGGTGGTACCTT-3'; Il22 reverse, 5'-CAGACGCAAGCATTTCTCAG-3'. Ahr forward, 5’-TGCACAAGGAGTGGACGA-3’; Ahr reverse, 5’-AGGAAGCTGGTCTGGGGTAT-3’. Expression was normalized to mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and presented as relative expression to control group by the 2^–ΔΔCt method.

Gene expression of human skin biopsies from microarray datasets

Microarray gene expression data of human skin biopsies were retrieved from Gene Expression Omnibus datasets (GSE16161, GSE32924, GSE36842, GSE32473 and GSE27887)^38–42. Patients information and skin sample have been described previously^38–40,42. In brief, skin biopsy specimens were collected from patients with moderate-to-severe AD (Scoring of Atopic Dermatitis 20-97) and healthy volunteers under institutional review board–approved protocols (written consent obtained). Patients (age 16-81, mean age 40 from three cohorts) with an acute exacerbation of chronic AD and without any therapy for more than 4 weeks were included. Biopsy specimens were obtained from acute lesional skin which was actively involved, and erythematous lesions with atopic dermatitis and were frozen in liquid nitrogen for RNA extraction^38–40,42. To standardize data across a wide range of experiments and to allow for the comparison of microarray data independent of the original hybridization intensities, gene expression levels were transformed to z-score values⁴³. P values were calculated by nonparametric Wilcoxon-Mann-Whitney test, paired nonparametric tests with post-hoc Dunn’s multiple comparisons or paired 2-way analysis of variance (ANOVA) test with post-hoc Bonferroni's multiple comparisons test. Correlations between expression levels of two genes were calculated by nonparametric Spearman correlation test.

Statistical analyses

All data were expressed as mean ± SEM or scatter dot-plots in which each dot represents one mouse, one AD patient or healthy individual. Statistical significance between two groups was examined by the Student’s t-test or Mann-Whitney test, while the one-way and two-way ANOVA with post-hoc Bonferroni’s multiple comparisons test were used to evaluate multiple groups unless otherwise indicated in figure legends. Statistical analyses were performed using Prism 6 software (GraphPad) and a P<0.05 was considered as statistically significant.

Results

PGE₂ promotes IL-22 production in vitro through its receptors EP2 and EP4

We have recently reported that PGE₂ promotes IL-22 production from group 3 innate lymphoid cells (ILC3s)³⁵. We thus evaluated whether PGE₂ also promoted IL-22 production from T-cells. To address this question, we isolated CD4⁺ T-cells from mouse spleens, activated with anti-CD3 and anti-CD28 antibodies (Abs), and co-cultured with various cytokines. Addition of exogenous PGE₂ enhanced Il22 gene expression with IL-6 or IL-23 alone, IL-6 + IL-23 (Th22 priming condition) or IL-6 + IL-23 + TGF-β (Th17 priming condition) (Fig 1A). IL-22 protein production in supernatants of primary cell cultures was also elevated by PGE₂ in the presence of IL-23 or IL-23 + IL-6 (Fig 1B). It is important to note that these data are in agreement with previous findings⁴⁴ that TGF-β inhibits IL-22 production even in the presence of PGE₂ (Fig 1A,B). Moreover, when same numbers of PGE₂-stimulated T-cells and control T-cells were washed and then re-stimulated with T-cell receptors (i.e. anti-CD3 and anti-CD28), PGE₂-stimulated T-cells produced more IL-22 than control T-cells even in the presence of TGF-β (Fig 1C), suggesting that PGE₂-treated cells have higher capability to produce IL-22 at the single cell level.

Figure 1. PGE₂ promotes T cell production of IL-22 through its receptors EP2 and EP4.

A, Il22 mRNA expression in naïve CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 (anti-CD3/CD28) antibodies (Abs) with indicated cytokines in the absence or presence of PGE₂ (100 nM) for 3 days. B, IL-22 levels in supernatants of cultures in A. C. IL-22 levels in supernatants of naïve CD4⁺ T cells were cultured as in A and then the same numbers of T cells were re-stimulated with anti-CD3/CD28 Abs for another 3 days. D, IL-22 levels in supernatants of naïve CD4⁺ T cells were stimulated with anti-CD3/CD28 Abs with IL-6+IL-23 with or without an EP2 agonist (butaprost, 1 μM) or EP4 agonist (L-902,688, 1 μM) for 3 days and then the same numbers of T cells were re-stimulated with anti-CD3/CD28 Abs for another 3 days. E, IL-22 levels in supernatants from Th17 cells re-stimulated with anti-CD3/CD28 Abs without or with IL-23 and various small molecule compounds activating or inhibiting PGE₂ receptors for 3 days. F, Il22 mRNA of re-stimulated Th17 cells with PGE₂ and/or IL-23 for 3 days. EP1/3 agonist 17-phenyl trinor PGE₂ was used at 1 μM while EP2 antagonist PF-04418948 and EP4 antagonist L-161,982 were used at 10 μM. All experiments were performed in duplicates or triplicates and repeated for 2-3 times independently. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

To define which PGE₂ receptor mediates IL-22 production, we cultured T-cells under Th22-priming conditions and selectively activated EP2 and EP4. Both EP2 and EP4 agonists individually mimicked augmentation of IL-22 production (Fig 1D). To further confirm the roles of PGE₂-EP2/EP4 signaling in inducing IL-22 production of T-cells, we also cultured differentiated Th17 T-cells and found that PGE₂ still promotes IL-23-driven IL-22 gene and protein expression (Fig 1E and 1F). Moreover, enhancement of IL-22 production by EP2 or EP4 agonists was prevented by co-treatment of antagonists against EP2 or EP4, respectively (Fig 1F); elevated IL-22 production by PGE₂ was also diminished by co-administration of EP2 and EP4 antagonists (Fig 1F). In addition, PGE₂ also increased IL-22 production in Th17 cells in the absence of other cytokines (Fig 1E and 1F), suggesting a potentially direct action of PGE₂ to promote Il22 gene expression. These data indicate the involvement of EP2 and EP4 in PGE₂ facilitation of IL-22 production from T cells in vitro.

Cyclic AMP promotes IL-22 production from T cells through the transcription factor aryl hydrocarbon receptor (AHR)

As both EP2 and EP4 receptors activate cAMP-PKA pathway in T cells³³, we next examined whether cAMP mediates PGE₂ facilitation of adaptive IL-22 production. Like PGE₂, db-cAMP promoted IL-22 production in a concentration-dependent manner in either presence or absence of IL-23 (Fig 2A), confirming a direct action of cAMP on IL-22 induction. The increased IL-22 production by cAMP was prevented by PKI, a PKA inhibitor, in a concentration-dependent manner (Fig 2B). Cyclic AMP also up-regulated Il22 gene expression in IL-23-stimulated T-cells, which was prevented by cycloheximide, an inhibitor for eukaryote protein synthesis (Fig 2C and 2D), suggesting that the PGE₂-cAMP pathway promoted adaptive IL-22 production through synthesis of new protein(s).

Figure 2. Cyclic AMP promotes IL-22 production from T cells through induction of aryl hydrocarbon receptor (Ahr).

A, IL-22 levels in supernatants from Th17 cells re-stimulated with anti-CD3/CD28 Abs without or with IL-23 and indicated concentrations of db-cAMP for 3 days. B, IL-22 levels in supernatants from Th17 cells re-stimulated with anti-CD3/CD28 Abs with IL-23, db-cAMP (100 μM) and indicated concentrations of PKI for 3 days. C,D, Il22 mRNA expression levels in Th17 cells re-stimulated with anti-CD3/CD28 Abs with IL-23 and db-cAMP in the absence (C) or presence (D) of cycloheximide (CHX, 1 μM) for 3 days. E, Ahr mRNA expression in naïve CD4⁺ T cells cultured with anti-CD3/CD28 Abs and indicated cytokines in the absence or presence of PGE₂ for 3 days. F-H, Ahr (F) and Il22 (G) mRNA expression and IL-22 levels (H) in supernatants of Th17 cells cultured with anti-CD3/CD28 Abs, IL-23, db-cAMP and CH-223191 (10 μM) for 3 days. All experiments were performed in duplicates or triplicates and repeated for 2-3 times independently. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. ns, non significant.

The transcription factor AHR has been reported to regulate IL-22 production in both T-cells and ILC3s³⁶. We thus investigated whether AHR contributes to PGE₂-cAMP signaling-dependent increase in adaptive IL-22 production. We first checked that both PGE₂ and cAMP up-regulated AHR gene expression in T-cells independently of cytokine stimuli (Fig 2E and 2F). Importantly, a small-molecule AHR inhibitor CH-223191 effectively suppressed cAMP-dependent IL-22 gene expression and protein production from T cells (Fig 2G and 2H) although it had no effect on AHR expression itself (Fig 2F). These results suggest that PGE₂-cAMP signaling promotes IL-22 production from T-cells through induction of AHR.

PGE₂-EP4 signaling promotes adaptive IL-22 production in vivo

To investigate whether PGE₂ promotes adaptive IL-22 production in vivo, we topically sensitized the abdominal skin of control mice and mice with specific EP4 deletion in T cells (EP4cKO mice³⁴) with DNFB and measured IL-22-producing T-cells in skin-draining LNs 5 days after sensitization using flow cytometry. Both percentages and absolute numbers of IL-22-producing T-cells were markedly reduced in EP4cKO compared to control mice (Fig 3A and 3B). Furthermore, EP4 deficiency reduced Il22 gene expression in skin-draining LN CD4⁺ T cells (Fig 3C). These data indicate that PGE₂-EP4 signaling promotes adaptive IL-22 production in vivo.

Figure 3. PGE₂-EP4 signaling in T cells facilitates IL-22 production in vivo.

A, Representative flow cytometry dot-plot of IL-22 and IL-17 expression in skin-draining LN CD4⁺ T cells from T cell-specific EP4 deficient (EP4cKO, n=9) mice or control mice (n=10) 5 days post sensitization with 0.5% DNFB. B, Percentages and numbers of IL-22⁺ CD4⁺ T cells. Each dot represents one mouse. C, Il22 mRNA expression levels in CD4⁺ T cells isolated from skin-draining LNs of EP4cKO (n=6) or control (n=6) mice. Each dot represents one mouse. *P<0.05.

Endogenous PGE₂-EP4 signaling in T cells promotes allergic contact dermatitis

We next investigated whether PGE₂ has a pathogenic role in development of allergic skin inflammation. To address this question, we used an animal model mimicking human ACD. We sensitized wild-type C57BL/6 mice with 3% oxalozone (OXA) on abdominal skin and after 5 days we repeatedly challenged mice with 0.6% OXA every two days for a total of 5 challenges. Indomethacin was administrated to mice in drinking water to inhibit endogenous PGE₂ production. Once DCs in the skin capture the antigen OXA, they migrate to skin dLNs and present antigen to T cells for their activation. Because the migration of antigen-capturing DCs to skin dLNs peaks between 18-24h requires PGE₂-EP4 signaling⁴⁵, we only treated mice with indomethacin 24 h post the OXA sensitization (Fig 4A). Consistent with previous reports¹¹, repeated challenge with OXA induced allergic skin inflammation (Fig 4B and 4C). Ears of OXA-challenged, indomethacin-treated mice showed decreased swelling compared to vehicle-treated mice (Fig 4B). Indomethacin had no effect on the thickness of vehicle (EtOH)-challenged ears. Compared to EtOH-challenged ears, histology of OXA-challenged ear skin showed features of eczema, namely parakeratosis, acanthosis and focal spongiosis with a dense and diffuse dermal infiltrate containing eosinophils (Fig 4C). However, in skin from the indomethacin-treated mice the epidermal changes are less pronounced, and the dermal infiltrates are markedly reduced (Fig 4C). Furthermore, whilst repeated OXA challenge recruited CD4⁺ and CD8⁺ T cells to ear-draining LNs (Fig 4D), indomethacin treatment reduced both this recruitment and the T-cell capacity to produce IL-22 (Fig 4E). To further investigated whether this pro-inflammatory effect of PGE2 on OXA-induced ACD development was mediated by EP4 and T cells, we performed this model on EP4cKO and control (EP4^fl/fl) mice. OXA induced significantly less ear skin inflammation in EP4cKO mice compared to control mice (Fig. 4F), suggesting that PGE₂-EP4 signaling in T cells promotes ACD development.

Figure 4. PGE₂ exacerbates atopic skin inflammation in the repeated oxalozone (OXA) challenge model.

A-E, WT C57BL/6 mice were sensitized with 3% OXA on abdominal skin on (day 0) and then challenged with 0.6% OXA or EtOH on ears 5 days later. Challenge with OXA was repeated once every two days for a total of 5 challenges. Ears and ear-draining LNs were collected 6 h after the last OXA challenge (n=4-5). A, Schematic representation of the experimental protocol. B, Ear thickness. C, Ear histology. Scale bar, 50 μM. D, CD4⁺ and CD8⁺ T cells in ear-draining LNs. E, IL-22 production by ear-draining LN cells cultured with soluble anti-CD3 or medium only in vitro for 3 days. F, Swelling of ears from control and EP4cKO mice (n=9-13) sensitized and repeatedly challenged with OXA as in A but without indomethacin treatment. *P<0.05; ***P<0.001 and ****P<0.0001.

PGE₂-dependent IL-22 production and signaling in human atopic lesional skin

Because ACD and AD potentially share common cellular mechanisms⁴ and the repeated hapten challenge-induced animal model is relevant to the acute phase of AD in humans¹¹, we next investigated whether PGE₂-dependent adaptive IL-22 production and signaling can be found in human atopic skin. We analyzed mRNA expression of genes related to PGE₂ metabolism (Fig 5A) and IL-22 signaling pathways in human lesional AD and normal skin biopsies. As reported previously³⁹, mRNA expression levels for Th17/Th22 related genes (IL17A, IL22, IL23R, AHR), IL-22-induced products (e.g. S100A7, S100A8, S100A9), and skin-homing chemokines (CCR4) were over-expressed in lesional AD compared to normal skin (Fig 5B). Strikingly, mRNA expression levels for PGE₂ synthases (PTGS2, PTGES, PTGES2) were also up-regulated in human atopic lesional skin, and expression levels for the PGE₂ degradation enzyme HPGD (hydroxyprostaglandin dehydrogenase 15-(NAD)) was in contrast down-regulated in lesional AD skin (Fig 5B). These gene expression data are consistent with previous reports showing increased PGE₂ levels in lesional AD skin²¹. There was a trend that expression of PTGER4 gene (encoding PGE₂ receptor EP4) was up-regulated in lesional AD skin, but expression of PTGER2 (encoding PGE₂ receptor EP2) was significantly down-regulated in lesional AD skin (Fig. 5B). Expression of Th2 cytokines (e.g. IL4, IL5, IL13) were not significantly up-regulated in lesional AD skin, however Th2 chemokines such as CCL26, CCL18, CCL22, CCL17, CCL11 and CCL5 were up-regulated in lesional AD skin compared to normal skin (Fig 5B). Interestingly, expression of IL-22 pathway genes showed strongly positive correlation with those of PTGES in biopsy samples from lesional AD but not normal skin (Fig 5C). There were no correlations between PTGES expression and Th2 cytokines (data not shown). However, weak correlations between PTGES gene expression and Th2 chemokines in lesional AD skin were observed (Fig 5D). These results suggest that PGE₂ signaling is activated and positively correlates with the IL-22 signaling pathway and, probably, the Th2 pathway in human atopic skin.

Figure 5. Over-expression of PGE₂ signaling genes in human lesional AD skin which positively correlate with expression of IL-22 signaling genes.

A, Schematic depicting synthesis and metabolism of PGE₂ mediated by COX2 (encoding by PTGS2), PGE synthases (encoding by PTGES or PTGES2) and 15-PGDH (encoding by HPGD), respectively. A PGE₂ receptor EP4 (encoding by PTGER4) is also shown. B, Heat map of expression profiles of Th2 cytokine and chemokine genes and genes related to IL-22 and PGE₂ pathways in human lesional AD skin (n=38) and normal skin (n=32). A color scale bar indicates the Z-score transformed values of microarray gene expression data³⁸–⁴⁰. Z-ratios represent the changes in gene expression levels between the normal and AD lesional groups⁴³. P values were calculated by nonparametric Wilcoxon-Mann-Whitney tests. Probes with the largest Z-ratios were chosen when several probes represented single genes. C,D, Correlations of PTGES gene expression versus expression of IL-22 pathway genes (C) or Th2 chemokines (D) in atopic lesional and normal skins. P value was calculated by nonparametric Spearman correlation test.

Finally, we investigated whether current therapies for atopic dermatitis modulate PGE₂ signaling in human AD. We analyzed changes in expression levels of IL-22 and PGE₂ signaling genes in lesional AD skin before and after treatments such as betamethasone (a corticosteroid) by re-analyzing a public microarray dataset⁴¹. Compared to baseline levels, treatments with betamethasone reduced IL22 gene expression in atopic lesional skin. Betamethasone also significantly reduced expression of IL-22-related genes such as AHR, S100A8, S100A9 (Fig 6A). Expression of PGE₂ synthases (e.g. PTGES, PTGES2) and EP4 receptor (PTGER4) was significantly downregulated by betamethasone treatment, while EP2 receptor (PTGER2) expression was not changed (Fig 6A). This observation is consistent with findings that betamethasone treatment reduces PGE₂ production^46,47. Interestingly, expression of PTGES, PTGES2 and PTGER4, but not PTGER2, positively correlated with IL22 expression in atopic biopsies at baseline, while expression of HPGD, which mediates PGE₂ degradation, negatively correlated with IL22 expression (Fig 6B). Furthermore, we have also reanalyzed gene expression of skin biopsies before and after treated with UVB from our previous dataset⁴². Expression of PGE₂ synthases PTGS1 and PTGES in lesional AD skin were down-regulated after treatment with UVB (Fig 6C). Interestingly, although IL-22 gene expression was not changed by UVB treatment, most likely due to the small number of samples, changes in expression of IL-22 activated genes (e.g. S100A8 and S100A9) positively correlated with changes in expression of PGE₂ synthases (i.e. PTGS1, PTGES and PTGES2) in atopic lesional, but not in non-lesional biopsies (Fig 6D and data not shown). Together, these data indicate that PGE₂, most likely through the EP4 receptor, plays a role in IL-22 regulation in human skin.

Figure 6. Steroid and UVB therapies down-regulate PGE₂ and IL-22 signaling pathway genes in human atopic lesional skin.

A, Expression profiles of genes related to PGE₂ and IL-22 pathways in human lesional AD skin (n=10) at baseline (AD Base) or after treatment with betamethasone for 3 weeks. The z-score transformed values of microarray gene expression data⁴¹ were used. P value was calculated by paired nonparametric tests with post-hoc Dunn’s multiple comparisons test. B, Correlations of PTGES, PTGES2, HPGD, PTGER4 gene expression versus that of IL22 gene in human atopic lesional skins at baseline. P value was calculated by nonparametric Spearman correlation test. C, Expression of genes related to PGE₂ and IL-22 signaling in human atopic lesional (AL, n=8) and non-lesional (ANL, n=7) skin before (PRE) or 12 weeks after (POST) treatment with UVB. The z-score transformed values of microarray gene expression data⁴² were used. P value was calculated by paired 2-way ANOVA test with post-hoc Bonferroni's multiple comparisons. D, Correlations of changes in expression of IL-22-induced genes (S100A8, S100A9) and PGE₂ synthase genes (PTGS1, PTGES and PTGES2) before (PRE) and after (POST) UVB therapy in atopic AL or NL skins. P value was calculated by nonparametric Spearman correlation test.

Discussion

The cytokine IL-22, notably produced by Th22 and Th17 T-cells, is emerging as a key player in AD^3,16,23, leading to a new therapy targeting IL-22 signaling in AD clinical trials (NCT01941537). Genome-wide association studies have identified that gene polymorphisms of the PGE₂ receptor EP4 were associated with T cell-mediated human autoimmune and allergic inflammatory diseases^48,49. However, the role of PGE₂ in promoting IL-22 production and development of AD remain to be determined. To address this, ideally a model which showed the features of AD would be used. However, as no single animal model can fully capture all aspects of the human AD profile⁵⁰ and there is acceptance that there are shared common cellular mechanisms between AD and ACD³, we have used a repeated hapten challenge ACD model to study the role of PGE₂ signaling in IL-22⁺ T cells and development of chronic skin inflammation. We also analysed gene expression in human AD lesional skin before and after common treatments for AD to examine the PGE₂-IL-22 aixs in the relevant human skin disease in both the chronic inflammatory phase and following therapeutic intervention.

We show that PGE₂ markedly stimulates IL-22 production from T-cells, which complements our previous findings that PGE₂ promotes innate IL-22 production from ILC3s³⁵. It is worth noting that PGE₂ promotes IL-22 production from freshly activated CD4⁺ T-cells under various conditions including Th17 and Th22-priming conditions. The effect of PGE₂ was mimicked by activation of EP2 and EP4 receptors and was prevented by antagonists against these receptors, confirming the involvement of these two receptors in adaptive IL-22 production in vitro. Further studies showed that the cAMP-PKA signaling pathway, activated by engagement of EP2 and EP4, mediates PGE₂ enhancement of IL-22 production through induction of newly expressed AHR. Our results thus uncover a new, targetable molecular mechanism for regulation of adaptive IL-22 by the PGE₂-cAMP-AHR axis.

The role of PGE₂ in promoting adaptive IL-22 production was also confirmed in vivo. T-cell specific EP4 deficiency led to large reduction of IL-22⁺ T-cells in skin-draining LNs in response to hapten sensitization and attenuated repeated OXA challenge-induced allergic skin inflammation in the mouse model of ACD, indicating a critical role of endogenous EP4 signaling in T-cells for IL-22 expression and function in vivo. This is similar to innate IL-22 expression which also requires PGE₂-EP4 signaling³⁵, suggesting a shared mechanism for regulating both the adaptive and innate IL-22 responses. Inhibition of endogenous PGE₂ production by a COX inhibitor successfully prevented accumulation of T-cells in ear-draining LNs, reduced T-cell production of IL-22 and attenuated allergic skin inflammation induced by repeated OXA challenges. Given that PGE₂ also promotes IL-17 production from both mouse and human Th17 T-cells^33,51 and that IL-17 may participate in creating the ACD phenotype^14–20, IL-17 may also be involved in the PGE₂-facilitated ACD pathogenesis.

Facilitation of IL-22 production by PGE₂ may contribute to several human inflammatory diseases such as AD and psoriasis⁵². Indeed, parallel up-regulations of IL-22 pathway genes, PGE₂ synthases were observed in human atopic lesional skin, whereas the PGE₂ degrading enzyme 15-PGDH (encoded by HPGD) was down-regulated in AD skin. This is consistent with previous observations showing increased PGE₂ levels in lesional AD skin²⁹. Moreover, our data indicate that IL-22 signaling positively correlated with PGE₂ signaling in atopic lesional skin, and these correlations were absent in both normal skin or in lesional AD skin following successful steroid or UVB treatments. These findings potentially suggest a PGE₂-dependent IL-22 production and signaling in human atopic skin.

Besides IL-22⁺ T-cells, Th2 cells (especially cutaneous lymphocyte antigen-positive population with skin-homing capacity) are expanded in severe AD⁵³. Th2 cells suppress major terminal differentiation proteins (i.e. filaggrin and loricrin) and predominates in the acute phase of AD through production of cytokines (e.g. IL4 and IL13) and chemokines (e.g. CCL17, CCL18, CCL26, etc)^40,54. The Th2 response is thus critical for AD pathogenesis. Inhibition of COX2 has been reported to enhance Th2 cytokine production and Th2 response to ovalbumin-induced epicutaneous sensitization³⁰, suggesting a potential inhibitory role of PGs in Th2 cell-driven allergic inflammation. In human lesional AD skin, however, there was no correlations between the PGE₂ pathway and Th2 cytokines and, in contrast, weak positive correlations between the PGE₂ pathway and Th2 chemokines, suggesting a possible promoting role of PGE₂ in Th2 response in AD. This may be because PGE₂ promotes IL-17 and IL-22 production^33–35,49 and subsequently IL-17 exacerbates Th2 type inflammation during the initiation of AD^11,55.

We acknowledge this report has several limitations. For example, our analysis of gene expression profiles in human skin biopsies were retrieved from public GEO datasets and confirmation of these findings was not performed by methods with higher sensitivity such as real-time PCR using fresh biopsies. Moreover, cytokine expression in protein levels in different T-cell subsets were not measured by flow cytometry in skin of patients and animals. Furthermore, although in mice, the OXA-repeated challenge-induced chronic allergic skin inflammation represents a robust model, additional animal models are required in attempt to fully mimic most pathogenic and physiologic processes during the development of human AD. In addition, due to limitation of resources, the effects of PGE₂ signaling in T cells, especially the involvement of both EP2 and EP4 in T cells, on initiation and progression of ACD could not be interrogated, e.g. using mice with T cell-specific deficiency of both EP2 and EP4 receptors. Future prospective studies are therefore required to further understand not only the role of PGE₂ during perpetuating ACD and AD skin inflammation but also during their onset.

In conclusion, our data ascertain that PGE₂ acts as a potent promoter of both adaptive and innate IL-22 production. We have previously found that PGE₂ signaling promotes DC production of IL-23, a cytokine essential for IL-22 expression, as well as for proliferation and maturation of Th17 T-cells³³. Taken together, our results highlight the PGE₂ signaling pathway as an important stimulus of T-cell responses and ACD and, possibly, AD development. Thus targeting PGE₂ synthesis or its receptors may represent a possible therapeutic strategy for the treatment of ACD and AD, and other inflammatory skin diseases such as psoriasis with an active role for IL-22, such as psoriasis.

Clinical Implications.

Allergic contact dermatitis is a common disabling disease characterized by elevated IL-22. The identification of a tightly regulated PGE₂ driven pathway controlling IL-22 dysfunction offers a novel target for therapeutic intervention.

Capsule Summary.

Prostaglandin E₂ promotes IL-22 production from T cells that mediates IL-22-driven development of allergic contact dermatitis.

Abbreviations used

ACD

Allergic contact dermatitis

AD

Atopic dermatitis

AHR

Aryl hydrocarbon receptor

ANOVA

Analysis of variance

cAMP

Cyclic adenosine monophosphate

CCR

Chemokine receptor

COX

Cyclooxygenases

Db-cAMP

Dibutyryl cAMP

DC

Dendritic cell

DNFB

Dinitrofluorobenzene

IL-22

Interleukin 22

ILC3

Group 3 innate lymphoid cells

LN

Lymph node

mPGES

Microsomal prostaglandin E synthases

OXA

Oxazolone

PGE₂

Prostaglandin E₂

PKA

Protein kinase A

Th

Helper T cells

UVB

Ultraviolet band B