Prostaglandin E2 directly inhibits the conversion of inducible regulatory T cells through EP2 and EP4 receptors via antagonizing TGF-β signalling

Background and Purpose Regulatory T (Treg) cells are essential for control of inflammatory processes by suppressing Th1 and Th17 cells. The bioactive lipid mediator prostaglandin E2 (PGE2) promotes inflammatory Th1 and Th17 cells and exacerbates T cell-mediated autoimmune diseases. However, the actions of PGE2 on the development and function of Treg cells, particularly under inflammatory conditions, are debated. In this study, we examined whether PGE2 had a direct action on T cells to modulate de novo differentiation of Treg cells. Experimental Approach We employed an in vitro T cell culture system of TGF-β-dependent Treg induction from naïve T cells. PGE2 and selective agonists for its receptors, and other small molecular inhibitors were used. Mice with specific lack of EP4 receptors in T cells were used to assess Treg cell differentiation in vivo. Human peripheral blood T cells from healthy individuals were used to induce differentiation of inducible Treg cells. Key Results TGF-β-induced Foxp3 expression and Treg cell differentiation in vitro was markedly inhibited by PGE2, which was due to interrupting TGF-β signalling. EP2 or EP4 agonism mimicked suppression of Foxp3 expression in WT T cells, but not in T cells deficient in EP2 or EP4, respectively. Moreover, deficiency of EP4 in T cells impaired iTreg cell differentiation in vivo. PGE2 also appeared to inhibit the conversion of human iTreg cells. Conclusion and Implications Our results show a direct, negative regulation of iTreg cell differentiation by PGE2, highlighting the potential for selectively targeting the PGE2-EP2/EP4 pathway to control T cell-mediated inflammation. What is already known PGE2 promotes inflammatory Th1 and Th17 cells and facilitates T cell-mediated immune inflammation, but the action of PGE2 on Treg cells is debated. What does this study add PGE2 directly acts on T cells to inhibit inducible Treg cell differentiation in vitro and in vivo through its receptors EP2 and EP4 and by antagonising TGF-β signalling. What is the clinical significance Therapeutically blocking the EP4 receptor may be beneficial for management of T cell-mediated autoimmune inflammation.


Introduction
Regulatory T cells (Tregs) are a subset of T lymphocytes that play essential roles not only in the maintenance of immune homeostasis but also in the control of inflammatory responses (Sakaguchi et al., 2020;Savage et al., 2020). Treg cells actively suppress immune responses against autologous and foreign antigens in vitro and in vivo. Evidence from many mouse models and human diseases indicates that eliminating Treg cell numbers or abrogation of their functions leads to a variety of immune-mediated pathologies, including autoimmunity (e.g. multiple sclerosis, active rheumatoid arthritis, and type 1 diabetes), allergies and graft rejection (Brusko et al., 2005;Möttönen et al., 2005;M. Schneider et al., 2006;Viglietta et al., 2004;Zhang et al., 2008). Treg cells are characterised as expression of the surface marker CD25 (i.e. IL-2 receptor a chain, IL-2Ra) and the master transcription factor Forkhead box P3 (Foxp3) and produce the anti-inflammatory cytokine IL-10 (Sakaguchi et al., 2020). Foxp3 controls both Treg cell development and their unique suppressive function (Fontenot et al., 2003;Gavin et al., 2007;Hori et al., 2003). Loss or mutation of Foxp3 expression links to a defective development of CD4 + CD25 + Treg cells, and in turn results in fatal autoimmune and inflammatory diseases, inducing a lymphoproliferative disorder in mice and leading to the IPEX (Immunodysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome in human (Bennett et al., 2001;Brunkow et al., 2001).
There are two main sub-groups of Treg cells in the body: natural (nTreg) and inducible Treg (iTreg) cells. Natural Treg cells arise in the thymus and can migrate into secondary lymphoid organs (spleen, lymph nodes, etc). In addition, iTreg cells can be developed in the periphery by conversion from naïve Foxp3 -T effector (Teff) cells. The cytokine transforming growth factor β (TGF-β) is a regulatory cytokine with an essential role in immune responses as well as in T cell tolerance (M. O. Li et al., 2006;Marie et al., 2006). TGF-β has both a direct role in regulating T effector cell differentiation, proliferation and apoptosis and an indirect role in the maintenance of immune homeostasis (Gorelik & Flavell, 2000;Gu et al., 2012). It has been well documented that TGF-β is required not only for the maintenance of the suppressive function and Foxp3 expression in nTregs but also for induction of Foxp3 expression in naïve CD4 + T cells and convert these cells into iTregs with a regulatory phenotype (W. Chen et al., 2003;Fu et al., 2004;Shanmugasundaram R. & Selvaraj RK., 2010). Lack or blockade of TGFβ signalling reduces Treg cell numbers and impairs suppressive functions, leading to development of autoimmune diseases (Polanczyk et al., 2019).
Prostaglandins (PGs) are a family of bioactive lipid mediators that are generated from arachidonic acid via the activities of cyclooxygenases (COXs) and selective PG synthases (Yao & Narumiya, 2019). PGs, including PGE2, PGD2, PGF2a, PGI2, and thromboxane A2, play essential roles in numerous physiological and pathophysiological processes through autocrine and/or paracrine manners. Among PGs, PGE2 is found in the highest amounts in most tissues and is best studied. PGE2 has diverse effects on the development, regulation, and activity of T cells through binding to its distinct G protein-coupled receptors (called EP1-4) (Yao & Narumiya, 2019). For example, PGE2 inhibits T cell receptor (TCR) signalling, activation and then reduces production of cytokines such as IL-2 and IFN-g through the EP2/EP4-dependent cAMP-PKA pathway (Brudvik & Taskén, 2012). However, PGE2 can also promote Th1 cell differentiation by inducing IL-12Rb1 expression through EP2/EP4-dependent cAMP and PI3K signalling (Yao et al., 2013). Moreover, PGE2 also fosters IL-23-dependent Th17 cell expansion and function by inducing IL-23R expression through EP4/EP2 and the cAMP pathway (J. Lee et al., 2018;Yao et al., 2009). Importantly, emergent studies using pharmacological approaches and transgenic animal models that target PGE2 receptors have demonstrated that the actions of PGE2 on T cells promotes immune-associated chronic inflammatory diseases in rodents and humans (including multiple sclerosis, rheumatoid arthritis, inflammatory skin and gut inflammation) (Q. Chen et al., 2010;Esaki et al., 2010;J. Lee et al., 2018;Robb et al., 2017;Schiffmann et al., 2014;Yao et al., 2009Yao et al., , 2013. While PGE2 was initially described to facilitate iTreg cell differentiation in vitro (Q. Chen et al., 2010;Esaki et al., 2010;J. Lee et al., 2018;Robb et al., 2017;Schiffmann et al., 2014;Yao et al., 2009Yao et al., , 2013, it has also been reported to inhibit Foxp3 induction and reduce Treg cell numbers (L. Chen et al., 2017;H. Li et al., 2017;Sahin & Sahin, 2020). We have recently reported a T cell-independent function of PGE2 on facilitation of Foxp3 + Treg cell responses in the intestine (Crittenden et al., 2021). However, whether and how PGE2 directly influences iTreg cell differentiation remains to be elucidated.
In this study, we have examined the direct actions of PGE2 in iTreg differentiation in vitro and in vivo using mice deficient in EP2 and EP4 receptors and highly selective small molecular reagents that target the respective PGE2 receptors. We found that PGE2 negatively regulated iTreg cell differentiation in vitro by inhibiting TGF-β-driven Foxp3 induction through EP2 and EP4. Lack of EP4 specifically in T cells increased Treg cell generation in vivo. The PGE2 pathway also appears to inhibit human iTreg cell differentiation. Our results have revealed that PGE2 directly acts on T cells to abrogate iTreg cell differentiation, which may contribute to foster T cell-mediated inflammation.

Animals
EP2 +/+ , EP2 -/- (Hizaki et al., 1999), EP4 +/+ , EP4 -/- (Segi et al., 1998), Lck Cre EP4 fl/fl (A. Schneider et al., 2004;Yao et al., 2013), Rag1 -/-, Foxp3 YFP-Cre (Rubtsov et al., 2008) and wildtype C57BL/6 mice were bred and maintained under specific pathogen-free conditions in accredited animal facilities at the University of Edinburgh and Kyoto University. Wild-type mice were purchased from Harlan UK. Age-(>7-weeks old) and sex-matched mice were used. Mice were randomly allocated into different groups and analysed individually. No mice were excluded from the analysis. All experiments were conducted in accordance with the UK Animals (Scientific Procedures) Act of 1986 with local ethical approval from the University of Edinburgh Animal Welfare and Ethical Review Body (AWERB) or approved by the Committee on Animal Research of Kyoto University Faculty of Medicine.

T cell transfer
Naive CD4 + CD25 − CD62L hi T cells were prepared from spleens of EP4 fl/fl or Lck Cre EP4 fl/fl mice by flow cytometry cell sorting. Cells (5 × 10 5 cells per mouse) were transferred intravenously into Rag1 -/mice. Mice were culled at 6 weeks after T cell transfer. Colons were collected for ex vivo analysis of lamina propria leukocytes.

DSS application
Wild type C57BL/6 mice were given drinking water with dextran sulfate sodium (DSS, 2% w/v) or DSS plus indomethacin (5 mg per kg body weight per day) for 5 consecutive days before colons were collected for in vitro analysis of T cells.

DNFB application
EP4 fl/fl and Lck Cre EP4 fl/fl mice were sensitised with 25 µl of 1% (w/v) Dinitrofluorobenzene (DNFB) in acetone/olive oil (4/1, v/v) on shaved abdominal skin on day 0. Skin draining lymph node cells were collected on day 5 for ex vivo analysis of T cells.

T cell isolation and in vitro culture
Mouse CD4 + CD25naïve T cells were isolated from spleens using Miltenyi Treg cell isolation kits. CD4 + CD25 -Foxp3(YFP)naïve T cells and CD4 + CD25 + Foxp3(YFP) + nTreg cells were isolated from Foxp3 YFP-Cre mouse spleens by flow cytometry. 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 (IL-2, rhTGF-b1) and other compounds as indicated for 3 days. Human CD4 + CD45RAnaïve T cells were isolated from peripheral blood of healthy individuals, stimulated with plate-bound anti-CD3 and anti-CD28, and then cultured with IL-2 (10 ng/ml) and/or rhTGF-b1 (10 ng/ml or indicated concentrations) for 3 days. PGE2 (1 µM or indicated concentrations) and its receptor agonists (1 µM) and other small molecular chemicals were added at the beginning of the culture or 24 hours later. Work with human blood cells was approved by the Centre for Inflammation Research (CIR) Blood Resource (AMREC Reference number 20-HV-069).

Isolation of intestinal lamina propria leukocytes
Intestinal lamina propria cells were isolated as described previously (Duffin et al., 2016).

Human gene expression analysis
We retrieved microarray data from Gene Expression Omnibus under an accession code (GSE71571) (Thomas et al., 2015). Raw data were normalized using the GC-RMA method (Wu et al., 2004). When multiple probe sets were present for a gene, the one with the largest variance was selected (Talloen et al., 2010). Change of the normalized expression levels for each gene by aspirin (i.e. aspirin-placebo) in colon biopsies was transformed into Z-score, which was used to estimate the alteration of PGE2 pathway in each patient in response to Aspirin administration. The signature score of PGE2 pathway was estimated using a method described previously (Bueno et al., 2016). Briefly, we curated a gene list representative of PGE2 signature including its synthases and receptors. The final list consisted of PTGS1, PTGS2, PTGES, PTGES2, PTGES3, PTGER2 and PTGER4. We weighted gene expression and computed a signature score per sample using singular-value decomposition. Pearson's correlation coefficient was used to measure the association between PGE2 signature and expression of Treg genes on a Z-score scale.

Statistical analysis
Data were expressed as mean ± SEM, and statistical significance was performed by unpaired Student's t test or analysis of variance (ANOVA) with post hoc Holm-Sidak's multiple comparisons test using Prism software (GraphPad). All P values <0.05 were considered as significant. Correlation analysis was calculated by Pearson's correlation coefficient (r).

PGE2 suppresses mouse iTreg differentiation in vitro.
We firstly examined whether PGE2 had an impact on iTreg differentiation in vitro. We isolated splenic CD4 + CD25naïve T cells from wild-type (WT) C57BL/6 mice, stimulated with anti-CD3 and anti-CD28 antibodies (Abs) and cultured with TGF-b to induce the differentiation of iTreg cells. We added different concentrations of PGE2 (0 to 1000 nM) at the beginning of TCR stimulation on day 0. TGF-b-induced Foxp3 expression in CD4 + T cells was suppressed by addition of PGE2 in a concentration-dependent manner ( Figure 1A, B). To avoid PGE2 inhibition of TCR activation when it was added at the same time of anti-CD3 stimulation (Yao et al., 2013), we tested the effect of PGE2 by postponing its time of addition to 24 h (day 1) after anti-CD3 stimulation. Under this condition, PGE2 still inhibited TGF-b-induced Foxp3 expression ( Figure 1A, B), suggesting that PGE2 prevents TGF-b-induced iTreg cell differentiation independently of its suppression on TCR activation.
To examine whether PGE2 affects the stability of Foxp3 expression on nTreg cells, we sorted splenic CD4 + CD25 + Foxp3(YFP) + nTreg cells from Foxp3 YFP-Cre mice and cultured with TGFb for 3 days. Addition of PGE2 did not affect total percentage of Foxp3 + cells, but appeared to reduce the mean fluorescence intensity (MFI) of Foxp3 (Figure 1E, F). Moreover, PGE2 treatment significantly reduced CD25 expression, leading to a reduction of the CD25 + Foxp3 + nTreg subpopulation (Figure 1E, F). Taken together, these results suggest that PGE2 represses both de novo iTreg cell differentiation and, to a less extent, Treg maintenance.

EP2 and EP4 receptors mediate PGE2 suppression of iTreg differentiation in vitro.
Next, we investigated which PGE2 receptors mediated the suppression of iTreg differentiation.
Given EP2 and EP4 activate the cyclic adenosine monophosphate (cAMP) and PI3K signalling pathways (Yao & Narumiya, 2019), we examined whether these pathways mediate the suppression of iTreg cell induction. We used dibutyryl cAMP (db-cAMP, a cell-permeable cAMP analogue) and isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor that blocks cAMP degradation) to increase the intracellular cAMP levels. Similar to PGE2, both db-cAMP and IBMX prevented TGF-b-dependent conversion of Foxp3 + iTreg cell ( Figure 2F).
Blockade of the cAMP pathway by a PKA inhibitor (H-89) or the PI3K pathway by LY-294002 repressed TGF-b-dependent Foxp3 expression ( Figure 2G). PGE2 had no additive suppression of Foxp3 induction with H-89, but did further reduced Foxp3 expression in the presence of LY-294002 ( Figure 2G). These results indicate that the cAMP/PKA, rather than PI3K, pathway is involved in PGE2-dependent inhibition of iTreg cell differentiation.
During iTreg differentiation, TGF-b firstly activates gene expression of its receptors (i.e. Tgfbr1 and Tgfbr2) on T cells, which were both repressed by the addition of PGE2 ( Figure   3C). TGF-b also stimulates gene expression of Smad6 and Smad7, endogenous inhibitors for TGF-b signalling, which were significantly further upregulated by PGE2 (Figure 3D). These results suggest an inhibitory effect of PGE2 on TGF-b signalling in T cells, as seen in other cell types (Lenicov et al., 2018;P. E. Thomas et al., 2007;Wettlaufer et al., 2017). To further study the possibility of PGE2 influence on TGF-b signalling, we used a small molecular ALK inhibitor which blocks the TGF-b/TGF-b receptor/Smad pathway. ALK inhibitor itself significantly repressed TGF-b-dependent iTreg cell induction, and addition of PGE2 had no additional effects on Foxp3 induction in the present of with the ALK inhibitor ( Figure 3E).

The transcription factor Foxo1 acts downstream of TGF-b receptors, and is responsible for
TGF-b responsiveness in iTreg cell differentiation (Kerdiles et al., 2010). The Foxo1 inhibitor (AS1842856) did not affect TGF-b-dependent Foxp3 induction, but it reversed PGE2 suppression of Foxp3 induction ( Figure 3F). These results suggest that PGE2 suppresses the process of iTreg differentiation by antagonizing TGF-b signalling.
In response to TCR engagement, activated T cells produce large amount of IL-2, which is also essential for iTreg cell differentiation through the transcription factor STAT5 (Davidson et al., 2007;Guo et al., 2013). As PGE2 strongly inhibits TCR activation and IL-2 production, we asked whether PGE2 suppresses iTreg cell induction via inhibiting IL-2-STAT5 signalling. We cultured T cells under the iTreg-skewing condition and used a STAT5 inhibitor (STAT5i). As expected, the STAT5 inhibitor suppressed iTreg cell conversion compared to vehicle control ( Figure 3G). However, PGE2 was still able to further down-regulate Foxp3 expression in the presence of STAT5 inhibitor ( Figure 3G). Thus, IL-2-STAT5 signalling is unlikely to be involved in PGE2 suppression of iTreg cell induction.

Lack of EP4 impairs iTreg cell differentiation in vivo
We have recently found that blockade of endogenous PGE2 production in naïve WT mice by inhibition of COX activities increased Foxp3 + Treg cell numbers in various organs (Crittenden et al., 2021). To examine whether blockade of endogenous PGE2 production also enhances Treg cell responses under inflammatory conditions, we used 2% DSS to induce acute colonic inflammation WT C57BL/6 mice. DSS treatment increased accumulation of total T cells in the colon, which was further enhanced by co-administration of indomethacin, a non-selective COX inhibitor ( Figure 4A). This is consistent with previous report that blocking COX activity exacerbated DSS-dependent intestinal inflammation (Duffin et al. 2016). Interestingly, indomethacin also significantly increased numbers of Foxp3 + Treg cells, but not Foxp3 -Teff cells, in inflamed colons (Figure 4B, C), which was in line with upregulated Foxp3 gene expression in the colon tissues ( Figure 4D). These results suggest that endogenous PG signalling represses Treg cell response under inflammatory conditions.
To further examine whether PGE2 signalling directly modulates Treg cell responses in vivo.
We crossed EP4-floxed mice to Lck-Cre mice to generate T cell-specific EP4 deficient mice (Lck Cre EP4 fl/fl ). Lck Cre EP4 fl/fl and control EP4 fl/fl mice had comparable nTregs in the thymus (Crittenden et al., 2021), suggesting that lack of EP4 signalling in T cells does not affect nTreg cell development in vivo. To examine whether PGE2 affects iTreg cell differentiation in vivo, we sorted naïve CD3 + CD4 + CD25 -CD62L + T cells from Lck Cre EP4 fl/fl and control EP4 fl/fl mice, and then transferred these cells into Rag1 -/mice that have no T and B cells (Figure 4E). Upon transfer, naïve T cells are activated, proliferated and differentiated into T effect cells (e.g. Th1 and Th17 cells) in the host mice and accumulated in the large intestines. Simultaneously, a small population of T cells are differentiated into Foxp3 + iTreg cells. Lack of EP4 signalling reduced total T cells migration to the colon and down-regulation of T cell activation evidenced by reduction of CD25 expression (Figure 4F). In contrast, differentiation of Foxp3 + Tregs in the host mouse colons from EP4-deficient naive T cells was greater than that from control EP4sufficient naïve T cells (Figure 4G). In agreement with our previous findings (Yao et al., 2013), Rag1 -/mice transferred with EP4-deficient naïve T cells had less IFN-g + Th1 cells compared to mice that were transferred with control naïve T cells, but EP4 deficiency had no influence on colonic IL-17 + Th17 cells in the host mice ( Figure 4H). To further confirm the effect of EP4 signalling on Treg responses in vivo, we sensitised Lck Cre EP4 fl/fl and control EP4 fl/fl mice with a hapten dinitrobenzfluorene (DNBF) on the abdominal skin and analysed T cells in skin-draining lymph nodes. Again, lack of EP4 signalling in T cells significantly increased Foxp3 + Treg cells but reduced Foxp3effector T cells in draining lymph nodes ( Figure 4I, J). Together, these results indicate that PGE2-EP4 signalling directly acts on T cells to impede iTreg cell differentiation in vivo.

Inhibition of human iTreg cell differentiation by PGE2
To corroborate our findings from mouse T cells, we examined whether PGE2 suppresses human iTreg cell differentiation. We isolated CD4 + CD45RAnaïve T cells from peripheral blood of healthy individuals, stimulated with anti-CD3 and anti-CD28 Abs, and cultured with IL-2 alone or IL-2 plus TGF-b. Addition of PGE2 had few effects on Foxp3 expression in T cells cultured with IL-2 from most donors. However, PGE2 suppressed TGF-b-dependent Foxp3 expression in T cells from 3 out of 4 donors (Figure 5A, B), suggesting that PGE2 may similarly inhibit TGF-b-dependent human iTreg cell differentiation.
We then asked whether the expression levels of PGE2 signalling pathway genes were correlated with Foxp3 gene expression in human tissues. We examined a public dataset from a clinical trial which measured gene expression of colon biopsies obtained from healthy individuals before and after administration of aspirin (325 mg/d, daily for 60 days) ( Thomas et al., 2015).
Changes in PGE2 pathway genes by aspirin treatment were negatively correlated with changes in Foxp3 gene expression ( Figure 5C). In contrast, changes in expression of HPGD (which mediates the metabolic inactivation of PGE2 to 15-keto PGE2) was positively correlated with changes in FOXP3 gene expression ( Figure 5C). These results suggest that the PGE2 pathway is associated with down-regulation of Foxp3 gene expression in healthy human gut tissues.

Discussion
In this report, we show that PGE2 directly acts on T cells to abrogate TGF-b signalling and iTreg cell differentiation, especially reducing the CD25 + Foxp3 + subpopulation. Although PGE2 did not affect percentages of the Foxp3 + population in nTreg cells cultured with TGF-b, it reduced Foxp3 expression at the single cell level. Importantly, PGE2 reduced CD25 expression in both iTreg and nTreg cells, indicating that PGE2 may lower Treg cell suppressive function. This finding is consistent with other reports, showing that PGE2 down-regulates Treg cell responses despite PGE2 initially being suggested to facilitate the induction of Foxp3 expression and iTreg cell differentiation in vitro (Baratelli et al., 2010;Sharma et al., 2005;Trinath et al., 2013). For example, PGE2 inhibited Foxp3 induction and Treg cell proliferation from mouse and human CD4 + CD25naïve T cells in vitro (B. P. L. Lee et al., 2009;H. Li et al., 2017;L. Li et al., 2019;Sahin & Sahin, 2020). In addition, PGE2 was also reported to suppress IL-27-dependent, IL-10-producing type 1 Treg cell differentiation (Hooper et al., 2017). Moreover, we have very recently discovered that PGE2 suppresses Treg cell accumulation in the intestine through a T cell-independent mechanism (Crittenden et al., 2021).
We have found here that PGE2 suppression of iTreg cell differentiation is mediated by PGE2 receptors EP2 and EP4 in vitro and that T cell-specific EP4 deficiency enhanced iTreg cell differentiation in vivo, suggesting that the receptor EP2 is dispensable for PGE2 suppression of iTreg development in vivo. This is consistent with previous findings of the actions of PGE2 on Th1 and Th17 cell functions in vivo (Yao et al., 2009(Yao et al., , 2013. Indeed, blockade of EP4 alone sufficiently diminishes Th1/Th17 cell-mediated immune inflammation such as multiple sclerosis, arthritis, and skin inflammation although blockade of EP2 may have additional effects under certain circumstances (Esaki et al., 2010;J. Lee et al., 2018;Yao et al., 2009Yao et al., , 2013. During iTreg cell differentiation, TCR engagement induces T cell activation and production of cytokines such as IL-2 which through activation of STAT5 boosts the induction of Foxp3 expression (Guo et al., 2013). Inhibition of STAT5 activity reduced Foxp3 expression during iTreg cell differentiation, which was further repressed by additional PGE2, excluding the possibility that PGE2 inhibits Foxp3 induction through the TCR-IL-2-STAT5 pathway.
Like actions on other T cell subsets, the inhibition of iTreg cell differentiation by PGE2 is also mediated by EP2/EP4-activated cAMP signalling. In Th1 and Th17 cells, cAMP signalling directly induces expression of IL-12Rb2 and IL-23R, key cytokine receptors for Th1 and Th17 cell differentiation, respectively (J. Lee et al., 2018;Yao et al., 2013). The mechanism for PGE2 inhibition of iTreg cell differentiation is through down-regulation of TGF-b signalling, possibly by reducing expression of TGF-b receptors. Engagement of TGF-b receptors results in phosphorylation of SMAD2/3 and formation of a complex with SMAD4. After translocation into the nuclear, the SMADs complex binds to the CREB/CBP/p300 complex, then in turn regulates transcription responses, including Foxp3 transcription in CD4 + CD25 -T cells (Bodor et al., 2007). Indeed, deficiency of CBP and p300 in Foxp3 + Treg cells impairs Treg cell stability and suppressive function, resulting in over-activation of effector T cells and autoimmune inflammation (Yujie Liu et al., 2014). The transcription factor CREB has also been implicated, as being essential for TCR-induced Foxp3 gene expression (Kim & Leonard, 2007). However, deficiency of CREB in T cells actually decreases Treg cell proliferation and survival and expands Th17 cell response, resulting in exacerbation of T cell-mediated autoimmune inflammation (Wang et al., 2017). Thus, PGE2-EP2/EP4 signalling activated CREB via cAMP-PKA signalling may contribute to our finding that PGE2 prevents TGF-bdependent induction of Ki-67 + Foxp3 + iTreg cells. Furthermore, the cAMP/PKA/CREB pathway has also been reported to antagonise the TGF-b/SMADs pathway in multiple cell types (Schiller et al., 2003). Likewise, we found that PGE2 suppressed expression of TGF-b receptor genes (e.g. Tgfbr1, Tgfbr2) but upregulated gene expression of TGF-b pathway inhibitors (e.g. Smad6, Smad7) in T cells. Lack of TGF-b or its receptors or interruption of TGF-b/SMAD signalling prevents Treg cell development (Yongzhong Liu et al., 2008). It is noteworthy that PGE2 also inhibited TGF-b/IL-6-induced Th17 cell differentiation although it markedly upregulated IL-23-driven Th17 cell expansion (Yao et al., 2009). Additionally, TGFβ-driven Foxp3 strongly down-regulates expression of endogenous inhibitors of TGF-β signalling such as SMAD6 and SMAD7 (Fantini et al., 2004), which interference with SMADspecific gene transactivation. Therefore, down-regulation of TGF-β receptors and upregulation of TGF-β signalling inhibitors by PGE2 may collaboratively lead to diminished TGF-b responsiveness during iTreg cell differentiation.
PGE2 signalling, especially through the EP4 receptor, is critical for T cell-mediated chronic autoimmune inflammation in numerous organs including skin, joint, brain and intestine etc (Yao & Narumiya, 2019). This was considered to be mediated by promoting inflammatory Th1 and Th17 cells. Our findings in this report suggest that inhibition of Treg cells may be also a mechanism involved in PGE2 exacerbation of immune inflammation. Especially, as PGE2 suppresses iTreg cell responses in vivo, evidenced by transfer of specific EP4 deficiency in T cells reducing Foxp3 + iTreg cells in colons of host Rag1 -/mice, which may partially contribute to reduced Th1 cell-medicated colonic inflammation (Yao et al., 2013). Furthermore, lack of EP4 in T cells reduced Foxp3 + Treg cell accumulation in draining lymph nodes in antigen sensitized mice, which may also contribute, at least in part, to reduced skin inflammation after challenge with the same antigen (Yao et al., 2009(Yao et al., , 2013. In addition to previous findings that PGE2 indirectly suppresses intestinal Tregs through modulation of the gut microbiota (Crittenden et al., 2021), our current results have revealed a direct action of PGE2 on T cells to negatively regulate Treg cell differentiation in vitro and in vivo through EP4 and EP2. These functions of PGE2 on Treg cells, together with its positive influences on Th17 and Th1 cells, contribute to facilitation of T cell-mediated tissue inflammation. PGE2 inhibition of Foxp3 expression was observed in not only mouse but human T cells during iTreg cell differentiation. Furthermore, negative correlations between the PGE2-EP4 pathway and Foxp3 gene expression was observed in healthy human subjects after use of aspirin which inhibits COX activity and PGE2 biosynthesis. Thus, therapeutically targeting PGE2-EP4 signalling in T cells may be beneficial for treating immune-mediated inflammation, partially due to modulation of Treg cells.  CD4 + CD25naïve T cells cultured with IL-2 and TGF-β1 with dm-PGE2 or selective agonists for each EP1-4 receptor for 3 days. (C,D) Percentages of Foxp3 + T cells in EP4 +/+ (C) or EP4 -/-(D) CD4 + CD25naïve T cells cultured with IL-2 and TGF-β1 with dm-PGE2 or selective agonists for each EP1-4 receptor for 3 days. (E) Percentages of Foxp3 + T cells in wild type C57BL/6 CD4 + CD25naïve T cells cultured with IL-2 and TGF-β1 in the absence or presence of PGE2, EP2 antagonist or EP4 antagonist or both EP2 and EP4 antagonists for 3 days. (F) Percentages of Foxp3 + T cells in wild type C57BL/6 CD4 + CD25naïve T cells cultured with IL-2 and TGF-β1 with db-cAMP or IBMX for 3 days. (G) Percentages of Foxp3 + T cells in wild type C57BL/6 CD4 + CD25naïve T cells cultured with IL-2 and TGF-β1 with PGE2, a PKA inhibitor (H-89) or a PI3K inhibitor (LY-294002) for 3 days. All experiments were performed in triplicates and repeated at least twice independently. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns, not significant. Percentages of live Foxp3 + T cells in CD4 + CD25naïve T cells cultured with IL-2 and indicated concentrations of TGF-β1 in the absence or presence of PGE2 for 3 days. (C,D) Expression of Tgfbr1, Tgfbr2, Smad6 and Smad7 genes in CD4 + CD25naïve T cells cultured with or without anti-CD3/CD28, TGF-β1 or PGE2 for 3 days. (E-G) Percentages of CD25 + Foxp3 + T cells in CD4 + CD25naïve T cells cultured with IL-2 and TGF-β1 in the absence or presence of PGE2 and inhibitors for ALK (ALKi, E), Foxo1 (Foxo1i, F) or STAT5 (STAT5i, G) for 3 days. Geometric mean fluorescent intensity (gMFI) of Foxp3 among Foxp3 + T cells (G). *P<0.05; **P<0.01; ***P<0.001. ns, not significant.  Representative flow cytometry dot-plot of Foxp3 and CD25 expression in CD4 + CD45RAnaïve T cells that were isolated from healthy human blood, stimulated with anti-CD3 and anti-CD28, and cultured IL-2 alone or IL-2 + TGF-β1 in the absence or presence of PGE2 for 3 days. (B) Accumulated percentages of CD25 + Foxp3 + human iTreg cells from four individual donors. (C) Microarray gene expression data from human colon biopsies in response to aspirin administration for 2 months in healthy individuals was analysed for the association of the PGE2 pathway signature gene expression with that of Treg-related genes. Correlations between the PGE2 signature scores or HPGD expression levels and Foxp3 gene expression from total tested samples (n=88). Raw gene expression data were retrieved from Gene Expression Omnibus GSE71571. Standardized expression values represent changes of gene expression levels before and after aspirin treatment and then transformed to Z-scores. Each dot represents one sample. Statistical analysis was calculated by two-tailed Pearson correlation coefficients (r), and a linear regression-fitting curve is shown as the red dotted line.