The Signaling Pathway of PGE2 and Its Regulatory Role in T Cell Differentiation

Prostaglandin E2 (PGE2) is a lipid mediator derived from the fatty acid arachidonic acid. As an essential inflammatory factor, PGE2 has a critical impact on immune regulation through the prostanoid E (EP) receptor pathway. T cells, including CD4+ and CD8+ T cell subsets, play crucial roles in the adaptive immune response. Previous studies have shown that PGE2 is involved in regulating CD4+ T cell differentiation and inflammatory cytokine production via the EP receptor pathway, thereby affecting the development of diseases mediated by CD4+ T cells. In this review, we summarize the signaling pathway of PGE2 and describe the relationship between PGE2 and T cell differentiation. Hence, this review may provide important evidence for immune therapies and may even promote the development of biomedicines.


Introduction
Prostaglandin (PG) is a lipid mediator family derived from the fatty acid arachidonic acid (AA). Due to differences in their molecular structures, PGs were classified into nine groups, including PGA, PGB, PGD, PGE, PGF, PGG, PGH, PGI, and PGJ. Among these molecules, PGE 2 is a kind of inflammatory factor that has been intensively studied. Although PGE 2 has a short half-life and a 90% degradation rate in pulmonary circulation [1], it plays a significant role in mediating inflammation and multiple physiological processes [2]. PGE 2 functions by prostanoid E (EP) receptors, which include four types of membrane-bound G proteincoupled receptors named EP1 to EP4 mediating the multiple signaling pathway. To date, a series of studies has demonstrated that PGE 2 regulates the differentiation, maturation, and activation of immune cells, especially T cells [3,4].
As essential components in the adaptive immune system, T cells are mainly divided into CD4 + helper T (Th) cells and CD8 + cytotoxic T lymphocytes (CTLs) [5]. After stimulation, CD4 + T cells can differentiate into a variety of effector subsets, including classical Th1 cells and Th2 cells, the subsequently defined Th17 cells, follicular helper T (Tfh) cells, and induced regulatory T (iTreg) cells. Th1 cells are characterized by their secretion of IFN-γ and are involved in cellular immunity, while Th2 cells produce IL-4, IL-5, IL-10, and IL-13 and are required for humoral immunity and allergic reactions. Th17 cells mainly secrete IL-17A, IL-17F, IL-21, and IL-22 and mediate inflammatory response. Tfh cells are a new subset of helper T cells that produce IL-21 and regulate the maturation of B cell responses. Treg cells are characterized by the expression of the forkhead transcription factor (Foxp3) and have essential roles in the maintenance of immune homeostasis by suppressing these effector T cell responses [6]. CTLs play their roles by directly killing infected cells and cancer cells. In this review, we provide a general overview of the metabolism of PGE 2 , the signaling pathways of EPs, and the regulatory role of PGE 2 in T cell differentiation. modified into prostaglandin or leukotrienes [8]. For prostaglandin synthesis, AA released from membranes is oxidized by the cyclooxygenase enzymes COX-1 and COX-2 [9,10]. The enzymatic effect of COX enzymes on AA involves two steps, one is the oxidation of AA to form prostaglandin G2 (PGG 2 ), and the other is the reduction of PGG 2 to form prostaglandin H2 (PGH 2 ). Depending on the specific enzymes, PGH 2 can be modified to five different prostaglandins, including PGD 2 , PGI 2 , PGF 2α , PGE 2 , and thromboxane A 2 (TXA 2 ) [2,7]. PGE 2 synthesis through COX is mediated by three distinct enzymes, cytosolic PGE synthase (cPGES), microsomal PGE synthase-1 (mPGES-1), and mPGES-2 [2].
Nearly all types of cells and tissues can secrete PGs. When PGs are secreted, however, they are quickly degraded by the lung or liver. The lung is the primary organ involved in PGE 2 metabolism [11,12]. PGE 2 metabolism mainly occurs via three mechanisms, leading to the inactive metabolite: (i) 15-hydroxyprostaglandin dehydrogenase (15-PGDH) converts a 15-hydroxyl group to a keto group for metabolism; (ii) 9-ketoreductase reduces 9-ketone to hydroxyl; and (iii) a side chain is inactivated by β-oxidation and/or ω-hydroxylation. The final metabolites of PGE 2 are eliminated from the body in the urine. The effects of PGE 2 are regulated by the balance between its COX-2-regulated synthesis and 15-PGDH-driven degradation [13].

PGE 2 Signaling and EP Receptors.
After PGE 2 is synthesized in the cytoplasm, it diffuses out of the cell through facilitated diffusion. Since PGE 2 is quickly degraded, it is distinct from typical hormones that act on distant target tissues but can only be produced and released locally. After being secreted from cells, the lipid mediator binds to membrane receptors, activates downstream signaling pathways, and exerts biological effects [14]. PGE 2 signals through four morphologically distinct G-protein-coupled receptors, namely, EP1, EP2, EP3, and EP4, and triggers divergent signaling pathways (Figure 2), thereby mediating its various biological functions. The mRNAs of the EP receptors also exhibit different expression patterns in a number of tissues, and activating each receptor subtype leads to distinct functional consequences [15]. EP3 and EP4 are high-affinity receptors and are expressed in all human tissues, whereas the activation of EP1 and EP2 occurs in only a few organs and requires significantly higher levels of PGE 2 [16]. EP receptors are also expressed on various immune cell membranes. EP2 and EP4 are the main receptors of PGE 2 involved in regulating the differentiation of CD4 + T cells [17]. By coupling with the activated G protein, it stimulates adenylate cyclase (AC) to activate the cAMP/ protein kinase A (PKA)/cAMP-responsive element-binding protein (CREB) signaling pathway and downstream molecules to participate in mediating proinflammatory responses and inhibiting PGE 2 activity [18]. It is well known that clear signal transduction of EPs is beneficial for therapeutic strategies against diseases related to the immune system.

EP1
Receptor. In humans, the EP1 receptor has the lowest affinity for PGE 2 [19] and couples to a Gq alpha subunit (Gαq). PGE 2 can result in smooth muscle contraction through this complex. The human single EP1 receptor consists of 402 amino acid residues, whereas the rat single EP1 receptor consists of 405 amino acid residues. The EP1 receptor has 7 hydrophilic transmembrane domains. An arginine residue exists in the 7 th domain, and this residue is an important PGE binding site. The phosphorylation of PGE 2 depends on cyclic adenosine monophosphate-(cAMP-) dependent protein kinase and protein kinase C (PKC). The Gαq subunit activates phosphoinositide-phospholipase C (PLC) and finally leads to an elevation in intracellular Ca 2+ and the activation of PKC, mediating gene transcription through the activation of the nuclear factor of activated T cells (NFAT), nuclear factor-kappaB (NF-κB), and the mitogen-activated protein kinase (MAPK) pathways [16]. EP1 receptor mRNA is highly expressed in the kidney, lung, and stomach [15]. EP1 has not been demonstrated to play a significant role in innate immunity but participates in controlling T cell differentiation [2,4].

EP2
Receptor. The human EP2 receptor consists of 358 amino acids [20]. This receptor is coupled to a Gs alpha subunit (Gαs). The long C-terminal tail of the EP1 receptor contains serine and threonine, the phosphorylation of which depends on cAMP-dependent protein kinase [15,21]. Gs proteins lead to an increase in cAMP and activation of PKA. A high concentration of intracellular cAMP can also activate both PKA and the exchange protein directly activated by cAMP (EPAC). Then, EPAC phosphorylates the  Figure 1: The process of PGE 2 synthesis. Arachidonic acid is released from the membrane by phospholipase A 2 . Cyclooxygenases (COX-1 and COX-2) produce prostaglandin H 2 (PGH 2 ) from arachidonic acid. PGH 2 is produced by prostaglandin synthase to produce PGI 2 , PGD 2 , PGE 2 , and PGF 2α .
2 Mediators of Inflammation transcription factor CREB [22]. In addition, EP2 receptor signaling leads to the inhibition of glycogen synthase kinase-3 (GSK-3), which inhibits the translocation of β-catenin into the nucleus [21]. The EP2 receptor participates in most of the immunoregulatory effects of PGE 2 in both the innate and adaptive immune responses. For example, PGE 2 in the supernatant of thyroid cancer cells inhibits the activity of NK cells through the EP2 and EP4 receptors [23]. In addition, PGE 2 promotes neurite outgrowth and suppresses cell proliferation by activating the EP2 receptor subtype, and the cAMP signaling pathway is involved in the PGE 2 -induced differentiation of NSC-34 cells [24].

EP3
Receptor. The EP3 receptor, which consists of 365 amino acid residues, is an abundantly and widely expressed EP receptor [15]. EP3 also contains two cAMP-dependent protein kinase phosphorylation sites. EP3 is the only EP receptor that includes multiple variants (α, β, and γ). During the transcription of the EP3 coding sequence, several types of mRNA spliceosomes are produced. The differentiation of these spliceosomes results in a change in the C-terminal tail [25]. It should be noted that these variants have a similar affinity for PGE 2 , but they have different signal transduction pathways, relative expression patterns, and desensitization properties [2]. EP3α and EP3β are coupled to Gi and inhibit adenylyl cyclase (AC) or cAMP, whereas EP3γ is coupled to Gs or Gi and stimulates cAMP production. Therefore, a high concentration of PGE 2 inhibits AC, while a low concentration of PGE 2 activates AC [26].
EP3 mRNA can be detected in almost all tissues. EP3 signaling participates in many metabolic processes. The selective inhibition of EP3 might be a potential approach for reducing chronic neuropathic pain [27]. Moreover, it has been reported that EP3 signaling is induced in placentas associated with unexplained recurrent pregnancy losses [28].

EP4
Receptor. The EP4 receptor signals through a pathway similar to that of the EP2 receptor. The EP4 receptor consists of 513 amino acids. Similar to EP2, EP4 couples to the Gαs protein and activates the cAMP/PKA pathway. cAMP is rapidly degraded by phosphodiesterase to limit the duration of the signal. PKA then phosphorylates target proteins in the cells [29]. EP2 exhibits higher sensitivity and can more effectively increase cAMP. However, the EP4 receptor has a higher affinity for PGE 2 than the EP2 receptor [21]. Compared to EP2, EP4 performs the remarkable function of activating the phosphatidylinositol 3 kinase (PI3K) signaling pathways. The subsequent phosphorylation is mediated via G-coupled receptor kinases or via the ability to bind Gi protein [30,31]. In addition, EP4 also stimulates noncanonical activation of the PI3K-Akt (also known as protein kinase B) and extracellular regulated kinase (ERK) pathways [16,29].
Among all four types of EP receptors, the maintenance of EP4 has received much attention [32] . PGE 2 -EP4 interaction causes muscle-specific stem cell expansion by triggering a cAMP/pCREB pathway that activates the proliferationinducing transcription factor Nurr1 [33]. Besides, activation of EP4 may mediate many cellular responses, such as the promotion of angiogenesis, proliferation, motility, and metastasis or the delay of tumor cell apoptosis [32].   Mediators of Inflammation differentiation of effector T cells, such as Th1, Th2, Th17, Treg, Tfh, and CTLs [3] (Figure 3).

PGE 2 and Th1
Cells. Th1 cells mainly secrete IFN-γ, IL-2, and TNF-α, mediating cellular immune responses and playing a key regulatory role in resistance to bacterial and viral infections. T-bet is the key transcription factor of this T cell subset. Previous experiments indicated that both EP2-and EP4mediated Th1 differentiation are inhibited by PI3K inhibitors [36]. This signaling pathway can decrease the cAMP level and then reduce the cAMP-mediated inhibition of T cell, indicating that PGE 2 could activate the differentiation of Th1 cells. Other researches suggested that PGE 2 selectively inhibits the differentiation of naïve CD4 + T cells into Th1 cells and noted that PGE 2 can also decrease the production of IL-12 by monocytes or dendritic cells, leading to an effect on the proliferation and differentiation of Th1 cells [37,38].

PGE 2 and Th2
Cells. Th2 cells, which mediate humoral immunity, mainly secrete IL-4, IL-5, IL-10, and IL-13. GATA-3 is considered the crucial transcription factor of Th2 cells, autoactivating its expression and driving epigenetic changes in the Th2 cytokine cluster (IL4, IL5, and IL13 genes) while suppressing the factors critical for regulating the Th1 pathway, such as signal transducer and activator of transcription factor 4 (STAT4) and the IL-12Rb2 chain [39]. PGE 2 inhibits the production of both IL-4 and IL-5 by Th2 clones [40]. Later, Bao et al. discovered that PGE 2 can enhance the IL-4/IFN-γ ratio in CD4 + T cell culture and polarize CD4 + T cells towards Th2 cells [41]. Hence, the promotion effect of PGE 2 mainly activates the differentiation of Th2 cells via the inhibition of Th1 cells.
2.9. PGE 2 and Th17 Cells. Th17 cell differentiation requires RORγt, a transcription factor that is induced by TGF-β in combination with the proinflammatory cytokines IL-6, IL-21, and IL-23, all of which activate STAT3 phosphorylation. The differentiation of naïve CD4 + T cells into Th17 cells is mediated by the EP2 or/and EP4 receptor leading to cAMP production. Moreover, PGE 2 increases the concentrations of IL-23R and IL-1βR. cAMP and other cytokines induce the expression of IL-23R via the EP2 receptor [17,42]. Klasen et al. found that the expression level of EP4 is significantly increased in Th17 cells from patients with ankylosing spondylitis (AS) compared to those in healthy individuals or rheumatoid arthritis (RA) patients and that the EP4 expression level in Th17 cells from AS patients has a positive correlation with disease activity [43]. Lee et al. declared that the T cellintrinsic EP2/EP4 signaling is critical in IL-23-driven generation of pathogenic Th17 cells and consequent pathogenesis in psoriasis [44].
2.10. PGE 2 and Tfh Cells. Tfh cells are defined as CD4 + T helper cells that express CD40L, chemokine receptor 5 (CXCR5), programmed death 1 (PD-1), and inducible T cell costimulator (ICOS). Bcl-6 is the characteristic transcription factor of Tfh cells [45]. Tfh cells migrate into follicles and interact with antigen-specific B cells to support their differentiation into memory B cells or plasma cells [46]. Tfh cells can secrete IL-21, which binds to IL-21R on the surface of B cells, assists B cell activation and proliferation, and induces self-antibody production. IL-21 is a potent differentiation factor for B cells. Tfh cell defects or hyperactivity can cause immune system dysfunction, leading to the occurrence of autoimmune diseases [47]. A recent study has shown that PGE 2 can facilitate antibody class switching in B cells by inducing Tfh cell differentiation [48]. We recently discovered that the serum concentration of prostaglandin E metabolite (PGEM) and the proportion of Tfh cells are increased in the collagen-induced arthritis mouse model compared with those in wild-type mice. In addition, there is a positive correlation between the concentration of serum PGEM and the population of Tfh cells in RA patients, suggesting that PGE 2 acts as a positive regulator of the process of Tfh differentiation [1]. However, the mechanism underlying the regulation of Tfh differentiation by PGE 2 still needs to be explored.
2.11. PGE 2 and Treg Cells. Tregs are immunosuppressive cells that mainly participate in immune homeostasis by acting as a major barrier to effective immunity against tumors and sterilizing immunity against chronic viral infections [35]. The membrane-bound IL-2 α-chain is a marker of Treg cells. There are two critical cytokines, namely, IL-2 and TGF-β, that drive the differentiation of Treg cells from naïve T cells. IL-2R is required for both the thymic and peripheral generation of Treg cells. TGF-β inhibits the recruitment of Dnmt1, the key maintenance DNA methyltransferase whose activity likely leads to silencing of the newly induced Foxp3 gene [49]. Foxp3 is a specific transcription factor that regulates the development of Treg cells and is essential for the immunosuppressive function of Treg cells, which is initially considered a specific marker of Treg cells. It has been reported that PGE 2 promotes the growth of Treg cells in both humans and mice via the EP2 or EP4 receptor [50]. Since PGE 2 can effectively suppress the EP/cAMP/PKA pathway, it also inhibits the differentiation of Treg cells. It is worth mentioning that PGE 2 signaling via EP2 expressed on human naïve CD4 + T cells suppresses Treg differentiation in vitro via the cAMP-PKA signaling pathway [3]. However, when UV was used to induce an immunosuppressive reaction, PGE 2 facilitated an increase in Treg cell numbers. Additionally, Kitipong et al. showed the impairment of the immunosuppressive effect of UV with an EP4 antagonist and the reversal of the indomethacin-induced impairment of immunosuppression by an EP4 agonist. Notably, treatment with an EP4 agonist alone, without UV irradiation, does not result in immunosuppression [50]. Additionally, PGE 2 inhibits the expression and production of IL-27 by activating conventional dendritic cells in vivo and in vitro, thus inhibiting the IL-27-induced differentiation and IL-10 production of murine CD4 + CD49b + LAG-3 + Foxp3 -Tr1 cells [51]. The imbalance of Th17/Treg may play a role in the progression of autoimmune diseases such as RA [52]; therefore, PGE 2 is expected to become one of the therapeutic directions.
2.12. PGE 2 and CD8 + CTLs. CTLs, which originate from naïve CD8 + T cells, can specifically recognize endogenous antigen peptides (i.e., MHC I complex) and induce the apoptosis of target cells. Li

Conclusions and Future Perspectives
In this review, we highlight the immunoregulatory roles of the lipid mediator PGE 2 in controlling the differentiation of T cells. The dysregulation of T cell differentiation leads to the occurrence of various diseases. Although PGE 2 exerts effects on different CD4 + T cell subsets, its role in Tfh cells and CD8 + T cells is not well known. The sophisticated interactions between PGE 2 and T cells still need to be further studied. Various cells can secrete this lipid mediator, whereas all cells can generate responses to the signaling of PGE 2 through the EP receptors. Importantly, studying the signaling pathways of PGE 2 may contribute to further elucidating the pathogenesis of diseases, and targeting PGE 2 may be used to develop personalized therapies for RA, AS, neuropathic pain, tumors, inflammation, and so on. PGE 2 may be considered the point of intersection of different human systems. Thus, understanding the pathway of PGE 2 would yield novel insights for designing more effective therapies for immune diseases.

Conflicts of Interest
The authors declare that they have no conflicts of interest.

Authors' Contributions
XN conceived and designed the manuscript. YA wrote the manuscript. JY helped with manuscript preparation and literature search. YA, JY, and XN edited and critically evaluated the manuscript.  Figure 3: Regulation of T cell subsets by PGE 2 . PGE 2 activates the differentiation of Th1 cells by decreasing the levels of cAMP; PGE 2 can enhance the IL-4/IFN-γ ratio in CD4 + T cell culture and regulates CD4 + T cell polarization towards Th2 cells; PGE 2 promotes the differentiation of naïve CD4 + T cells into Th17 cells via the EP2 or EP4 receptor and cAMP pathway; PGE 2 may promote Tfh differentiation; PGE 2 suppresses the EP/cAMP/PKA pathway and inhibits Treg cell differentiation; PGE 2 inhibits the antiviral activity of CTLs and the survival of patients receiving cancer immunotherapy.