Effects Of PGE2 On The Differentiation Of Treg/Th17 Cells And Its Significance On The Pathogenesis Of Collagen-induced Arthritis

It is well known that na?ve CD4+ T cells may polarize into different T helper cell lineages (Th1, Th2) which then control T effector cell responses. Recently, a subset of interleukin (IL)-17-producing T (Th17) cells distinct from Th1 or Th2 cells has been identified and shown to play a crucial role in the induction of autoimmune tissue injury. In contrast, CD4+CD25+Foxp3+ regulatory T (Treg) cells inhibit autoimmunity and protect against tissue injury. The cytokine transforming growth factor-β(TGF-β) converts na?ve T cells into Treg cells, however in the presence of interleukin-6 (IL-6), TGF-βhas also been found to promote the differentiation of na?ve T lymphocytes into Th17 cells. Once differentiated, Th17 cells and Treg cells are characterized by the expression of specific transcription factors, the forkhead box P3 (Foxp3) for Treg and orphan nuclear receptor RORγt for Th17 cells.PGE2 is a lipid inflammatory mediator synthesized from the arachidonic acid pathway involving cyclooxygenases (COX). The effects of PGE2 on the immune response are complex, depending on the cell type. In terms of effects on T cells, many previous studies indicated that PGE2 functioned as an anti-inflammatory agent that inhibited T cell proliferation and the release of Th1 cytokines whereas promotes Th2 differentiation and responses. Rheumatoid arthritis is an autoimmune disease characterized by systemic and local inflammation resulting in cartilage and bone destruction. PGE2 plays important role in the pathogenesis of rheumatoid arthritis. In animal models, the role of PGE2 was confirmed in PGE2 receptor EP2/EP4-deficient mice, which exhibit a reduced incidence and severity of disease. Clinically, the importance of PGE2 is emphasized by the extensive use of nonsteroidal anti-inflammatory drugs (NSAIDs). Currently, there is an abundance of evidence supporting that CD4+CD25+Foxp3+ Treg cells and Th17 cells are crucial participants in rheumatoid arthritis.Based on these results, we proposed that PGE2 might play essential role in the pathogenesis of rheumatoid arthritis through controlling the differentiation of Treg and Th17 cells. In this study we used the MACS-purified CD4+CD62L+ T cells as Th0 cells polarizing to CD4+CD25+Foxp3+ Treg cells and Th17 cells to investigate the effects of PGE2 on the differentiation of Treg and Th17 cells. We also examined the effects of EP2 antagonist and EP4 antagonist on the disease severity and the balance of Treg/Th17 cells in collagen-induced arthritis (CIA). 1 Expression of PGE2 receptor on the murine CD4+CD62L+ T (Th0) cellsFor CD4+CD62L+ na?ve T cell isolation, spleens of C57BL/6 mice were removed, teased into cell-single suspensions and filtered through a 30μm Pre-Separation Filter. CD4+CD62L+ T cells were isolated by MACS selection according to the manufacturer's protocol. CD25+ cells were depleted in the isolating program. The purity of the sorted cells was determined by Flow cytometry analysis. After CD4+CD62L+ T cells were incubated with anti-EP1/EP2/EP3/EP4 antibody and anti-rabbit IgG-FITC, the expression of EP1-4 was analyzed by flow cytometry. The expression of EP1-4 mRNA was analyzed by RT-PCR. Results: (1) The purity of the sorted CD4+CD62L+ T cells was > 90%. (2) EP1-4 were expressed on CD4+ CD62L+ na?ve T cells at different levels, EP2 had the strongest expression. (3) EP1-4 mRNA were expressed on CD4+ CD62L+ na?ve T cells at different levels. 2 Effects and receptor pathway of PGE2 on the differention of Treg and Th17 cellsWe used MACS-purified CD4+CD62L+ T cells to study the effects of PGE2 on the differentiation of Treg cells. In this study, CD4+CD62L+ T cells were stimulated under Treg-promoting conditions with plate-bound anti-CD3εAb, soluble anti-CD28 Ab and TGF-β1. Data showed that after 72h the proportion of CD25+Foxp3+ cells appeared to (28.65±6.83)% analyzed by flow cytometry, freshly isolated CD4+CD62L+ T cells contained less than 2% Foxp3+ cells. The inhibitory action of PGE2 was dose-dependent in a concentration range of 0.01-1μM (P<0.05). EP1 agonist 17-phenyl trinor PGE2 and EP3 agonist sulprostone have no significant effect on the quantity of CD25+Foxp3+ T cells (P>0.05). EP4 agonist PGE1 alchol partially simulated the effect of PGE2 (P<0.05), while EP2 agonist butaprost almost completely simulated the inhibitory effect of PGE2 on the quantity of CD25+ Foxp3+ T cells (P<0.05). We also examined the Foxp3 mRNA expression during the differentiation program of Treg cells from CD4+CD62L+ T cells. Data showed that mRNA expression for Foxp3 peaked at 36 hr. Consistent with the flow cytometry result, the mRNA expression of Foxp3 was also dose-dependently decreased in the presence of PGE2 (P<0.05). Sulprostone have no significant effect on the mRNA expression of Foxp3 (P>0.05). 17-phenyl trinor PGE2 and PGE1 alchol partially simulated the effect of PGE2 (P<0.05), while EP2 agonist almost completely simulated the inhibitory effect of PGE2 on the mRNA expression of Foxp3 (P<0.05). Our results indicated that PGE2 dose-dependently inhibited the differentiation of Treg cells through EP2 and EP4 receptor signaling.CD4+CD62L+ T cells were stimulated under Treg-promoting conditions with plate-bound anti-CD3εAb, soluble anti-CD28 Ab and TGF-β1 plus IL-6. Data showed that 72h later TGF-β1 and IL-6 induced a significant IL-17 secretion to (677.89±87.73)pg/ml, but CD4+CD62L+ T cells without stimulation did not secret IL-17. PGE2 dose-dependently decreased IL-17 secrection in a concentration range of 0.01-1μM (P<0.05). 17-phenyl trinor PGE2 and sulprostone have no significant effect on the secrection of IL-17 (P>0.05). PGE1 alchol partially simulated the effect of PGE2 (P<0.05), while EP2 agonist almost completely simulated the inhibitory effect of PGE2 on the IL-17 secrection (P<0.05). We also examined the RORγt mRNA expression during the differentiation program of Th17 cells from CD4+CD62L+ T cells. Data showed that mRNA expression for RORγt peaked at 48 hr. Consistent with the flow cytometry result, the mRNA expression of RORγt was also dose-dependently decreased in the presence of PGE2 (P<0.05). Sulprostone have no significant effect on the mRNA expression of RORγt (P>0.05). 17-phenyl trinor PGE2 and PGE1 alchol partially simulated the effect of PGE2 (P<0.05), while EP2 agonist almost completely simulated the inhibitory effect of PGE2 on the mRNA expression of RORγt (P<0.05). Our results indicated that PGE2 dose-dependently inhibited the differentiation of Th17 cells through EP2 and EP4 receptor signaling.3 Effects of PGE2 on disease severity and the balance of Treg/Th17 in collagen-induced arthritisGroups (CIA model group, AH6809 group, L161982 group, DMSO control group, DMF control group) of 6-12 female DBA/1 mice were immunized subcutaneously (SC) in the base of the tail with type II collagen (200μg) emulsified in Freund's complete adjuvant. On day 21 after primary immunization, mice were boosted SC with the same agent. AH6809 and L161982 therapies were consisted of the intraperitoneal (IP) administration on 14 consecutive days after the secondary immunization. Spleens were recovered from the CIA group at day 28 postimmunization, cells were stimulated in vitro with CⅡto observe the effects of exogenous PGE2 on CD4+CD25+Foxp3+ cells and IL-17 secrection. Spleens and draining lymph nodes were recovered at day 35 postimmunization, quantity of CD4+CD25+ Foxp3+ cells and serum IL-17 were determined to observe the effects of EP2 and EP4 antagonist on the balance of Treg/Th17 in CIA.Exogenous PGE2 dose-dependently decreased the quantity of CD4+ CD25+Foxp3+ cells in a concentration range of 0.01-1μM (P<0.05) on the spleen cells recovered from the CIA group at day 28 postimmunization. 17-phenyl trinor PGE2 and sulprostone have no significant effect on the quantity of CD25+Foxp3+ T cells (P>0.05). Butaprost partially simulated the effect of PGE2 (P<0.05), but AH6809 (EP2 antagonist) did not reversed the inhibitory effect of PGE2. PGE1 alchol almost completely simulated the inhibitory effect of PGE2 on the quantity of CD25+Foxp3+ T cells (P<0.05), L161982 (EP4 antagonist) almost completely reversed this effect. Exogenous PGE2 dose-dependently decreased the supernatants IL-17 level in a concentration range of 0.01-1μM (1μM group P<0.05) on the spleen cells recovered from the CIA group at day 28 postimmunization. 17-phenyl trinor PGE2, butaprost and sulprostone have no significant effect on the secrection of IL-17 (P>0.05), AH6809 (EP2 antagonist) did not reverse the inhibitory effect of PGE2. PGE1 alchol almost completely simulated the inhibitory effect of PGE2 on the on the secrection of IL-17 (P<0.05), L161982 (EP4 antagonist) almost completely reversed this effect. Our results indicated that exogenous PGE2 dose-dependently inhibited Treg differentiation on the spleen cells recovered from the CIA group at day 28 postimmunization through EP2 and EP4 receptor signaling and inhibited Th17 differentiation through EP4 receptor signaling.90% mice immunized with type II collagen developed into CIA, clinical signs of the disease first appeared at day 26 and peaked at day 35 after the first immunization. Histologic examination of paws at day 35 showed the fibrovascular synovial and periarticular proliferation, erosion of articular cartilage, intra-articular exudates. The quantity of CD25+Foxp3+ T cells in spleens and draining inguinal lymph nodes isolated from CIA mice were significant lower than in those from normal DBA/1mice (P<0.05). Serum IL-17 level of CIA mice was significant higher than in normal DBA/1mice (P<0.05).The intraperitoneal injection of L161982 but not AH6809 to CIA mice decreased paw edema and swelling and alleviated the histologic manifest at day 35 after the first immunization. L161982-treated CIA but not AH6809-treated CIA mice had significantly higher percentages of CD4+CD25+Foxp3+ cells in both draining inguinal lymph nodes and spleens compared with control CIA mice (P<0.05). L161982-treated but not AH6809-treated CIA mice had significantly lower level of serum IL-17 compared with control CIA mice (P<0.05).Conclusions: In this study we used the MACS-purified CD4+CD62L+ T cells as Th0 cells polarizing to CD4+CD25+Foxp3+ Treg cells or Th17 cells and investigated the effects of PGE2 on the differentiation of Treg and Th17 cells. We also examined the effects of EP2 antagonist and EP4 antagonist on disease severity and the balance of Treg/Th17 cells in CIA. The conclusions were as follows: (1) EP1-4 were expressed on CD4+CD62L+ na?ve T cells at different levels and EP2 showed the strongest expression. (2) PGE2 dose-dependently inhibited the differentiation of Treg and Th17 cells from CD4+CD62L+ na?ve T cells through EP2 and EP4 receptor signaling. (3) Exogenous PGE2 dose-dependently inhibited the differentiation of Treg and Th17 on the spleen cells recovered from the CIA mice at day 28 postimmunization through EP2 and EP4 receptor signaling. (4) The intraperitoneal injection of L161982 but not AH6809 to CIA mice decreased disease severity and alleviated the histologic manifest. (5) The intraperitoneal injection of L161982 but not AH6809 to CIA mice decreased the serum IL-17 level and increased the percentages of CD4+CD25+Foxp3+ cells in both spleens and draining inguinal lymph nodes. In summary, the present study indicates that PGE2 inhibits differentiation of both Treg and Th17 cells from Th0 cells. The pro-inflammatory activity of PGE2 in collagen-induced arthritis might play essential role in the pathogenesis of RA through controlling the differentiation of Treg and Th17 cells. The capacity of PGE2 to suppress the generation of immunosuppressive Treg cells and may promote the differentiation of pathogenic Th17 cells through EP4 signaling in collagen-induced arthritis suggests a new therapeutic strategy targeting EP4 receptor in the prevention and treatment of rheumatoid arthritis.