PGE2对Treg/Th17细胞分化的调节及对胶原诱导性小鼠关节炎发病的影响
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摘要
既往认为T辅助细胞分为Th1(T help cell 1)和Th2(T help cell 2)型,最近研究发现初始T辅助细胞(naive CD4+T cell,Th0)至少可以分化为4种亚群,除Th1和Th2外,还有调节性T细胞(regulatory T cell, Treg)和Th17(IL-17 producing T cell)细胞。Th0细胞在抗原呈递细胞分泌的TGF-β作用下可分化为调节性T细胞(Treg),若同时存在TGF-β和IL-6则Th0分化为Th17细胞。Foxp3 (foxhead box protein 3)和RORγt (orphan nuclear receptor)分别是控制Th0向Treg和Th17分化的关键性转录因子。Th17细胞能够介导前炎症反应,与自身免疫性疾病关系密切。调节性T细胞在阻止自身免疫反应和维持机体免疫平衡方面发挥重要作用。
类风湿关节炎(rheumatoid arthritis,RA)是一种以慢性侵蚀性周围关节炎为主要表现的自身免疫性疾病,病因及发病机制尚不完全清楚。胶原诱导的关节炎(collagen induced arthritis,CIA)小鼠是目前公认的研究RA较为理想的动物模型。越来越多的研究表明Th17细胞和CD4+ Treg细胞在RA发病中占有重要地位。各种免疫活性细胞参与RA发病,病程中细胞因子网络失衡并产生大量炎症介质,其中花生四烯酸代谢产物前列腺素E2(prostaglandin E2, PGE2)是相当重要的致炎因子。动物实验表明阻断PGE2受体或敲除PGE2受体的CIA小鼠病情明显缓解。PGE2的受体(EP1-4)分布广泛,研究认为PGE2具有明确的免疫调节作用。但其调控Th1/Th2平衡的作用存在争议,认为PGE2既有促炎作用又有抗炎活性,多数研究表明PGE2抑制Th0向Th1分化而促进其向Th2方向分化。但PGE2对Treg和Th17分化的影响鲜有报道。
基于上述研究我们认为炎症介质PGE2可能对Treg/Th17细胞的分化具有调控作用,并可能通过此调节作用参与RA的发病过程。本课题在细胞水平以Th0作为靶细胞研究了PGE2对Treg和Th17细胞分化的影响及及其受体学机制,在整体水平应用PGE2受体阻断剂观察对CIA小鼠病情的影响探讨了PGE2是否可通过影响Treg和Th17细胞分化而参与RA发病,以进一步揭示炎症介质PGE2在RA发病中的免疫调节作用。
1 PGE2受体在CD4+CD62L+ na?ve T (Th0)细胞的表达
本部分研究应用MiniMACS系统,磁珠分选C57/BL6小鼠脾CD4+ CD62L+ (Th0)细胞流式细胞术鉴定纯度。分选后的细胞分别加入抗EP1、EP2、EP3和EP4的多克隆抗体和荧光二抗,流式细胞仪检测EP1-4在Th0的表达,RT-PCR技术检测EP1-4 mRNA在Th0的表达情况。结果显示:(1)细胞经磁珠分选后,经流式细胞术鉴定CD4+CD62L+细胞纯度在90%以上。(2)经流式细胞术检测,前列腺素E2的4个受体EP1、EP2、EP3、EP4在CD4+CD62L+ na?ve T细胞上均有不同程度的表达,其中EP2表达最强,EP4表达最弱。(3) RT-PCR技术检测到EP1、EP2、EP3、EP4 mRNA在CD4+CD62L+ na?ve T细胞上均有表达。该结果为进一步研究PGE2对Th0分化的调节作用提供了结构基础。
2 PGE2对Th0向Treg和Th17细胞定向分化的调节作用及受体学途径
本部分研究应用磁珠分选的Th0细胞,在抗CD3/CD28存在的条件下,TGF-β1作用72h后,应用流式细胞术检测,发现(28.65+6.83)%细胞同时表达CD25和Foxp3 (P<0.05);应用实时荧光定量RT-PCR技术检测转录因子Foxp3 mRNA的表达,发现在36h达到高峰,说明成功诱导了Treg细胞的产生。PGE2能剂量依赖性降低CD25+Foxp3+细胞数量,其中1μM PGE2作用最强(0.01, 0.1, 1μM三个剂量组均P<0.05),表明PGE2可明显抑制Th0细胞向Treg细胞分化的数量。诱导Th0向Treg细胞分化,加入EP1-4受体的特异性激动剂(浓度均为10μM),研究PGE2产生上述作用的受体途径,发现17-phenyl trinor PGE2 (EP1激动剂)、sulprostone (EP3激动剂)对CD25 + Foxp3 +细胞数量无明显影响(P>0.05) ,PGE1-alchol (EP4激动剂)部分模拟了PGE2降低CD25+Foxp3+细胞数量的作用(P<0.05),而butaprost (EP2激动剂)几乎完全模拟了PGE2的上述作用(P<0.05)。诱导Th0向Treg细胞分化,加入不同剂量的PGE2,36h后应用荧光定量RT-PCR技术检测Foxp3 mRNA的表达,发现PGE2剂量依赖性抑制了Foxp3 mRNA的表达,其中1μM PGE2作用最强(0.01, 0.1, 1μM三个剂量组均P<0.05),表明PGE2在mRNA水平可抑制Th0细胞向Treg细胞分化。诱导Th0向Treg细胞分化,加入EP1-4受体的激动剂(浓度均为10μM,研究PGE2产生上述作用的受体途径,发现sulprostone对Foxp3 mRNA的表达无明显影响(P>0.05),17-phenyl trinor PGE2和PGE1-alchol部分模拟了PGE2抑制Foxp3 mRNA表达的作用(P<0.05),而butaprost几乎完全模拟了PGE2的上述作用(P<0.05)。上述结果表明PGE2产生抑制Th0向Treg细胞方向分化的作用,该作用可能通过EP2和EP4受体途径介导。
Th0细胞在抗CD3/CD28存在的条件下,TGFβ1和IL-6作用72h后应用ELISA法检测,发现IL-17含量明显升高,达到(677.89+87.73)ng/L (P<0.05);应用实时荧光定量RT-PCR技术检测转录因子RORγt mRNA的表达,发现在48h达到高峰,说明成功诱导了Th17细胞的产生。PGE2能剂量依赖性降低IL-17的含量,其中1μM PGE2作用最强(0.01, 0.1, 1μM三个剂量组均P<0.05),表明PGE2在蛋白水平可能抑制Th0细胞向Th17细胞分化(P<0.05)。诱导Th0向Th17细胞分化,加入EP1-4受体的激动剂(浓度均为10μM),研究PGE2产生上述作用的受体途径,发现17-phenyl trinor PGE2、sulprostone对IL-17分泌无明显影响(P>0.05), PGE1-alchol部分模拟了PGE2降低IL-17分泌的作用(P<0.05),而butaprost几乎完全模拟了PGE2的上述作用(P<0.05)。诱导Th0向Th17细胞分化,加入不同剂量的PGE2,48h后应用荧光定量RT-PCR技术检测RORγt mRNA的表达,发现PGE2剂量依赖性抑制了RORγt mRNA的表达,其中1μM PGE2作用最强(0.01, 0.1, 1μM三个剂量组均P<0.05),表明PGE2在mRNA水平可抑制Th0细胞向Th17细胞分化(P<0.05)。诱导Th0向Treg细胞分化,加入EP1-4受体的激动剂(浓度均为10μM),研究PGE2产生上述作用的受体途径,发现sulprostone对RORγt mRNA的表达无明显影响(P>0.05),17-phenyl trinor PGE2和PGE1-alchol部分模拟了PGE2抑制RORγt mRNA表达的作用(P<0.05),而butaprost几乎完全模拟了PGE2的上述作用(P<0.05)。上述结果表明PGE2可能抑制Th0向Th17细胞方向的分化,该作用可能通过EP2和EP4受体途径介导。
3 PGE2对CIA小鼠Treg/Th17细胞的影响
选用雌性DBA/1小鼠50只,随机分为空白对照组(6只)和CIA单纯造模组(12只),AH6809(EP2受体阻断剂)处理组(9只),L161982(EP4受体阻断剂)处理组(9只),DMF溶剂对照组(7只),DMSO溶剂对照组(7只)。适应环境3天后尾根部注射Ⅱ型胶原及完全弗氏佐剂的乳化剂100μl (含CⅡ200μg),d21后再次尾根部加强免疫。AH6809和L161982处理组分别给相应的药物,剂量均为5mg/Kg/d,从第二次CⅡ免疫后次日开始腹腔注射连续14天。溶剂对照组分别给予相应的溶剂DMF+PBS和DMSO+PBS,注射方式相同。造模28天时分离CIA造模组小鼠的脾细胞,裂解红细胞后制成单细胞悬液,1×106cell/孔种于24孔板,与CⅡ(50μg/ml)共同孵育,观察PGE2及其受体激动剂、阻断剂对Treg和Th17细胞的影响。首次免疫后第35天观察各组发病情况及关节病理改变,取各处理组脾及腹股沟引流淋巴结细胞流式细胞术以CD4+ T细胞开窗测定CD25+Foxp3+ Treg细胞占CD4+T细胞的比例,ELISA法测定血清IL-17含量观察AH6809和L161982对CIA小鼠病情和Treg/Th17平衡的影响。
结果表明外源性PGE2能剂量依赖性降低造模28天分离的脾细胞CD25+Foxp3+细胞占CD4+T细胞的比例,其中1μM PGE2作用最强(0.01, 0.1, 1μM三个剂量组均P<0.05)。17-phenyl trinor PGE2、sulprostone对CD25+Foxp3+细胞比例无明显影响(P>0.05),butaprost部分模拟了PGE2降低CD25+Foxp3+细胞比例的作用(P<0.05),AH6809未能拮抗外源性PGE2对CD25+Foxp3+细胞比例的影响。PGE1-alchol几乎完全模拟了PGE2的上述作用(P<0.05),L161982对外源性PGE2降低CD25+Foxp3+细胞比例的作用产生了明显的拮抗作用。上述结果表明外源性PGE2抑制造模第28天CIA小鼠脾细胞CD4+ Treg细胞的分化,此作用可能通过EP2和EP4受体介导。另外,外源性PGE2能剂量依赖性减少造模28天分离的脾细胞IL-17分泌量,其中1μM PGE2作用明显(P<0.05)。17-phenyl trinor PGE2、sulprostone对IL-17分泌量无明显影响(P>0.05),butaprost部分模拟了PGE2降低IL-17分泌量的作用,但无明显统计学意义(P>0.05),AH6809未能拮抗外源性PGE2对IL-17含量的影响。而PGE1-alchol几乎完全模拟了PGE2的上述作用(P<0.05),L161982对外源性PGE2降低IL-17分泌量的作用产生了明显的拮抗作用。上述结果表明外源性PGE2可能抑制免疫第28天CIA小鼠脾细胞Th17细胞的分化,且此作用通过EP4介导。
首次免疫后第27天CIA组小鼠踝关节及足趾开始出现肿胀,随着时间的延长出现踝关节、足趾肿痛及活动受限等典型改变,在造模第35天左右病情达到高峰,造模率可达到90%。造模第35天取踝关节和足趾关节进行组织病理学检测:CIA小鼠关节腔内有大量的炎性细胞浸润、滑膜增生形成血管翳,侵犯关节软骨和骨。首次免疫后第35天检测造模鼠脾和腹股沟引流淋巴结CD25+Foxp3+细胞占CD4+T细胞的比例,发现腹股沟引流淋巴结CD25+Foxp3+细胞占CD4+T细胞的比例较脾明显升高(P<0.05),造模组小鼠脾和腹股沟引流淋巴结CD25+Foxp3+细胞比例均较空白对照组明显减少(P<0.05),表明CIA小鼠CD4+调节性T细胞数量较正常鼠明显减少。首次免疫后第35天测定CIA小鼠血清,发现造模组小鼠血清IL-17含量较空白对照组明显增高(P<0.05),表明CIA小鼠Th17细胞可能较正常鼠明显增多。
AH6809和L161982对CIA疾病开始时间无明显影响,各组均在第27-28天开始发病,发病率各组之间无明显差异。造模组关节评分和溶剂对照DMF、DMSO组无明显差异,AH6809组关节评分较造模组略低,但无明显统计学差异(P>0.05),而L161982组关节评分较造模组明显降低(P<0.05)。AH6809组关节组织病理与造模组无明显差异,而L161982组在关节腔内炎性细胞浸润、滑膜增生及关节软骨和骨侵犯的组织病理评分上较造模组明显减轻(P<0.05)。上述结果表明EP4受体阻断剂明显减轻了CIA小鼠的病情,而EP2受体阻断剂对CIA病情无明显影响。造模组小鼠脾和腹股沟引流淋巴结CD25+Foxp3+细胞占CD4+T细胞比例与溶剂对照DMF组、溶剂对照DMSO组无明显差异,AH6809组较造模组略高,但无明显统计学差异(P>0.05),而L161982组CD25+Foxp3+细胞占CD4+T细胞比例较造模组明显升高(P<0.05)。上述结果表明EP4受体阻断剂增加了CIA小鼠脾和引流淋巴结CD4+ Treg细胞的数量,而EP2受体阻断剂对此无明显影响。另外,造模组小鼠血清IL-17含量与溶剂对照DMF组、溶剂对照DMSO组无明显差异,AH6809组较造模组略低,但无明显统计学差异(P>0.05),而L161982组血清IL-17的含量较造模组明显减低(P<0.05)。结果表明EP4受体阻断剂可能减少CIA发病过程中Th17细胞的数量,而EP2受体阻断剂对此无明显影响。
结论:本研究在细胞水平以Th0作为靶细胞研究了PGE2对Treg和Th17细胞分化的影响及及其受体学途径,在整体水平应用PGE2受体阻断剂观察对CIA小鼠病情和Treg/Th17细胞平衡的影响,得出以下结论:(1)小鼠脾来源的Th0细胞表面存在PGE2的四种受体亚型,其中EP2表达最强,EP4表达最弱。(2)细胞学实验表明PGE2抑制Th0向Treg细胞方向的分化;PGE2同样抑制Th0向Th17细胞的分化,上述作用通过EP2和EP4受体介导。(3)外源性PGE2抑制造模28天分离的脾细胞中CD4+ Treg细胞和Th17细胞的分化,该作用主要通过EP4受体介导。(4) EP2受体阻断剂和EP4受体阻断剂对CIA发病率和起病时间无明显影响,EP4受体阻断剂明显减轻CIA病情,但EP2受体阻断剂对病情无明显影响。(5)造模组小鼠脾和引流淋巴结中CD4+ Treg细胞数量明显减少,EP4受体阻断剂明显升高脾和引流淋巴结CD4+ Treg细胞数量,而EP2受体阻断剂对此无明显影响。造模组小鼠血清IL-17含量明显增多,EP4受体阻断剂明显降低IL-17分泌量,而EP2受体阻断剂对此无明显影响。本研究表明PGE2对Treg/Th17分化具有明确的调节作用,细胞实验显示PGE2对Th0向Treg和Th17的分化均具有抑制作用,但动物实验显示内源性PGE2通过EP4受体促进IL-17的产生同时抑制Treg细胞的分化而发挥明显的促炎作用,PGE2可通过调节Treg/Th17细胞平衡参与RA发病,EP4受体可能是RA的更精确的治疗靶点。
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) cells
For 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 cells
We 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 arthritis
Groups (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.
类风湿关节炎(rheumatoid arthritis,RA)是一种以慢性侵蚀性周围关节炎为主要表现的自身免疫性疾病,病因及发病机制尚不完全清楚。胶原诱导的关节炎(collagen induced arthritis,CIA)小鼠是目前公认的研究RA较为理想的动物模型。越来越多的研究表明Th17细胞和CD4+ Treg细胞在RA发病中占有重要地位。各种免疫活性细胞参与RA发病,病程中细胞因子网络失衡并产生大量炎症介质,其中花生四烯酸代谢产物前列腺素E2(prostaglandin E2, PGE2)是相当重要的致炎因子。动物实验表明阻断PGE2受体或敲除PGE2受体的CIA小鼠病情明显缓解。PGE2的受体(EP1-4)分布广泛,研究认为PGE2具有明确的免疫调节作用。但其调控Th1/Th2平衡的作用存在争议,认为PGE2既有促炎作用又有抗炎活性,多数研究表明PGE2抑制Th0向Th1分化而促进其向Th2方向分化。但PGE2对Treg和Th17分化的影响鲜有报道。
基于上述研究我们认为炎症介质PGE2可能对Treg/Th17细胞的分化具有调控作用,并可能通过此调节作用参与RA的发病过程。本课题在细胞水平以Th0作为靶细胞研究了PGE2对Treg和Th17细胞分化的影响及及其受体学机制,在整体水平应用PGE2受体阻断剂观察对CIA小鼠病情的影响探讨了PGE2是否可通过影响Treg和Th17细胞分化而参与RA发病,以进一步揭示炎症介质PGE2在RA发病中的免疫调节作用。
1 PGE2受体在CD4+CD62L+ na?ve T (Th0)细胞的表达
本部分研究应用MiniMACS系统,磁珠分选C57/BL6小鼠脾CD4+ CD62L+ (Th0)细胞流式细胞术鉴定纯度。分选后的细胞分别加入抗EP1、EP2、EP3和EP4的多克隆抗体和荧光二抗,流式细胞仪检测EP1-4在Th0的表达,RT-PCR技术检测EP1-4 mRNA在Th0的表达情况。结果显示:(1)细胞经磁珠分选后,经流式细胞术鉴定CD4+CD62L+细胞纯度在90%以上。(2)经流式细胞术检测,前列腺素E2的4个受体EP1、EP2、EP3、EP4在CD4+CD62L+ na?ve T细胞上均有不同程度的表达,其中EP2表达最强,EP4表达最弱。(3) RT-PCR技术检测到EP1、EP2、EP3、EP4 mRNA在CD4+CD62L+ na?ve T细胞上均有表达。该结果为进一步研究PGE2对Th0分化的调节作用提供了结构基础。
2 PGE2对Th0向Treg和Th17细胞定向分化的调节作用及受体学途径
本部分研究应用磁珠分选的Th0细胞,在抗CD3/CD28存在的条件下,TGF-β1作用72h后,应用流式细胞术检测,发现(28.65+6.83)%细胞同时表达CD25和Foxp3 (P<0.05);应用实时荧光定量RT-PCR技术检测转录因子Foxp3 mRNA的表达,发现在36h达到高峰,说明成功诱导了Treg细胞的产生。PGE2能剂量依赖性降低CD25+Foxp3+细胞数量,其中1μM PGE2作用最强(0.01, 0.1, 1μM三个剂量组均P<0.05),表明PGE2可明显抑制Th0细胞向Treg细胞分化的数量。诱导Th0向Treg细胞分化,加入EP1-4受体的特异性激动剂(浓度均为10μM),研究PGE2产生上述作用的受体途径,发现17-phenyl trinor PGE2 (EP1激动剂)、sulprostone (EP3激动剂)对CD25 + Foxp3 +细胞数量无明显影响(P>0.05) ,PGE1-alchol (EP4激动剂)部分模拟了PGE2降低CD25+Foxp3+细胞数量的作用(P<0.05),而butaprost (EP2激动剂)几乎完全模拟了PGE2的上述作用(P<0.05)。诱导Th0向Treg细胞分化,加入不同剂量的PGE2,36h后应用荧光定量RT-PCR技术检测Foxp3 mRNA的表达,发现PGE2剂量依赖性抑制了Foxp3 mRNA的表达,其中1μM PGE2作用最强(0.01, 0.1, 1μM三个剂量组均P<0.05),表明PGE2在mRNA水平可抑制Th0细胞向Treg细胞分化。诱导Th0向Treg细胞分化,加入EP1-4受体的激动剂(浓度均为10μM,研究PGE2产生上述作用的受体途径,发现sulprostone对Foxp3 mRNA的表达无明显影响(P>0.05),17-phenyl trinor PGE2和PGE1-alchol部分模拟了PGE2抑制Foxp3 mRNA表达的作用(P<0.05),而butaprost几乎完全模拟了PGE2的上述作用(P<0.05)。上述结果表明PGE2产生抑制Th0向Treg细胞方向分化的作用,该作用可能通过EP2和EP4受体途径介导。
Th0细胞在抗CD3/CD28存在的条件下,TGFβ1和IL-6作用72h后应用ELISA法检测,发现IL-17含量明显升高,达到(677.89+87.73)ng/L (P<0.05);应用实时荧光定量RT-PCR技术检测转录因子RORγt mRNA的表达,发现在48h达到高峰,说明成功诱导了Th17细胞的产生。PGE2能剂量依赖性降低IL-17的含量,其中1μM PGE2作用最强(0.01, 0.1, 1μM三个剂量组均P<0.05),表明PGE2在蛋白水平可能抑制Th0细胞向Th17细胞分化(P<0.05)。诱导Th0向Th17细胞分化,加入EP1-4受体的激动剂(浓度均为10μM),研究PGE2产生上述作用的受体途径,发现17-phenyl trinor PGE2、sulprostone对IL-17分泌无明显影响(P>0.05), PGE1-alchol部分模拟了PGE2降低IL-17分泌的作用(P<0.05),而butaprost几乎完全模拟了PGE2的上述作用(P<0.05)。诱导Th0向Th17细胞分化,加入不同剂量的PGE2,48h后应用荧光定量RT-PCR技术检测RORγt mRNA的表达,发现PGE2剂量依赖性抑制了RORγt mRNA的表达,其中1μM PGE2作用最强(0.01, 0.1, 1μM三个剂量组均P<0.05),表明PGE2在mRNA水平可抑制Th0细胞向Th17细胞分化(P<0.05)。诱导Th0向Treg细胞分化,加入EP1-4受体的激动剂(浓度均为10μM),研究PGE2产生上述作用的受体途径,发现sulprostone对RORγt mRNA的表达无明显影响(P>0.05),17-phenyl trinor PGE2和PGE1-alchol部分模拟了PGE2抑制RORγt mRNA表达的作用(P<0.05),而butaprost几乎完全模拟了PGE2的上述作用(P<0.05)。上述结果表明PGE2可能抑制Th0向Th17细胞方向的分化,该作用可能通过EP2和EP4受体途径介导。
3 PGE2对CIA小鼠Treg/Th17细胞的影响
选用雌性DBA/1小鼠50只,随机分为空白对照组(6只)和CIA单纯造模组(12只),AH6809(EP2受体阻断剂)处理组(9只),L161982(EP4受体阻断剂)处理组(9只),DMF溶剂对照组(7只),DMSO溶剂对照组(7只)。适应环境3天后尾根部注射Ⅱ型胶原及完全弗氏佐剂的乳化剂100μl (含CⅡ200μg),d21后再次尾根部加强免疫。AH6809和L161982处理组分别给相应的药物,剂量均为5mg/Kg/d,从第二次CⅡ免疫后次日开始腹腔注射连续14天。溶剂对照组分别给予相应的溶剂DMF+PBS和DMSO+PBS,注射方式相同。造模28天时分离CIA造模组小鼠的脾细胞,裂解红细胞后制成单细胞悬液,1×106cell/孔种于24孔板,与CⅡ(50μg/ml)共同孵育,观察PGE2及其受体激动剂、阻断剂对Treg和Th17细胞的影响。首次免疫后第35天观察各组发病情况及关节病理改变,取各处理组脾及腹股沟引流淋巴结细胞流式细胞术以CD4+ T细胞开窗测定CD25+Foxp3+ Treg细胞占CD4+T细胞的比例,ELISA法测定血清IL-17含量观察AH6809和L161982对CIA小鼠病情和Treg/Th17平衡的影响。
结果表明外源性PGE2能剂量依赖性降低造模28天分离的脾细胞CD25+Foxp3+细胞占CD4+T细胞的比例,其中1μM PGE2作用最强(0.01, 0.1, 1μM三个剂量组均P<0.05)。17-phenyl trinor PGE2、sulprostone对CD25+Foxp3+细胞比例无明显影响(P>0.05),butaprost部分模拟了PGE2降低CD25+Foxp3+细胞比例的作用(P<0.05),AH6809未能拮抗外源性PGE2对CD25+Foxp3+细胞比例的影响。PGE1-alchol几乎完全模拟了PGE2的上述作用(P<0.05),L161982对外源性PGE2降低CD25+Foxp3+细胞比例的作用产生了明显的拮抗作用。上述结果表明外源性PGE2抑制造模第28天CIA小鼠脾细胞CD4+ Treg细胞的分化,此作用可能通过EP2和EP4受体介导。另外,外源性PGE2能剂量依赖性减少造模28天分离的脾细胞IL-17分泌量,其中1μM PGE2作用明显(P<0.05)。17-phenyl trinor PGE2、sulprostone对IL-17分泌量无明显影响(P>0.05),butaprost部分模拟了PGE2降低IL-17分泌量的作用,但无明显统计学意义(P>0.05),AH6809未能拮抗外源性PGE2对IL-17含量的影响。而PGE1-alchol几乎完全模拟了PGE2的上述作用(P<0.05),L161982对外源性PGE2降低IL-17分泌量的作用产生了明显的拮抗作用。上述结果表明外源性PGE2可能抑制免疫第28天CIA小鼠脾细胞Th17细胞的分化,且此作用通过EP4介导。
首次免疫后第27天CIA组小鼠踝关节及足趾开始出现肿胀,随着时间的延长出现踝关节、足趾肿痛及活动受限等典型改变,在造模第35天左右病情达到高峰,造模率可达到90%。造模第35天取踝关节和足趾关节进行组织病理学检测:CIA小鼠关节腔内有大量的炎性细胞浸润、滑膜增生形成血管翳,侵犯关节软骨和骨。首次免疫后第35天检测造模鼠脾和腹股沟引流淋巴结CD25+Foxp3+细胞占CD4+T细胞的比例,发现腹股沟引流淋巴结CD25+Foxp3+细胞占CD4+T细胞的比例较脾明显升高(P<0.05),造模组小鼠脾和腹股沟引流淋巴结CD25+Foxp3+细胞比例均较空白对照组明显减少(P<0.05),表明CIA小鼠CD4+调节性T细胞数量较正常鼠明显减少。首次免疫后第35天测定CIA小鼠血清,发现造模组小鼠血清IL-17含量较空白对照组明显增高(P<0.05),表明CIA小鼠Th17细胞可能较正常鼠明显增多。
AH6809和L161982对CIA疾病开始时间无明显影响,各组均在第27-28天开始发病,发病率各组之间无明显差异。造模组关节评分和溶剂对照DMF、DMSO组无明显差异,AH6809组关节评分较造模组略低,但无明显统计学差异(P>0.05),而L161982组关节评分较造模组明显降低(P<0.05)。AH6809组关节组织病理与造模组无明显差异,而L161982组在关节腔内炎性细胞浸润、滑膜增生及关节软骨和骨侵犯的组织病理评分上较造模组明显减轻(P<0.05)。上述结果表明EP4受体阻断剂明显减轻了CIA小鼠的病情,而EP2受体阻断剂对CIA病情无明显影响。造模组小鼠脾和腹股沟引流淋巴结CD25+Foxp3+细胞占CD4+T细胞比例与溶剂对照DMF组、溶剂对照DMSO组无明显差异,AH6809组较造模组略高,但无明显统计学差异(P>0.05),而L161982组CD25+Foxp3+细胞占CD4+T细胞比例较造模组明显升高(P<0.05)。上述结果表明EP4受体阻断剂增加了CIA小鼠脾和引流淋巴结CD4+ Treg细胞的数量,而EP2受体阻断剂对此无明显影响。另外,造模组小鼠血清IL-17含量与溶剂对照DMF组、溶剂对照DMSO组无明显差异,AH6809组较造模组略低,但无明显统计学差异(P>0.05),而L161982组血清IL-17的含量较造模组明显减低(P<0.05)。结果表明EP4受体阻断剂可能减少CIA发病过程中Th17细胞的数量,而EP2受体阻断剂对此无明显影响。
结论:本研究在细胞水平以Th0作为靶细胞研究了PGE2对Treg和Th17细胞分化的影响及及其受体学途径,在整体水平应用PGE2受体阻断剂观察对CIA小鼠病情和Treg/Th17细胞平衡的影响,得出以下结论:(1)小鼠脾来源的Th0细胞表面存在PGE2的四种受体亚型,其中EP2表达最强,EP4表达最弱。(2)细胞学实验表明PGE2抑制Th0向Treg细胞方向的分化;PGE2同样抑制Th0向Th17细胞的分化,上述作用通过EP2和EP4受体介导。(3)外源性PGE2抑制造模28天分离的脾细胞中CD4+ Treg细胞和Th17细胞的分化,该作用主要通过EP4受体介导。(4) EP2受体阻断剂和EP4受体阻断剂对CIA发病率和起病时间无明显影响,EP4受体阻断剂明显减轻CIA病情,但EP2受体阻断剂对病情无明显影响。(5)造模组小鼠脾和引流淋巴结中CD4+ Treg细胞数量明显减少,EP4受体阻断剂明显升高脾和引流淋巴结CD4+ Treg细胞数量,而EP2受体阻断剂对此无明显影响。造模组小鼠血清IL-17含量明显增多,EP4受体阻断剂明显降低IL-17分泌量,而EP2受体阻断剂对此无明显影响。本研究表明PGE2对Treg/Th17分化具有明确的调节作用,细胞实验显示PGE2对Th0向Treg和Th17的分化均具有抑制作用,但动物实验显示内源性PGE2通过EP4受体促进IL-17的产生同时抑制Treg细胞的分化而发挥明显的促炎作用,PGE2可通过调节Treg/Th17细胞平衡参与RA发病,EP4受体可能是RA的更精确的治疗靶点。
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) cells
For 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 cells
We 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 arthritis
Groups (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.
引文
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