MTX和HCQ对PGI2类似物调节Treg/Th17细胞分化的影响
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摘要
CD4+T细胞是适应性免疫应答系统的主要组分，在调节抗原特异性免疫应答中发挥重要作用。一旦被活化之后，初始CD4+T细胞（na veCD4+T cell）可分化成具有不同功能的各种T细胞亚群，包括Th1（T helpcell1）, Th2（T help cell2）, Th17（IL-17producing T cell）细胞和调节性T细胞（regulatory T cell, Treg），目前广泛认为Th17和Treg细胞的分化平衡在类风湿关节炎（rheumatoid arthritis, RA）等自身免疫病的发生发展中起更重要的作用。Th17细胞因其可分泌IL-17（interleukin17）而命名，表达特异性转录因子RORC（orphan nuclear receptor C），具有促炎症反应的作用，且与组织特异性自身免疫性疾病的免疫应答相关。Treg细胞表达CD25和FoxP3（foxhead box protein3），具有抑制T细胞增殖、抑制抗原呈递细胞（antigen presenting cell, APC）的功能、抑制促炎性细胞因子及抗体的分泌等作用，在抑制移植物耐受、阻止自身免疫反应和维持机体免疫平衡稳态等方面发挥重要功能。抗原提呈过程中，T细胞的分化方向主要决取于三个因素：T细胞抗原受体（T cells receptor, TCR）的刺激强度、细胞因子的种类和APCs所分泌的其他调节介质，如前列腺素（prostaglandin, PGs）。
PGs包括PGD2，PGE2，PGF2，PGI2和TXA2，对免疫系统有明确的调节作用，一直以来PGE2被认为是引起RA的首要PG。但目前的研究结果显示，在胶原诱导的关节炎（collagen induced arthritis，CIA）小鼠的疾病进展中，PGI2和PGE2发挥同等重要的作用。已有包括我室在内的一些研究致力于PGE2对Treg和Th17细胞分化的调节作用，结果显示PGE2可通过调节Treg/Th17细胞平衡参与RA发病。但是，关于PGI2调节Treg和Th17分化的作用尚不明确。
PGI2通过结合G蛋白偶联受体IP发挥其生物学作用，IP受体活化可引起胞内环磷酸腺苷（cyclic adenosine monophosphate, cAMP）水平升高及下游蛋白激酶A（protein kinaseA, PKA）活化。另外，转录活化子5（signal transducers and transcription activators5, STAT5）和转录活化子3（STAT3）分别是调节T细胞向Treg和Th17细胞分化的关键核转录因子。有趣的是，STAT5活化可促进Treg细胞而抑制Th17分化，而STAT3磷酸化可促进RORC而抑制FoxP3表达。而且，cAMP-PKA信号通路的活化有可能导致STAT3和STAT5磷酸化水平的变化。然而，cAMP-PKA信号通路是否参与了PGI2调节Treg和Th17细胞分化的过程，以及PGI2对STAT3和STAT5的活化有无调节作用尚无文献报道。
甲氨蝶呤（methotrexate, MTX）和羟氯喹（hydroxychloroquine, HCQ）都是常用的慢作用抗风湿药（disease-modifying antirheumatic drug,DMARDs），二者单独或联合使用对RA有明显疗效，但其作用机制尚未完全阐明。研究结果表明MTX和HCQ治疗RA的机制都有可能与Treg细胞有关，而Treg和Th17细胞在分化上相互抑制。如果PGI2确实可以影响Treg和Th17细胞分化，比如说导致二者分化失衡，那么MTX和HCQ对PGI2的作用结果会有什么影响呢？
基于上述理论和研究结果，我们认为炎性介质PGI2可能对Treg/Th17分化具有调控作用，进而参与RA的发病和进展，cAMP-PKA信号通路,STAT3和STAT5分子可能参与了该调节过程，而MTX和HCQ可能通过影响PGI2对Treg/Th17分化的调节作用而治疗RA。据此，本课题使用磁珠分选健康人外周血来源的na ve CD4+T细胞，分别体外诱导其向Treg和Th17细胞分化，检测PGI2类似物对Treg和Th17细胞分化的调节作用，并探索cAMP-PKA信号通路, STAT3和STAT5分子在其调节过程中的作用，同时观察MTX和HCQ对PGI2所致的上述调节作用及相关信号转导通路的影响，以进一步探索PGI2在RA发病中的免疫调节机制及MTX和HCQ的治疗机制。
1PGI2类似物对Th0向Treg和Th17细胞分化的调节作用
目的：Treg和Th17细胞的分化平衡在RA等自身免疫病的发生发展中起重要作用，PGE2可通过调节Treg/Th17细胞平衡参与RA发病。在CIA小鼠的疾病进展中，PGI2和PGE2作用相似。故本部分研究使用磁珠分选健康人外周血来源的na ve CD4+T细胞，体外分别诱导其向Treg和Th17细胞分化，检测PGI2类似物对Th0细胞向Treg和Th17细胞分化的调节作用。
方法：免疫磁珠法分选健康人外周血来源的CD4+CD45RA+T细胞，流式细胞术鉴定纯度。（1）首先检测PGI2受体IP的表达：使用RT-PCR、流式细胞术和荧光免疫共聚焦技术检测na ve CD4+T表面是否有IP受体的表达。（2）Treg和Th17细胞诱导成功的鉴定：收集分选后的细胞，接种于anti-CD3预先包被的24孔板，加入anti-CD28、TGF-β1和IL-2诱导Th0向Treg分化；或加入anti-CD28、TGF-β1、IL-6、IL-1β和IL-23诱导Th0向Th17分化，诱导培养7天。分别于0d、1d、3d、5d和7d收集细胞，RT-PCR检测FoxP3或RORC mRNA的相对表达；在7d收集细胞，用流式细胞术测定CD25+FoxP3+T或CD4+IL-17+T的细胞数量，并用ELISA方法测定Th17细胞上清中IL-17A含量。（3）PGI2类似物（伊洛前列素，Iloprost）对Th0向Treg和Th17细胞分化的调节作用及其受体学机制：将收集的细胞各分为六组：Control组（单纯诱导组）；Iloprost（0.1μM、1μM、10μM）组；IP拮抗剂组（CAY10449）；CAY10449+Iloprost（10μM）组，按照上述诱导方案分别培养7天，收集细胞，用RT-PCR检测FoxP3或RORC mRNA的表达；流式细胞术测定CD25+FoxP3+T或CD4+IL-17+T的细胞数量；用ELISA方法测定Th17细胞上清中IL-17A含量。
结果：（1）RT-PCR结果显示，人外周血来源的na ve CD4+T表达一定水平的IP mRNA，流式细胞术和免疫共聚焦的结果进一步证实了IP蛋白的表达。（2）诱导培养7d后，使得（30.83±5.77）%细胞同时表达CD25和FoxP3，CD4+IL-17+T细胞比例达到（5.39±0.88）%。RT-PCR结果显示，FoxP3或RORC mRNA的表达均具时间依从性，且均在7d达到高峰，说明成功诱导了Treg和Th17细胞的分化。（3）Iloprost能剂量依赖性抑制FoxP3mRNA的表达和CD25+FoxP3+T细胞增殖（P<0.05）；同时，Iloprost可剂量依赖性增加RORC mRNA的表达水平和CD4+IL-17+T细胞的数量（P<0.05），另外，Iloprost也促进了IL-17A的分泌（P<0.05）。（4）使用CAY10449拮抗IP受体之后，Iloprost的上述作用在很大程度上被阻断。
以上结果提示，PGI2类似物通过与na ve CD4+T细胞表面的IP受体结合而抑制Treg细胞分化，同时促进Th17细胞分化。我室之前的研究结果发现，PGE2可通过调节Treg/Th17细胞平衡参与RA发病。综合分析这些实验结果，提示前列腺素通过调节Treg和Th17分化而导致Treg/Th17分化失衡，在诸如类风湿性关节炎等自身免疫性疾病的发病中发挥重要作用。
2MTX和HCQ对PGI2类似物调节Treg/Th17细胞分化的影响
目的： MTX和HCQ都是常用的DMARDs，二者单独或联合使用对RA有明显疗效，但其作用机制尚未完全阐明，研究表明MTX和HCQ的治疗机制可能与Treg细胞有关。本部分研究体外诱导Th0向分别向Treg和Th17细胞分化，观察单独和联合应用MTX和HCQ对Treg和Th17细胞分化的调节作用，及其对Iloprost所引起的Treg/Th17分化失衡产生的影响，以期进一步揭示MTX和HCQ的免疫调节机制。
方法：CD4+CD45RA+T细胞的分选同第一部分。（1）MTX和HCQ单独及联合作用对Treg和Th17细胞分化的影响：将收集的细胞各分为八组：Control组（单纯诱导组）；MTX（1nM、10nM、100nM）组；HCQ（1nM、10nM、100nM）组；MTX（100nM）+HCQ（100nM）组，按照上述Treg和Th17细胞诱导方案分别培养7天，收集细胞，用RT-PCR检测FoxP3或RORC mRNA的表达；流式细胞术测定CD25+FoxP3+T或CD4+IL-17+T的细胞数量；用ELISA方法测定Th17分化细胞上清中IL-17A含量。（2）MTX和HCQ单独及联合作用对Iloprost所引起的Treg/Th17分化失衡产生的影响：将收集的细胞各分为七组：Control组（单纯诱导组）；Iloprost（10μM）组；MTX（100nM）组；HCQ（100nM）组；Iloprost+MTX组；Iloprost+HCQ组；Iloprost+MTX+HCQ组，按照上述Treg和Th17细胞诱导方案分别培养7天，收集细胞，用RT-PCR检测FoxP3或RORC mRNA的表达；流式细胞术测定CD25+FoxP3+T或CD4+IL-17+T的细胞数量；用ELISA方法测定Th17分化细胞上清中IL-17A含量。
结果：（1）单独HCQ和MTX+HCQ联合作用均能增加FoxP3mRNA的表达和CD25+FoxP3+T细胞的数量（P<0.05），且降低RORC mRNA的表达水平和CD4+IL-17+T细胞的数量（P<0.05），HCQ的上述作用具剂量依赖性。另外，单独HCQ和MTX+HCQ联合作用均抑制了IL-17A的分泌（P<0.05）。而单独MTX对上述指标均无明显影响（P>0.05）。单独HCQ和MTX+HCQ联合的作用效应相比基本无显著差异（P>0.05）。（2）MTX和HCQ单独及联合作用均可上调Iloprost所抑制的FoxP3mRNA的表达和CD25+FoxP3+T细胞的数量（P<0.05），且下调Iloprost所促进的RORCmRNA的表达和CD4+IL-17+T细胞的数量及IL-17A的分泌（P<0.05）。相对而言，二者的联合作用强于单独作用。
以上结果表明，MTX和HCQ单独及联合作用均可不同程度地上调被PGI2类似物抑制的Treg细胞分化，而下调被PGI2类似物促进的Th17细胞分化，二者的联合作用强于单独作用，推测MTX和HCQ可能通过纠正PGs所致的Treg/Th17失衡而发挥治疗RA等自身免疫病的作用。
3PGI2类似物调节Treg/Th17细胞分化的信号转导机制及MTX和HCQ对该通路的影响
目的：PGI2通过结合G蛋白偶联受体IP发挥其生物学作用，IP受体活化可引起胞内cAMP水平升高及下游PKA活化。STAT3和STAT5分别是调节Th0细胞向Treg和Th17分化的关键核转录因子。然而，cAMP-PKA信号通路的活化有可能导致STAT3和STAT5磷酸化水平的变化。本部分研究探索Iloprost对T细胞分化的调节过程中，cAMP-PKA信号通路、STAT3和STAT5分子所发挥的作用，以及MTX及HCQ对Iloprost所活化的信号转导通路的影响，以进一步探索PGI2在RA发病中的免疫调节机制及MTX和HCQ的治疗机制。
方法：CD4+CD45RA+T细胞的分选同第一部分。（1）收集分选的细胞，分别按照Treg和Th17细胞诱导方案培养7天，将诱导成功的Treg和Th17细胞分别分为七组： Control组（单纯诱导组）； Iloprost组；CAY10449+Iloprost组； Iloprost+MTX组； Iloprost+HCQ组；Iloprost+MTX+HCQ组；db-cAMP组，用免疫荧光法测定胞内cAMP含量。（2）cAMP-PKA信号通路在Iloprost调节Treg和Th17细胞分化中的作用：将分选后收集的细胞各分为四组：Control组（单纯诱导组）；Iloprost组；db-cAMP组；H-89+Iloprost组，按照上述Treg和Th17细胞诱导方案分别培养7天，收集细胞，用RT-PCR检测FoxP3或RORC mRNA的表达；流式细胞术测定CD25+FoxP3+T或CD4+IL-17+T细胞数量；用ELISA方法测定Th17分化细胞上清中IL-17A含量。（3）收集分选的细胞，加入anti-CD3和anti-CD28抗体，将细胞分别分为八组： Control组； Iloprost组；CAY10449+Iloprost组； Iloprost+MTX组； Iloprost+HCQ组；Iloprost+MTX+HCQ组；db-cAMP组；H-89+Iloprost组，于37℃、5%CO2培养箱培养20h，然后加入3μl IL-2或IL-6，37℃孵育30min，用Phowsflow技术分别检测各组细胞中STAT5或STAT3的磷酸化水平。
结果：（1）Iloprost可分别使得诱导培养7天的Treg和Th17细胞内的cAMP水平增加近4倍和6倍多（P<0.05），作为阳性对照的db-cAMP也产生了类似的结果，而IP拮抗剂却可以非常有效地阻断Iloprost对cAMP含量产生的影响。MTX和HCQ单独及联合作用均不同程度地降低了Iloprost所上调的Treg和Th17胞内cAMP水平（P<0.05），其中MTX+HCQ组和单独HCQ组的抑制作用较强。（2）db-cAMP模拟了Iloprost降低CD25+FoxP3+T细胞数量和FoxP3mRNA水平，及增加CD4+IL-17+T细胞数量和RORC转录水平以及IL-17A分泌的作用（P<0.05），而采用特异的PKA抑制剂H-89阻断cAMP-PKA信号通路，可大大削弱Iloprost对Treg和Th17细胞分化的调节作用。（3）Iloprost促进了IL-6所致的STAT3磷酸化，但抑制了IL-2所致的STAT5磷酸化，CAY10449可以明显削弱Iloprost的上述作用效应。MTX和HCQ单独及联合作用均不同程度地增加了Iloprost所下调的STAT5磷酸化水平，且降低Iloprost所上调的STAT3磷酸化水平（P<0.05）。（4）db-cAMP模拟了Iloprost抑制IL-2所致的STAT5磷酸化，及促进IL-6所致的STAT3磷酸化的作用。而用H-89阻断cAMP-PKA之后再加入Iloprost，p-STAT5水平明显增加，p-STAT3水平明显降低，与Iloprost组相比，差异均具有统计学意义（P<0.05）。
以上结果表明，PGI2-IP可能通过上调cAMP-PKA信号通路，从而上调p-STAT3而下调p-STAT5表达，进而促进Th17细胞而抑制Treg细胞分化。而MTX和HCQ单独及联合作用均可显著削弱PGI2-IP在上述信号通路中的调节作用，推测MTX和HCQ可能通过拮抗PGs与其受体结合后引发的cAMP-PKA-STAT3/STAT5信号通路，而纠正PGs所致的Treg/Th17失衡，从而发挥治疗RA等自身免疫病的作用。
结论
本研究使用磁珠分选健康人外周血来源的na ve CD4+T细胞，体外分别诱导其向Treg和Th17细胞分化，检测PGI2类似物对Treg和Th17细胞分化的调节作用，并且探索cAMP-PKA信号通路, STAT3和STAT5分子在其调节过程中的作用，同时观察MTX和HCQ对PGI2所致的上述调节作用及相关信号转导通路的影响，得出以下结论：
1本实验首次在细胞水平证实PGI2类似物抑制Treg细胞体外分化，同时促进Th17细胞分化，二者的共同作用可诱导外周血T细胞亚群的紊乱，进而促进T细胞介导的自身免疫性疾病的发生发展。
2PGI2类似物上述作用主要由IP受体介导，可能通过其上调cAMP-PKA信号通路，从而上调STAT3磷酸化而下调STAT5的磷酸化所致。
3MTX和/或HCQ均可削弱PGI2类似物对上述信号通路的调节作用，进而逆转PGI2类似物对Treg和Th17细胞分化的影响，也就是说MTX和HCQ可能通过纠正Treg/Th17分化失衡而发挥治疗RA等自身免疫性疾病的作用。
总之，PGI2-IP通过抑制Treg细胞分化和促进Th17细胞分化在RA等自身免疫性疾病的发病中发挥重要的促炎作用。MTX和HCQ可通过多靶点发挥治疗作用，其中主要的途径之一是通过纠正PGs所致的Treg/Th17分化失衡而抑制T细胞介导的自身免疫反应和炎症反应。本研究为进一步完善PGs在自身免疫性疾病中的免疫调节作用及MTX和HCQ的治疗机制提供了重要的理论依据。
CD4+T cells are the major components of the adaptive immune systemthat play a prime role in the sustenance and regulation of the antigen-specificimmune response. On activation, naive CD4+T cells differentiate into varioussubsets with distinct functions，including Th1, Th2, Th17and Treg cells. Thebalance between Treg and Th17, has been hypothesized as the more importantfactor than Th1/Th2balance in the development/prevention of autoimmunediseases, including RA. Th17cells, which express the lineage-specifictranscription factor retinoic acid-related orphan receptor C (RORC), aredefined by their ability to produce IL-17. Th17cells have a pro-inflammatoryrole and are associated with immune responses to tissue-specific autoimmunediseases. Tregs that coexpress foxhead box protein3(FoxP3) and CD25, playimportant roles in suppression of inflammatory and autoimmune responses bycontact-dependent suppression or releasing anti-inflammatory cytokines(IL-10and TGF-β1). During Ag presentation, the direction of T celldifferentiation is determined by the strength of TCR stimulation and a varietyof cytokines and other mediators produced by APCs, such as prostanoids,which may influence their balance.
Prostanoids, including PGD2, PGE2, PGF2, PGI2, and TXA2. It has longbeen thought that PGE2is the primary PGs responsible for inflammation inRA. But new evidence indicate that PGI2works as much as PGE2in theprogression of CIA. Some studies,including our previous researchhypothesized that PGE2might play essential role in the pathogenesis of RAby controlling Treg and Th17differentiation. However, the effect of PGI2onTreg and Th17differentiation are not very clear.
PGI2exerts its functions by selective binding to the G-protein-coupled IPreceptor. Stimulation of the IP receptor leads to an increase in intracellular cAMP and activation of downstream protein kinase A (PKA). On the otherhand, STAT3and STAT5protein are key nuclear transcription factors thatregulate T cell differentiation into Treg and Th17cells respectively. However,published evidences show that PGE2regulated phophorylation of STAT3orSTAT5by increasing cAMP. To our best known, we are lacking in potentevidence about the role of PGI2on STAT3and STAT5.
Methotrexate (MTX) and hydroxychloroquine (HCQ) are commondisease modifying anti-rheumatic drug (DMARDs), both single or combineduse of MTX and HCQ has obvious curative effect on autoimmune diseases,but its mechanism has not been fully elucidated. However, published datasuggested that regulation of Treg/Th17differentiation might be involved inthe treatment of autoimmune diseases. Evidence indicated that MTX andHCQ blocked the synthesis of endogenous PGs by inhibiting COX-2andPLA2and PLC, so we speculated that MTX and HCQ may also influence theeffect of exogenous PGs, and regulate the differentiation of Treg/Th17, thusplay an important role on T-cell mediated diseases.
Based on these results, we proposed that PGI2might play essential rolein the pathogenesis of RA through controlling the differentiation of Treg andTh17cells，cAMP-PKA signaling，STAT3and STAT5probably be involved inthese process, and MTX and HCQ may influence the effects of PGI2onTreg/Th17differentiation in the treatment of RA. In this study, we usedpurified na ve CD4+T cells from human peripheral blood and in vitroactivated and differentiated the cells under Treg-and Th17-polarizingconditions. We determined the effects of PGI2analog on the differentiation ofTreg and Th17cells and examined the role of cAMP-PKA signaling, STAT3and STAT5in the PGI2analog-mediated effects. We also examined the effectsof MTX and HCQ on regulatory action of PGI2analog and the signaltransduction pathways related to PGI2analog, in order to explore themechanism of PGI2on the pathogenesis of RA, and the therapeuticmechanism of MTX and HCQ.
Part1Prostaglandin I2-IP signaling regulates human Treg and Th17cellsdifferentiation in vitro
Objective: The balance between Treg and Th17, has been hypothesizedas an important factor in the development of autoimmune diseases, includingRA. PGE2might play essential role in the pathogenesis of RA by controllingTreg and Th17differentiation. New evidence indicate that PGI2works asmuch as PGE2in the progression of CIA. In this part，we activated anddifferentiated na ve CD4+T cells under Treg-and Th17-polarizing conditionsin vitro, and determined the effects of PGI2analog on the differentiation ofTreg and Th17cells.
Methods: Na ve CD4+T cells from human peripheral blood were isolatedby MACS selection, and the purity of the sorted cells was determined by flowcytometry analysis.(1) The expression of the PGI2receptors IP on surface ofna ve CD4+T cells were examined by RT-PCR, flow cytometry and confocalimmunofluorescence.(2) Naive CD4+T cells (5×105/well) were culturedunder Treg-and Th17-polarizing condition for7days. Relative expressionlevels of RORC or FoxP3mRNA were measured at the0,1,3,5,7day byquantitative real-time RT-PCR. The proportion of CD4+IL-17+T cells orCD25+FoxP3+T cells were analyzed by flow cytometry at7day, and theproduction of IL-17A in culture supernatants were detected by ELISA.(3) Todetermine the effect of PGI2analog on T cell differentiation, Iloprost wereadded at10-fold serial-diluted concentrations (0.1M,1M and10M) to theculture medium at the beginning of the cell culture. In some experiments, cellswere pretreated with CAY10449(10M) for30min at37℃before incubationwith Iloprost. Cultured the cells under Treg-and Th17-polarizing conditionfor7days, Relative expression levels of RORC or FoxP3mRNA weremeasured by RT-PCR, The proportion of CD4+IL-17+T cells or CD25+FoxP3+T cells were analyzed by flow cytometry, and the production of IL-17A inculture supernatants were detected by ELISA.
Results:(1) Freshly isolated na ve CD4+T cells constitutively expressedmoderate level of IP mRNA. Cell surface expression of IP protein wasconfirmed by flow cytometry and confocal immunofluorescence.(2) After7 days cultivation, the proportion of CD4+IL-17+T cells appeared to(5.39±0.88)%, which is (0.16±0.06)%before induced. And the proportion ofCD25+FoxP3+T cells appeared to (30.83±5.77)%versus (0.38±0.47)%beforeinduced. The expression of RORC and FoxP3mRNA were time-dependent,and all peaked at7d.(3) Iloprost inhibited the percentage of CD25+FoxP3+Tcells and the expression of FoxP3mRNA, but increased the proportion ofCD4+IL-17+T cells and the expression of RORC mRNA, these effects ofIloprost were in dose-dependent manner, peaking at10M. In addition,Iloprost also increased the level of IL-17A in culture supernatants. But whenwe blocked IP by CAY10449, the effects of Iloprost were almost completelyabolished.
These results indicated that PGI2analog suppresses Treg differentiationand facilitates Th17differentiation through its IP receptor. These resultsprovide further support for our previous studies, which revealed that PGs playa crucial role in the adaptive immune response of autoimmune diseases bysuppressing Treg generation and promoting Th17cells.
Part2The effect of MTX and HCQ on the regulation of PGI2analog onTreg/Th17differentiation
Objective: MTX and HCQ are common DMARDs, both single orcombined use of MTX and HCQ has obvious curative effect on autoimmunediseases, but its mechanism has not been fully elucidated. Evidences indicatedthat regulation of Treg/Th17differentiation might be involved in the treatmentof RA. In this part, we determined the effects of MTX and HCQ on theregulation of PGI2analog on Treg/Th17differen-tiation.
Methods: The MACS selection of na ve CD4+T cells were the same aspart1.(1) The effect of MTX, HCQ and MTX+HCQ on the regulation ofTreg and Th17differention. Naive CD4+T cells were cultured under Treg-orTh17-polarizing condition for7days.10-fold serial-diluted concentrations ofMTX or HCQ (1nM,10nM and100nM) or MTX+HCQ were added at to theculture medium at the beginning of the cell culture.(2) To determine the effectof MTX and HCQ on the regulation of Iloprost on Treg/Th17differentiation, Iloprost plus MTX or HCQ or MTX+HCQ were added to culture mediumtogether, Relative expression levels of RORC or FoxP3mRNA weremeasured by RT-PCR, The proportion of CD4+IL-17+T cells or CD25+FoxP3+T cells were analyzed by flow cytometry, and the production of IL-17A inculture supernatants were detected by ELISA.
Results:(1) HCQ and MTX+HCQ increased the percentage ofCD25+FoxP3+T cells and the expression of FoxP3mRNA, but decreased theproportion of CD4+IL-17+T cells and the expression of RORC mRNA, theseeffects of HCQwere in dose-dependent manner. In addition, HCQ andMTX+HCQ also inhibited the level of IL-17A in culture supernatants. SingleMTX shows no influence on Treg/Th17differentiation.(2) MTX, HCQ andMTX+HCQ increased the inhibited percentage of CD25+FoxP3+T cells andthe expression of FoxP3mRNA induced by Iloprost, and decreased theproportion of CD4+IL-17+T cells, the expression of RORC mRNA andIL-17A secretion which were promoted by Iloprost. Relatively, the effects ofMTX+HCQ were more abvious.
These results indicated that MTX, HCQ and MTX+HCQ antagonize theeffects of the regulation of PGI2analog on Treg/Th17differentiation, whichprobably is the treatment mechanism of MTX and HCQ on autoimmunediseases, such as RA.
Part3The signal transduction in the PGI2analog-mediated effects andthe influence of MTX and HCQ on the pathway.
Objective: Stimulation of the IP receptor leads to an increase inintracellular cAMP and activation of downstream PKA. STAT5and STAT3protein are key nuclear transcription factors that regulate T cell differentiationinto Treg and Th17cells respectively. However, published evidences showthat PGE2regulated phophorylation of STAT3or STAT5by increasing cAMP.In this part, We examined the role of cAMP-PKA signaling, STAT3andSTAT5in the PGI2analog-mediated effects, and the effects of MTX and HCQon the pathways related to PGI2.
Methods: The MACS selection of na ve CD4+T cells were the same as part1.(1) Seven-day polarised Treg and Th17cells were assayed for cAMPaccumulation using a LANCE cAMP kit.(2) The roles of the cAMP-PKApathway in the Iloprost-mediated effects were further investigated withdb-cAMP and H-89. Relative expression levels of RORC or FoxP3mRNAwere measured by RT-PCR, The proportion of CD4+IL-17+T cells orCD25+FoxP3+cells were analyzed by flow cytometry, and the production ofIL-17A in culture supernatants were detected by ELISA.(3) Each group ofCD4+T cells were activatied by anti-CD3and anti-CD28for20h, thenstimulated by3μl IL-2or IL-6, we detected the phosphorylated STAT3orSTAT5by flow cytometry.
Results:(1) Intracellular cAMP levels increased by four-and six-fold inthe T cells polarised under Treg and Th17conditions, respectively, upon theaddition of Iloprost. As a positive control, db-cAMP had a similar effect.Additionally, CAY10449effectively interrupted the effects of Iloprost oncAMP levels in both conditions. However, MTX, HCQ and MTX+HCQinhibited the cAMP levels increased by Iloprost.(2) The blockade ofcAMP-dependent PKA with the H-89inhibitor abrogated the inhibited Tregdifferentiation and the enhanced Th17differentiation by Iloprost, anddb-cAMP induced similar effects to Iloprost.(3) Iloprost increased thephosphorylation of STAT3induced by IL-6and decreased the phosphorylationof STAT5in response to IL-2, CAY10449attenuated the effects of Iloprostunder both conditions. However, MTX, HCQ and MTX+HCQ also weakenedthe effects of Iloprost on p-STAT5and p-STAT3to different entent.(4)db-cAMP imited the effects of Iloprost on p-STAT5and p-STAT3, while, H-89abolished above effects obviously.
These results indicated that PGI2-IP reduces the phosphorylation ofSTAT5and promotes the phosphorylation of STAT3, likely by upregulat-ingcAMP-PKA signalling, thus suppressing Treg differentiation and facilitatingTh17differentiation. MTX, HCQ and MTX+HCQ antagonize the Iloprost-mediated effects on the signal transduction pathway.
CONCLUSIONS
In present study, we used purified na ve CD4+T cells from humanperipheral blood and in vitro activated and differentiated the cells under Th17-and Treg-polarizing conditions. We determined the effects of PGI2analog onthe differentiation of Treg and Th17cells and examined the role ofcAMP-PKA signaling, STAT3and STAT5in the PGI2analog-mediated effects.We also examined the effects of MTX and HCQ on regulatory action of PGI2and the signal transduction pathways related to PGI2. The conclusions were asfollows:
1PGI2analog suppresses Treg differentiation and facilitates Th17differentiation through its IP receptor.
2MTX, HCQ and MTX+HCQ increased the inhibited Treg differentia-tion and decreased facilitates Th17differentiation by Iloprost. Relatively, theeffect of MTX+HCQ were more abvious.
3PGI2-IP reduces the phosphorylation of STAT5and promotes thephosphorylation of STAT3, likely by upregulating cAMP-PKA signalling, thussuppressing Treg differentiation and facilitating Th17differentiation. MTX,HCQ and MTX+HCQ antagonize the Iloprost-mediated effects on the signaltransduction pathway.
In summary, PGI2-IP plays a crucial role in the adaptive immuneresponse of autoimmune diseases by suppressing Treg and promoting Th17cells generation. The effect of MTX and HCQ was exerted at multiple targets,primarily being associated with down-regulation T cell mediated autoimmuneresponses and inflammation by retrieveing the imbalance of Treg/Th17causedby PGs. These findings provide a preliminary rationale for improving themechanism of PGI2on the pathogenesis of RA, and the therapeuticmechanism of MTX and HCQ.
PGs包括PGD2，PGE2，PGF2，PGI2和TXA2，对免疫系统有明确的调节作用，一直以来PGE2被认为是引起RA的首要PG。但目前的研究结果显示，在胶原诱导的关节炎（collagen induced arthritis，CIA）小鼠的疾病进展中，PGI2和PGE2发挥同等重要的作用。已有包括我室在内的一些研究致力于PGE2对Treg和Th17细胞分化的调节作用，结果显示PGE2可通过调节Treg/Th17细胞平衡参与RA发病。但是，关于PGI2调节Treg和Th17分化的作用尚不明确。
PGI2通过结合G蛋白偶联受体IP发挥其生物学作用，IP受体活化可引起胞内环磷酸腺苷（cyclic adenosine monophosphate, cAMP）水平升高及下游蛋白激酶A（protein kinaseA, PKA）活化。另外，转录活化子5（signal transducers and transcription activators5, STAT5）和转录活化子3（STAT3）分别是调节T细胞向Treg和Th17细胞分化的关键核转录因子。有趣的是，STAT5活化可促进Treg细胞而抑制Th17分化，而STAT3磷酸化可促进RORC而抑制FoxP3表达。而且，cAMP-PKA信号通路的活化有可能导致STAT3和STAT5磷酸化水平的变化。然而，cAMP-PKA信号通路是否参与了PGI2调节Treg和Th17细胞分化的过程，以及PGI2对STAT3和STAT5的活化有无调节作用尚无文献报道。
甲氨蝶呤（methotrexate, MTX）和羟氯喹（hydroxychloroquine, HCQ）都是常用的慢作用抗风湿药（disease-modifying antirheumatic drug,DMARDs），二者单独或联合使用对RA有明显疗效，但其作用机制尚未完全阐明。研究结果表明MTX和HCQ治疗RA的机制都有可能与Treg细胞有关，而Treg和Th17细胞在分化上相互抑制。如果PGI2确实可以影响Treg和Th17细胞分化，比如说导致二者分化失衡，那么MTX和HCQ对PGI2的作用结果会有什么影响呢？
基于上述理论和研究结果，我们认为炎性介质PGI2可能对Treg/Th17分化具有调控作用，进而参与RA的发病和进展，cAMP-PKA信号通路,STAT3和STAT5分子可能参与了该调节过程，而MTX和HCQ可能通过影响PGI2对Treg/Th17分化的调节作用而治疗RA。据此，本课题使用磁珠分选健康人外周血来源的na ve CD4+T细胞，分别体外诱导其向Treg和Th17细胞分化，检测PGI2类似物对Treg和Th17细胞分化的调节作用，并探索cAMP-PKA信号通路, STAT3和STAT5分子在其调节过程中的作用，同时观察MTX和HCQ对PGI2所致的上述调节作用及相关信号转导通路的影响，以进一步探索PGI2在RA发病中的免疫调节机制及MTX和HCQ的治疗机制。
1PGI2类似物对Th0向Treg和Th17细胞分化的调节作用
目的：Treg和Th17细胞的分化平衡在RA等自身免疫病的发生发展中起重要作用，PGE2可通过调节Treg/Th17细胞平衡参与RA发病。在CIA小鼠的疾病进展中，PGI2和PGE2作用相似。故本部分研究使用磁珠分选健康人外周血来源的na ve CD4+T细胞，体外分别诱导其向Treg和Th17细胞分化，检测PGI2类似物对Th0细胞向Treg和Th17细胞分化的调节作用。
方法：免疫磁珠法分选健康人外周血来源的CD4+CD45RA+T细胞，流式细胞术鉴定纯度。（1）首先检测PGI2受体IP的表达：使用RT-PCR、流式细胞术和荧光免疫共聚焦技术检测na ve CD4+T表面是否有IP受体的表达。（2）Treg和Th17细胞诱导成功的鉴定：收集分选后的细胞，接种于anti-CD3预先包被的24孔板，加入anti-CD28、TGF-β1和IL-2诱导Th0向Treg分化；或加入anti-CD28、TGF-β1、IL-6、IL-1β和IL-23诱导Th0向Th17分化，诱导培养7天。分别于0d、1d、3d、5d和7d收集细胞，RT-PCR检测FoxP3或RORC mRNA的相对表达；在7d收集细胞，用流式细胞术测定CD25+FoxP3+T或CD4+IL-17+T的细胞数量，并用ELISA方法测定Th17细胞上清中IL-17A含量。（3）PGI2类似物（伊洛前列素，Iloprost）对Th0向Treg和Th17细胞分化的调节作用及其受体学机制：将收集的细胞各分为六组：Control组（单纯诱导组）；Iloprost（0.1μM、1μM、10μM）组；IP拮抗剂组（CAY10449）；CAY10449+Iloprost（10μM）组，按照上述诱导方案分别培养7天，收集细胞，用RT-PCR检测FoxP3或RORC mRNA的表达；流式细胞术测定CD25+FoxP3+T或CD4+IL-17+T的细胞数量；用ELISA方法测定Th17细胞上清中IL-17A含量。
结果：（1）RT-PCR结果显示，人外周血来源的na ve CD4+T表达一定水平的IP mRNA，流式细胞术和免疫共聚焦的结果进一步证实了IP蛋白的表达。（2）诱导培养7d后，使得（30.83±5.77）%细胞同时表达CD25和FoxP3，CD4+IL-17+T细胞比例达到（5.39±0.88）%。RT-PCR结果显示，FoxP3或RORC mRNA的表达均具时间依从性，且均在7d达到高峰，说明成功诱导了Treg和Th17细胞的分化。（3）Iloprost能剂量依赖性抑制FoxP3mRNA的表达和CD25+FoxP3+T细胞增殖（P<0.05）；同时，Iloprost可剂量依赖性增加RORC mRNA的表达水平和CD4+IL-17+T细胞的数量（P<0.05），另外，Iloprost也促进了IL-17A的分泌（P<0.05）。（4）使用CAY10449拮抗IP受体之后，Iloprost的上述作用在很大程度上被阻断。
以上结果提示，PGI2类似物通过与na ve CD4+T细胞表面的IP受体结合而抑制Treg细胞分化，同时促进Th17细胞分化。我室之前的研究结果发现，PGE2可通过调节Treg/Th17细胞平衡参与RA发病。综合分析这些实验结果，提示前列腺素通过调节Treg和Th17分化而导致Treg/Th17分化失衡，在诸如类风湿性关节炎等自身免疫性疾病的发病中发挥重要作用。
2MTX和HCQ对PGI2类似物调节Treg/Th17细胞分化的影响
目的： MTX和HCQ都是常用的DMARDs，二者单独或联合使用对RA有明显疗效，但其作用机制尚未完全阐明，研究表明MTX和HCQ的治疗机制可能与Treg细胞有关。本部分研究体外诱导Th0向分别向Treg和Th17细胞分化，观察单独和联合应用MTX和HCQ对Treg和Th17细胞分化的调节作用，及其对Iloprost所引起的Treg/Th17分化失衡产生的影响，以期进一步揭示MTX和HCQ的免疫调节机制。
方法：CD4+CD45RA+T细胞的分选同第一部分。（1）MTX和HCQ单独及联合作用对Treg和Th17细胞分化的影响：将收集的细胞各分为八组：Control组（单纯诱导组）；MTX（1nM、10nM、100nM）组；HCQ（1nM、10nM、100nM）组；MTX（100nM）+HCQ（100nM）组，按照上述Treg和Th17细胞诱导方案分别培养7天，收集细胞，用RT-PCR检测FoxP3或RORC mRNA的表达；流式细胞术测定CD25+FoxP3+T或CD4+IL-17+T的细胞数量；用ELISA方法测定Th17分化细胞上清中IL-17A含量。（2）MTX和HCQ单独及联合作用对Iloprost所引起的Treg/Th17分化失衡产生的影响：将收集的细胞各分为七组：Control组（单纯诱导组）；Iloprost（10μM）组；MTX（100nM）组；HCQ（100nM）组；Iloprost+MTX组；Iloprost+HCQ组；Iloprost+MTX+HCQ组，按照上述Treg和Th17细胞诱导方案分别培养7天，收集细胞，用RT-PCR检测FoxP3或RORC mRNA的表达；流式细胞术测定CD25+FoxP3+T或CD4+IL-17+T的细胞数量；用ELISA方法测定Th17分化细胞上清中IL-17A含量。
结果：（1）单独HCQ和MTX+HCQ联合作用均能增加FoxP3mRNA的表达和CD25+FoxP3+T细胞的数量（P<0.05），且降低RORC mRNA的表达水平和CD4+IL-17+T细胞的数量（P<0.05），HCQ的上述作用具剂量依赖性。另外，单独HCQ和MTX+HCQ联合作用均抑制了IL-17A的分泌（P<0.05）。而单独MTX对上述指标均无明显影响（P>0.05）。单独HCQ和MTX+HCQ联合的作用效应相比基本无显著差异（P>0.05）。（2）MTX和HCQ单独及联合作用均可上调Iloprost所抑制的FoxP3mRNA的表达和CD25+FoxP3+T细胞的数量（P<0.05），且下调Iloprost所促进的RORCmRNA的表达和CD4+IL-17+T细胞的数量及IL-17A的分泌（P<0.05）。相对而言，二者的联合作用强于单独作用。
以上结果表明，MTX和HCQ单独及联合作用均可不同程度地上调被PGI2类似物抑制的Treg细胞分化，而下调被PGI2类似物促进的Th17细胞分化，二者的联合作用强于单独作用，推测MTX和HCQ可能通过纠正PGs所致的Treg/Th17失衡而发挥治疗RA等自身免疫病的作用。
3PGI2类似物调节Treg/Th17细胞分化的信号转导机制及MTX和HCQ对该通路的影响
目的：PGI2通过结合G蛋白偶联受体IP发挥其生物学作用，IP受体活化可引起胞内cAMP水平升高及下游PKA活化。STAT3和STAT5分别是调节Th0细胞向Treg和Th17分化的关键核转录因子。然而，cAMP-PKA信号通路的活化有可能导致STAT3和STAT5磷酸化水平的变化。本部分研究探索Iloprost对T细胞分化的调节过程中，cAMP-PKA信号通路、STAT3和STAT5分子所发挥的作用，以及MTX及HCQ对Iloprost所活化的信号转导通路的影响，以进一步探索PGI2在RA发病中的免疫调节机制及MTX和HCQ的治疗机制。
方法：CD4+CD45RA+T细胞的分选同第一部分。（1）收集分选的细胞，分别按照Treg和Th17细胞诱导方案培养7天，将诱导成功的Treg和Th17细胞分别分为七组： Control组（单纯诱导组）； Iloprost组；CAY10449+Iloprost组； Iloprost+MTX组； Iloprost+HCQ组；Iloprost+MTX+HCQ组；db-cAMP组，用免疫荧光法测定胞内cAMP含量。（2）cAMP-PKA信号通路在Iloprost调节Treg和Th17细胞分化中的作用：将分选后收集的细胞各分为四组：Control组（单纯诱导组）；Iloprost组；db-cAMP组；H-89+Iloprost组，按照上述Treg和Th17细胞诱导方案分别培养7天，收集细胞，用RT-PCR检测FoxP3或RORC mRNA的表达；流式细胞术测定CD25+FoxP3+T或CD4+IL-17+T细胞数量；用ELISA方法测定Th17分化细胞上清中IL-17A含量。（3）收集分选的细胞，加入anti-CD3和anti-CD28抗体，将细胞分别分为八组： Control组； Iloprost组；CAY10449+Iloprost组； Iloprost+MTX组； Iloprost+HCQ组；Iloprost+MTX+HCQ组；db-cAMP组；H-89+Iloprost组，于37℃、5%CO2培养箱培养20h，然后加入3μl IL-2或IL-6，37℃孵育30min，用Phowsflow技术分别检测各组细胞中STAT5或STAT3的磷酸化水平。
结果：（1）Iloprost可分别使得诱导培养7天的Treg和Th17细胞内的cAMP水平增加近4倍和6倍多（P<0.05），作为阳性对照的db-cAMP也产生了类似的结果，而IP拮抗剂却可以非常有效地阻断Iloprost对cAMP含量产生的影响。MTX和HCQ单独及联合作用均不同程度地降低了Iloprost所上调的Treg和Th17胞内cAMP水平（P<0.05），其中MTX+HCQ组和单独HCQ组的抑制作用较强。（2）db-cAMP模拟了Iloprost降低CD25+FoxP3+T细胞数量和FoxP3mRNA水平，及增加CD4+IL-17+T细胞数量和RORC转录水平以及IL-17A分泌的作用（P<0.05），而采用特异的PKA抑制剂H-89阻断cAMP-PKA信号通路，可大大削弱Iloprost对Treg和Th17细胞分化的调节作用。（3）Iloprost促进了IL-6所致的STAT3磷酸化，但抑制了IL-2所致的STAT5磷酸化，CAY10449可以明显削弱Iloprost的上述作用效应。MTX和HCQ单独及联合作用均不同程度地增加了Iloprost所下调的STAT5磷酸化水平，且降低Iloprost所上调的STAT3磷酸化水平（P<0.05）。（4）db-cAMP模拟了Iloprost抑制IL-2所致的STAT5磷酸化，及促进IL-6所致的STAT3磷酸化的作用。而用H-89阻断cAMP-PKA之后再加入Iloprost，p-STAT5水平明显增加，p-STAT3水平明显降低，与Iloprost组相比，差异均具有统计学意义（P<0.05）。
以上结果表明，PGI2-IP可能通过上调cAMP-PKA信号通路，从而上调p-STAT3而下调p-STAT5表达，进而促进Th17细胞而抑制Treg细胞分化。而MTX和HCQ单独及联合作用均可显著削弱PGI2-IP在上述信号通路中的调节作用，推测MTX和HCQ可能通过拮抗PGs与其受体结合后引发的cAMP-PKA-STAT3/STAT5信号通路，而纠正PGs所致的Treg/Th17失衡，从而发挥治疗RA等自身免疫病的作用。
结论
本研究使用磁珠分选健康人外周血来源的na ve CD4+T细胞，体外分别诱导其向Treg和Th17细胞分化，检测PGI2类似物对Treg和Th17细胞分化的调节作用，并且探索cAMP-PKA信号通路, STAT3和STAT5分子在其调节过程中的作用，同时观察MTX和HCQ对PGI2所致的上述调节作用及相关信号转导通路的影响，得出以下结论：
1本实验首次在细胞水平证实PGI2类似物抑制Treg细胞体外分化，同时促进Th17细胞分化，二者的共同作用可诱导外周血T细胞亚群的紊乱，进而促进T细胞介导的自身免疫性疾病的发生发展。
2PGI2类似物上述作用主要由IP受体介导，可能通过其上调cAMP-PKA信号通路，从而上调STAT3磷酸化而下调STAT5的磷酸化所致。
3MTX和/或HCQ均可削弱PGI2类似物对上述信号通路的调节作用，进而逆转PGI2类似物对Treg和Th17细胞分化的影响，也就是说MTX和HCQ可能通过纠正Treg/Th17分化失衡而发挥治疗RA等自身免疫性疾病的作用。
总之，PGI2-IP通过抑制Treg细胞分化和促进Th17细胞分化在RA等自身免疫性疾病的发病中发挥重要的促炎作用。MTX和HCQ可通过多靶点发挥治疗作用，其中主要的途径之一是通过纠正PGs所致的Treg/Th17分化失衡而抑制T细胞介导的自身免疫反应和炎症反应。本研究为进一步完善PGs在自身免疫性疾病中的免疫调节作用及MTX和HCQ的治疗机制提供了重要的理论依据。
CD4+T cells are the major components of the adaptive immune systemthat play a prime role in the sustenance and regulation of the antigen-specificimmune response. On activation, naive CD4+T cells differentiate into varioussubsets with distinct functions，including Th1, Th2, Th17and Treg cells. Thebalance between Treg and Th17, has been hypothesized as the more importantfactor than Th1/Th2balance in the development/prevention of autoimmunediseases, including RA. Th17cells, which express the lineage-specifictranscription factor retinoic acid-related orphan receptor C (RORC), aredefined by their ability to produce IL-17. Th17cells have a pro-inflammatoryrole and are associated with immune responses to tissue-specific autoimmunediseases. Tregs that coexpress foxhead box protein3(FoxP3) and CD25, playimportant roles in suppression of inflammatory and autoimmune responses bycontact-dependent suppression or releasing anti-inflammatory cytokines(IL-10and TGF-β1). During Ag presentation, the direction of T celldifferentiation is determined by the strength of TCR stimulation and a varietyof cytokines and other mediators produced by APCs, such as prostanoids,which may influence their balance.
Prostanoids, including PGD2, PGE2, PGF2, PGI2, and TXA2. It has longbeen thought that PGE2is the primary PGs responsible for inflammation inRA. But new evidence indicate that PGI2works as much as PGE2in theprogression of CIA. Some studies,including our previous researchhypothesized that PGE2might play essential role in the pathogenesis of RAby controlling Treg and Th17differentiation. However, the effect of PGI2onTreg and Th17differentiation are not very clear.
PGI2exerts its functions by selective binding to the G-protein-coupled IPreceptor. Stimulation of the IP receptor leads to an increase in intracellular cAMP and activation of downstream protein kinase A (PKA). On the otherhand, STAT3and STAT5protein are key nuclear transcription factors thatregulate T cell differentiation into Treg and Th17cells respectively. However,published evidences show that PGE2regulated phophorylation of STAT3orSTAT5by increasing cAMP. To our best known, we are lacking in potentevidence about the role of PGI2on STAT3and STAT5.
Methotrexate (MTX) and hydroxychloroquine (HCQ) are commondisease modifying anti-rheumatic drug (DMARDs), both single or combineduse of MTX and HCQ has obvious curative effect on autoimmune diseases,but its mechanism has not been fully elucidated. However, published datasuggested that regulation of Treg/Th17differentiation might be involved inthe treatment of autoimmune diseases. Evidence indicated that MTX andHCQ blocked the synthesis of endogenous PGs by inhibiting COX-2andPLA2and PLC, so we speculated that MTX and HCQ may also influence theeffect of exogenous PGs, and regulate the differentiation of Treg/Th17, thusplay an important role on T-cell mediated diseases.
Based on these results, we proposed that PGI2might play essential rolein the pathogenesis of RA through controlling the differentiation of Treg andTh17cells，cAMP-PKA signaling，STAT3and STAT5probably be involved inthese process, and MTX and HCQ may influence the effects of PGI2onTreg/Th17differentiation in the treatment of RA. In this study, we usedpurified na ve CD4+T cells from human peripheral blood and in vitroactivated and differentiated the cells under Treg-and Th17-polarizingconditions. We determined the effects of PGI2analog on the differentiation ofTreg and Th17cells and examined the role of cAMP-PKA signaling, STAT3and STAT5in the PGI2analog-mediated effects. We also examined the effectsof MTX and HCQ on regulatory action of PGI2analog and the signaltransduction pathways related to PGI2analog, in order to explore themechanism of PGI2on the pathogenesis of RA, and the therapeuticmechanism of MTX and HCQ.
Part1Prostaglandin I2-IP signaling regulates human Treg and Th17cellsdifferentiation in vitro
Objective: The balance between Treg and Th17, has been hypothesizedas an important factor in the development of autoimmune diseases, includingRA. PGE2might play essential role in the pathogenesis of RA by controllingTreg and Th17differentiation. New evidence indicate that PGI2works asmuch as PGE2in the progression of CIA. In this part，we activated anddifferentiated na ve CD4+T cells under Treg-and Th17-polarizing conditionsin vitro, and determined the effects of PGI2analog on the differentiation ofTreg and Th17cells.
Methods: Na ve CD4+T cells from human peripheral blood were isolatedby MACS selection, and the purity of the sorted cells was determined by flowcytometry analysis.(1) The expression of the PGI2receptors IP on surface ofna ve CD4+T cells were examined by RT-PCR, flow cytometry and confocalimmunofluorescence.(2) Naive CD4+T cells (5×105/well) were culturedunder Treg-and Th17-polarizing condition for7days. Relative expressionlevels of RORC or FoxP3mRNA were measured at the0,1,3,5,7day byquantitative real-time RT-PCR. The proportion of CD4+IL-17+T cells orCD25+FoxP3+T cells were analyzed by flow cytometry at7day, and theproduction of IL-17A in culture supernatants were detected by ELISA.(3) Todetermine the effect of PGI2analog on T cell differentiation, Iloprost wereadded at10-fold serial-diluted concentrations (0.1M,1M and10M) to theculture medium at the beginning of the cell culture. In some experiments, cellswere pretreated with CAY10449(10M) for30min at37℃before incubationwith Iloprost. Cultured the cells under Treg-and Th17-polarizing conditionfor7days, Relative expression levels of RORC or FoxP3mRNA weremeasured by RT-PCR, The proportion of CD4+IL-17+T cells or CD25+FoxP3+T cells were analyzed by flow cytometry, and the production of IL-17A inculture supernatants were detected by ELISA.
Results:(1) Freshly isolated na ve CD4+T cells constitutively expressedmoderate level of IP mRNA. Cell surface expression of IP protein wasconfirmed by flow cytometry and confocal immunofluorescence.(2) After7 days cultivation, the proportion of CD4+IL-17+T cells appeared to(5.39±0.88)%, which is (0.16±0.06)%before induced. And the proportion ofCD25+FoxP3+T cells appeared to (30.83±5.77)%versus (0.38±0.47)%beforeinduced. The expression of RORC and FoxP3mRNA were time-dependent,and all peaked at7d.(3) Iloprost inhibited the percentage of CD25+FoxP3+Tcells and the expression of FoxP3mRNA, but increased the proportion ofCD4+IL-17+T cells and the expression of RORC mRNA, these effects ofIloprost were in dose-dependent manner, peaking at10M. In addition,Iloprost also increased the level of IL-17A in culture supernatants. But whenwe blocked IP by CAY10449, the effects of Iloprost were almost completelyabolished.
These results indicated that PGI2analog suppresses Treg differentiationand facilitates Th17differentiation through its IP receptor. These resultsprovide further support for our previous studies, which revealed that PGs playa crucial role in the adaptive immune response of autoimmune diseases bysuppressing Treg generation and promoting Th17cells.
Part2The effect of MTX and HCQ on the regulation of PGI2analog onTreg/Th17differentiation
Objective: MTX and HCQ are common DMARDs, both single orcombined use of MTX and HCQ has obvious curative effect on autoimmunediseases, but its mechanism has not been fully elucidated. Evidences indicatedthat regulation of Treg/Th17differentiation might be involved in the treatmentof RA. In this part, we determined the effects of MTX and HCQ on theregulation of PGI2analog on Treg/Th17differen-tiation.
Methods: The MACS selection of na ve CD4+T cells were the same aspart1.(1) The effect of MTX, HCQ and MTX+HCQ on the regulation ofTreg and Th17differention. Naive CD4+T cells were cultured under Treg-orTh17-polarizing condition for7days.10-fold serial-diluted concentrations ofMTX or HCQ (1nM,10nM and100nM) or MTX+HCQ were added at to theculture medium at the beginning of the cell culture.(2) To determine the effectof MTX and HCQ on the regulation of Iloprost on Treg/Th17differentiation, Iloprost plus MTX or HCQ or MTX+HCQ were added to culture mediumtogether, Relative expression levels of RORC or FoxP3mRNA weremeasured by RT-PCR, The proportion of CD4+IL-17+T cells or CD25+FoxP3+T cells were analyzed by flow cytometry, and the production of IL-17A inculture supernatants were detected by ELISA.
Results:(1) HCQ and MTX+HCQ increased the percentage ofCD25+FoxP3+T cells and the expression of FoxP3mRNA, but decreased theproportion of CD4+IL-17+T cells and the expression of RORC mRNA, theseeffects of HCQwere in dose-dependent manner. In addition, HCQ andMTX+HCQ also inhibited the level of IL-17A in culture supernatants. SingleMTX shows no influence on Treg/Th17differentiation.(2) MTX, HCQ andMTX+HCQ increased the inhibited percentage of CD25+FoxP3+T cells andthe expression of FoxP3mRNA induced by Iloprost, and decreased theproportion of CD4+IL-17+T cells, the expression of RORC mRNA andIL-17A secretion which were promoted by Iloprost. Relatively, the effects ofMTX+HCQ were more abvious.
These results indicated that MTX, HCQ and MTX+HCQ antagonize theeffects of the regulation of PGI2analog on Treg/Th17differentiation, whichprobably is the treatment mechanism of MTX and HCQ on autoimmunediseases, such as RA.
Part3The signal transduction in the PGI2analog-mediated effects andthe influence of MTX and HCQ on the pathway.
Objective: Stimulation of the IP receptor leads to an increase inintracellular cAMP and activation of downstream PKA. STAT5and STAT3protein are key nuclear transcription factors that regulate T cell differentiationinto Treg and Th17cells respectively. However, published evidences showthat PGE2regulated phophorylation of STAT3or STAT5by increasing cAMP.In this part, We examined the role of cAMP-PKA signaling, STAT3andSTAT5in the PGI2analog-mediated effects, and the effects of MTX and HCQon the pathways related to PGI2.
Methods: The MACS selection of na ve CD4+T cells were the same as part1.(1) Seven-day polarised Treg and Th17cells were assayed for cAMPaccumulation using a LANCE cAMP kit.(2) The roles of the cAMP-PKApathway in the Iloprost-mediated effects were further investigated withdb-cAMP and H-89. Relative expression levels of RORC or FoxP3mRNAwere measured by RT-PCR, The proportion of CD4+IL-17+T cells orCD25+FoxP3+cells were analyzed by flow cytometry, and the production ofIL-17A in culture supernatants were detected by ELISA.(3) Each group ofCD4+T cells were activatied by anti-CD3and anti-CD28for20h, thenstimulated by3μl IL-2or IL-6, we detected the phosphorylated STAT3orSTAT5by flow cytometry.
Results:(1) Intracellular cAMP levels increased by four-and six-fold inthe T cells polarised under Treg and Th17conditions, respectively, upon theaddition of Iloprost. As a positive control, db-cAMP had a similar effect.Additionally, CAY10449effectively interrupted the effects of Iloprost oncAMP levels in both conditions. However, MTX, HCQ and MTX+HCQinhibited the cAMP levels increased by Iloprost.(2) The blockade ofcAMP-dependent PKA with the H-89inhibitor abrogated the inhibited Tregdifferentiation and the enhanced Th17differentiation by Iloprost, anddb-cAMP induced similar effects to Iloprost.(3) Iloprost increased thephosphorylation of STAT3induced by IL-6and decreased the phosphorylationof STAT5in response to IL-2, CAY10449attenuated the effects of Iloprostunder both conditions. However, MTX, HCQ and MTX+HCQ also weakenedthe effects of Iloprost on p-STAT5and p-STAT3to different entent.(4)db-cAMP imited the effects of Iloprost on p-STAT5and p-STAT3, while, H-89abolished above effects obviously.
These results indicated that PGI2-IP reduces the phosphorylation ofSTAT5and promotes the phosphorylation of STAT3, likely by upregulat-ingcAMP-PKA signalling, thus suppressing Treg differentiation and facilitatingTh17differentiation. MTX, HCQ and MTX+HCQ antagonize the Iloprost-mediated effects on the signal transduction pathway.
CONCLUSIONS
In present study, we used purified na ve CD4+T cells from humanperipheral blood and in vitro activated and differentiated the cells under Th17-and Treg-polarizing conditions. We determined the effects of PGI2analog onthe differentiation of Treg and Th17cells and examined the role ofcAMP-PKA signaling, STAT3and STAT5in the PGI2analog-mediated effects.We also examined the effects of MTX and HCQ on regulatory action of PGI2and the signal transduction pathways related to PGI2. The conclusions were asfollows:
1PGI2analog suppresses Treg differentiation and facilitates Th17differentiation through its IP receptor.
2MTX, HCQ and MTX+HCQ increased the inhibited Treg differentia-tion and decreased facilitates Th17differentiation by Iloprost. Relatively, theeffect of MTX+HCQ were more abvious.
3PGI2-IP reduces the phosphorylation of STAT5and promotes thephosphorylation of STAT3, likely by upregulating cAMP-PKA signalling, thussuppressing Treg differentiation and facilitating Th17differentiation. MTX,HCQ and MTX+HCQ antagonize the Iloprost-mediated effects on the signaltransduction pathway.
In summary, PGI2-IP plays a crucial role in the adaptive immuneresponse of autoimmune diseases by suppressing Treg and promoting Th17cells generation. The effect of MTX and HCQ was exerted at multiple targets,primarily being associated with down-regulation T cell mediated autoimmuneresponses and inflammation by retrieveing the imbalance of Treg/Th17causedby PGs. These findings provide a preliminary rationale for improving themechanism of PGI2on the pathogenesis of RA, and the therapeuticmechanism of MTX and HCQ.
引文
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