【作者】 张永红；

【导师】 廖爱华；

【作者基本信息】 华中科技大学 ， 妇产科学， 2019， 博士

【摘要】 第一部分PD-1和PD-L1在早孕期蜕膜巨噬细胞和绒毛中表达的研究【目的】通过检测早孕期正常妊娠(Normal pregnancy,NP)妇女和反复自然流产(Recurrent miscarriages,RM)患者蜕膜组织中M1和M2型蜕膜巨噬细胞(Decidual macrophage,DM)百分比,以及程序性死亡受体(Programmed cell death,PD)-1及其配体(PD-1 ligand,PD-L)-1在DM的表达水平,PD-L1在NP妇女和RM患者绒毛组织的表达水平,探讨早孕期母-胎界面DM表型与PD-1/PD-L1信号通路之间的关系。【方法】以NP妇女(n=20)为对照组,RM患者(n=16)为病例组,收集流产后蜕膜组织,采用流式细胞技术检测早孕期NP妇女和RM患者蜕膜组织中M1(CD14+CD80+/CD14+CD86+/CD14+CD80+CD86+)和M2(CD14+CD163+/CD14+CD206+/CD14+CD163+CD206+)型DM百分比,以及PD-1和PD-L1在DM的表达水平;同时采集NP妇女(n=19)和RM患者(n=15)绒毛组织,采用定量逆转录聚合酶链式反应(Quantitative reverse transcription polymerase chain reaction,q RT-PCR)和Western Blot技术分别检测绒毛组织中PD-L1 m RNA和蛋白表达水平。【结果】早孕期NP妇女和RM患者蜕膜中均存在M1和M2型DM:NP妇女中DM以M2型为主,RM患者中DM以M1型为主;与NP妇女相比,RM患者中M1型(CD14+CD80+/CD14+CD86+/CD14+CD80+CD86+)DM百分比增加(P<0.01),M2型(CD14+CD163+/CD14+CD206+)DM百分比则明显减少(P<0.05);与NP妇女相比,RM患者中M1(CD14+CD80+/CD14+CD86+/CD14+CD80+CD86+)和M2(CD14+CD163+/CD14+CD206+)型DM中PD-1表达水平明显减少(P<0.05);与NP妇女相比,RM患者中M2(CD14+CD163+)型DM中PD-L1表达水平明显减少(P<0.05);与NP妇女相比,RM患者绒毛组织表达PD-L1 m RNA和蛋白表达水平明显降低(P<0.001)。【结论】与其他分子标记相比,CD14+CD86+和CD14+CD206+是分别界定早孕期M1和M2型DM的最佳分子标记;RM患者中M1型DM百分比增加,M2型DM百分比降低,也许与其表面PD-1表达降低,以及绒毛组织中PD-L1表达降低有关。第二部分PD-1/PD-L1信号通路调控早孕期巨噬细胞分化和功能的研究【目的】通过体外激活或阻断巨噬细胞分化过程中PD-1/PD-L1信号通路,检测早孕期单核细胞在体外分化为巨噬细胞的表型和功能,探讨PD-1/PD-L1信号通路对早孕期巨噬细胞分化和功能的调控效应。【方法】收集早孕期NP妇女(n=50)外周血,采用免疫磁珠试剂盒分选CD14+单核细胞,经重组人粒细胞-巨噬细胞集落刺激因子(Recombinate human granulocyte-macrophage colony-stimulating factor,rh GM-CSF)(50 ng/m L)体外活化,单独培养或与PD-L1 Fc(10μg/m L)、anti-PD-L1 m Ab(10μg/m L)和anti-PD-1 m Ab(10μg/m L)共培养7天,采用流式细胞技术检测M1(CD14+CD86+)和M2(CD14+CD206+)型巨噬细胞百分比,以及巨噬细胞吞噬功能;同时,采用q RT-PCR方法检测巨噬细胞分化相关转录因子(Interferon regulatory factor,IRF)-4,5和细胞因子白介素(Interleukin,IL)-1β和肿瘤坏死因子(Tumor necrosis factor,TNF)-αm RNA表达水平。【结果】与对照组相比,M2型巨噬细胞百分比在PD-L1 Fc处理组明显上调(P<0.05),M1型巨噬细胞百分比则明显降低(P<0.01);anti-PD-1 m Ab明显上调M1型巨噬细胞百分比(P<0.001),M2型巨噬细胞百分比则明显降低(P<0.01);anti-PD-L1m Ab则对M1和M2型巨噬细胞百分比影响不大(P>0.05)。与对照组相比,巨噬细胞吞噬功能在PD-L1 Fc处理组明显增强(P<0.01),anti-PD-1 m Ab处理组中巨噬细胞吞噬功能明显减弱(P<0.001),而anti-PD-L1 m Ab对巨噬细胞吞噬功能影响不大(P>0.05)。同时,在PD-L1 Fc处理组,M2型巨噬细胞转录因子IRF4 m RNA表达明显增加(P<0.05),M1型巨噬细胞转录因子IRF5 m RNA表达则明显降低(P<0.01);anti-PD-1 m Ab阻断PD-1信号传导,则明显抑制IRF4 m RNA表达(P<0.05),促进IL-1β和TNF-αm RNA表达水平(P<0.05);anti-PD-L1 m Ab则对IRF4、IRF5、IL-1β和TNF-αm RNA表达水平无明显调控效应(P>0.05)。【结论】PD-1/PD-L1信号通路活化,促进早孕期单核细胞向M2型巨噬细胞分化,增强巨噬细胞吞噬功能,抑制促炎型细胞因子IL-1β和TNF-α转录;阻断PD-1/PD-L1信号通路,则促进M1型巨噬细胞分化,抑制巨噬细胞吞噬功能,并增强促炎型细胞因子IL-1β和TNF-α转录。因此,PD-1/PD-L1信号通路对早孕期巨噬细胞分化和功能存在调控效应。第三部分PD-1/PD-L1信号通路调控早孕期巨噬细胞分化和功能的分子机制【目的】通过体外激活或阻断PD-1/PD-L1信号通路,体外诱导早孕期单核细胞分化为巨噬细胞,检测诱导分化的巨噬细胞中物质(葡萄糖、脂肪酸和谷氨酰胺)代谢相关基因和信号传导分子的m RNA表达水平,阐明PD-1/PD-L1信号通路调控早孕期巨噬细胞分化和功能的分子机制。【方法】收集早孕期NP妇女(n=8)外周血,采用免疫磁珠试剂盒分选CD14+单核细胞,经rh GM-CSF(50 ng/m L)体外活化,单独培养或与anti-PD-1 m Ab(10μg/m L)共培养7天,采用q RT-PCR技术检测分化后巨噬细胞中PD-1/PD-L1信号通路相关分子磷脂酰肌醇-3-激酶(Phosphatidylinositol-3-kinase,PI3K)、蛋白激酶B(Protein kinase B,AKT)、哺乳动物雷帕霉素靶蛋白(Mammalian target of Rapamycin,m-TOR)和丝裂原活化蛋白激酶/细胞外信号调节激酶(Mitogen-activated protein kinase/extracellular signal-regulated kinase,MEK)、细胞外信号调节激酶(Extracellular signal-regulated kinase,ERK)m RNA的表达水平,以及葡萄糖、脂肪酸和谷氨酰胺相关转运体和分解代谢关键酶m RNA的表达水平。【结果】与对照组相比,anti-PD-1 m Ab处理组巨噬细胞中葡萄糖转运体(Glucose transporter,Glut)-1 m RNA表达水平,以及葡萄糖酵解关键酶己糖激酶(Hexokinase,HK)-2和丙酮酸脱氢酶激酶(Pyruvate dehydrogenase kinase,PDK)-1 m RNA表达水平明显升高(P<0.01),乳酸脱氢酶(Lactate dehydrogenase,LDH)-1 m RNA表达水平略升高但无明显差异(P>0.05);与对照组相比,anti-PD-1 m Ab处理组巨噬细胞中氨基酸代谢中谷氨酰胺转运体(Sodium-coupled neutral amino acid transporter,SNAT)-1和2 m RNA表达水平明显升高(P<0.01);与对照组相比,anti-PD-1 m Ab处理组巨噬细胞中脂肪酸合酶(Fatty acid synthase,FASN)m RNA表达水平明显升高(P<0.05),而脂肪酸转运体肉毒碱棕榈酰转移酶(Carnitine palmitoyltransferase,CPT)1A m RNA表达水平无明显变化(P>0.05);同时,采用anti-PD-1 m Ab阻断PD-1信号传导促进巨噬细胞中信号传导分子PI3K、AKT、m-TOR和MEK、ERK m RNA的表达(P<0.01)。【结论】阻断PD-1/PD-L1信号通路,诱导早孕期巨噬细胞向M1型分化,也许是通过促进细胞无氧糖酵解和抑制细胞脂肪酸氧化实现的,并且受PI3K/AKT/m-TOR和MEK/ERK信号分子调控。第四部分滋养细胞来源的可溶性PD-L1促进巨噬细胞向M2型分化【目的】采集早孕期NP妇女绒毛组织,分离人原代滋养细胞,并进一步分析人滋养细胞系和人原代滋养细胞产生可溶性PD-L1(Soluble PD-L1,s PD-L1)的水平;建立滋养细胞条件性培养基(Trophoblast conditioned medium,TCM)诱导单核细胞分化为巨噬细胞的实验模型,通过阻断PD-1/PD-L1信号通路,检测M1和M2型巨噬细胞百分比和细胞因子分泌水平。【方法】收集早孕期NP妇女绒毛组织,采用酶消化法分离滋养细胞并体外培养,获取人滋养细胞系(Swan 71细胞和3A细胞)和人原代滋养细胞低血清培养条件下培养上清和细胞,采用Western Blot和Simple Plex方法分析膜PD-L1(Membrane PD-L1,m PD-L1)和s PD-L1在滋养细胞的基础表达水平;同时采集具有正常生育力的妇女外周血,免疫磁珠法分选CD14+单核细胞,建立TCM诱导单核细胞分化为巨噬细胞的实验模型,采用anti-PD-1 m Ab(10μg/m L)阻断PD-1/PD-L1信号通路,检测培养7天后M1和M2型巨噬细胞百分比和细胞因子分泌水平。【结果】m PD-L1和s PD-L1在Swan 71细胞均表达;与m PD-L1相比,s PD-L1分泌水平随时间进展逐渐增加(P<0.01),在3A和人原代滋养细胞中均存在与Swan 71细胞相似的s PD-L1的分泌曲线(P<0.01);TCM诱导巨噬细胞分化模型发现,TCM诱导M2型巨噬细胞分化,而anti-PD-1 m Ab处理组中M2型巨噬细胞明显减少;此外,anti-PD-1 m Ab处理组中巨噬细胞经脂多糖(Lipopolysaccharide,LPS)活化后,分泌更高水平的促炎细胞因子,如IL-6和TNF-α。【结论】滋养细胞组成性分泌s PD-L1,体外阻断PD-1受体,减弱TCM对巨噬细胞分化为M2型的促进作用;这些数据表明滋养细胞来源的s PD-L1参与M2型巨噬细胞的分化。第五部分阻断PD-1信号传导对正常妊娠小鼠胚胎吸收和子宫巨噬细胞表型的调控效应【目的】建立正常妊娠和流产倾向小鼠模型,采用PD-1抗体阻断正常妊娠小鼠中PD-1/PD-L1信号传导,检测各妊娠小鼠胚胎吸收率,子宫和脾脏M1和M2型巨噬细胞百分比,以及PD-1在子宫和脾脏巨噬细胞的表达,在体探讨PD-1/PD-L1信号通路对妊娠结局和巨噬细胞分化的调控作用。【方法】采用CBA/J♀(n=8)和BALB/c♂建立正常妊娠小鼠模型,随机分为两组,分别于妊娠4.5天、6.5天和8.5天给予anti-PD-1 m Ab或等量无菌PBS,均为腹腔注射;采用CBA/J♀(n=4)和DBA/2♂建立流产倾向小鼠模型。于妊娠第10.5天,处死孕鼠,计数胚胎吸收率,分离孕鼠子宫和脾脏,采用流式细胞技术检测子宫和脾脏中M1和M2型巨噬细胞百分比,以及PD-1在子宫和脾脏巨噬细胞的表达水平。【结果】与正常妊娠CBA/J×BALB/c小鼠相比,anti-PD-1 m Ab处理的CBA/J×BALB/c妊娠小鼠和CBA/J×DBA/2流产倾向小鼠中胚胎吸收率明显升高(P<0.05),而anti-PD-1 m Ab处理的CBA/J×BALB/c妊娠小鼠和CBA/J×DBA/2流产倾向小鼠组胚胎吸收率无明显差异(P>0.05)。与健康妊娠CBA/J×BALB/c小鼠相比,子宫M2型巨噬细胞百分比和M1/M2比值在anti-PD-1 m Ab处理的CBA/J×BALB/c妊娠小鼠和CBA/J×DBA/2流产倾向小鼠明显降低(P<0.05),而anti-PD-1 m Ab处理的CBA/J×BALB/c妊娠小鼠和CBA/J×DBA/2流产倾向小鼠组间则无明显差异(P>0.05)。子宫M1型巨噬细胞百分比在三组之间没有明显差异(P>0.05);脾脏M1和M2型巨噬细胞百分比在三组之间也没有明显差异(P>0.05),但与正常妊娠小鼠相比,子宫和脾脏M1/M2比值在anti-PD-1 m Ab处理的CBA/J×BALB/c妊娠小鼠和CBA/J×DBA/2流产倾向小鼠明显升高(P<0.05)。与正常妊娠CBA/J×BALB/c小鼠相比,子宫巨噬细胞PD-1表达水平在anti-PD-1 m Ab处理的CBA/J×BALB/c妊娠小鼠和CBA/J×DBA/2流产倾向小鼠中明显降低(P<0.05);而其在anti-PD-1 m Ab处理的CBA/J×BALB/c妊娠小鼠和CBA/J×DBA/2流产倾向小鼠间则无明显差异(P>0.05)。与正常妊娠CBA/J×BALB/c小鼠相比,PD-1在脾脏巨噬细胞表达水平在CBA/J×DBA/2流产倾向小鼠中明显降低(P<0.05),而与anti-PD-1 m Ab处理的CBA/J×BALB/c妊娠小鼠间无明显差异(P>0.05)。此外,与正常妊娠CBA/J×BALB/c小鼠相比,子宫PD-1+M2型巨噬细胞百分比在的anti-PD-1 m Ab处理CBA/J×BALB/c妊娠小鼠和CBA/J×DBA/2流产倾向小鼠明显降低(P<0.05),而anti-PD-1 m Ab处理的CBA/J×BALB/c妊娠小鼠和CBA/J×DBA/2流产倾向小鼠之间无明显差异(P>0.05)。脾脏PD-1+M2型巨噬细胞在三组之间无明显差异(P>0.05)。【结论】PD-1/PD-L1信号传导减弱或缺失与胚胎吸收率升高和子宫M1/M2比值升高相关,即阻断PD-1信号传导导致胚胎吸收率升高,同时伴有子宫M2型巨噬细胞减少。母-胎界面PD-1表达缺失,也许是导致流产倾向小鼠胚胎吸收率升高和子宫M1/M2比值升高的关键因素。更多还原

【Abstract】 Part 1 The profile of decidual macrophages and PD-1 and PD-L1 expression at the maternal-fetal interface during early pregnancy[Purpose] To characterize the profile of decidual macrophage(DM)and PD-1 and PD-L1 expression in first trimester decidual samples and villus obtained from normal pregnant(NP)women undergoing elective terminations and from patients with recurrent miscarriages(RM).[Methods] The profile of M1(CD14+CD80+/CD14+CD86+/CD14+CD80+CD86+)and M2(CD14+CD163+/CD14+CD206+/CD14+CD163+CD206+)DM,and the expression of programmed cell death(PD)-1 and PD-L1 in DM from women with NP(n=20)and RM(n=16)were measured by flow cytometry.PD-L1 expression in human villi from women with NP(n=19)and RM(n=15)was determined by quantitative real time-polymerase chain reaction(q RT-PCR)and western blot.[Results] In decidual samples obtained from NP and RM,we identified both M1(CD14+CD80+/CD14+CD86+/CD14+CD80+CD86+)and M2(CD14+CD163+/CD14+CD206+ /CD14+CD163+CD206+)DM;although the M2 was the dominant phenotype in NP,the M1 in RM.The dominant subset for M1 was CD14+CD86+ and CD14+CD206+ for M2 DM.The M1 population is significantly higher in RM samples compared to the control NP(P < 0.01).Similarly,the M2 population is diminished in RM patients compared to control NP(P < 0.01).In RM,M1 DM positive for PD-1 was significantly lower than those observed in NPs as well as for M2 DM(P < 0.05).PD-L1 was also expressed in M1 and M2 DM,the percentage of M1/PD-L1+DMs was higher than that of M2/PD-L1+DM in NP and RM(P < 0.05).The percentage of PD-L1+CD14+CD163+ and PD-L1+CD14+CD206+ were lower in RM compared to NP(P < 0.05).Compared to women with NP,lower levels of villus PD-L1 were found in women with RM(P < 0.001).[Conclusions] CD14+CD86+ and CD14+CD206+ are the best markers to define M1 and M2 DM in early pregnancy,respectively.Lower levels of PD-1 and PD-L1 might contribute to higher percentage of M1 and lower percentage of M2 DM in women with RM.Part 2 The modulatory effects of the PD-1/PD-L1 axis on macrophage differentiation and function during early pregnancy[Purpose] To determine the modulatory effects of the PD-1/PD-L1 axis on macrophage differentiation and function during early pregnancy.[Methods] An in vitro model consisting of peripheral CD14+ monocytes isolated from women with NP(n=50)was used.With PD-1/PD-L1 axis activation(PD-L1 Fc(10 μg/m L))or blockade(anti-PD-L1 m Ab(10 μg/m L)and anti-PD-1 m Ab(10 μg/m L)),CD14+ monocytes were treated with recombinatant human granulocyte-macrophage colony-stimulating factor(rh GM-CSF)for 7d.Then the profile of differentiated macrophages and their phagocytotic activity were measured by flow cytometry.The m RNA levels of genes potentially underlying macrophage polarization modulated by PD-1 signaling were determined by q RT-PCR.[Results] Activation of the PD-1 pathway by PD-1 agonist(PD-L1 Fc)promoted the polarization of GM-CSF-differentiated macrophages towards the M2 phenotype(P < 0.01).Inhibition of PD-1 with PD-1 blocking antibody enhanced the polarization towards the M1 phenotype(P < 0.001).The inhibition of PD-L1 has no significant effect on macrophage polarization(P > 0.05).Compared with control macrophages,PD-L1 Fc increased macrophages’ phagocytic activity(P < 0.01).This was profoundly inhibited by anti-PD-1 m Abs(P < 0.001).Anti-PD-L1 m Ab treatment has no inhibitory effect on phagocytic activity(P > 0.05).PD-L1 Fc enhanced the expression of interferon regulatory factor(IRF)-4 m RNA while inhibition of PD-1 promoted IRF5 m RNA(P < 0.05).Higher levels of interleukin(IL)-1β m RNA and tumor necrosis factor(TNF)-α m RNA were induced by anti-PD-1 m Ab(P < 0.05).[Conclusions] Activation of the PD-1 pathway was responsible for the polarization of macrophages towards the M2 phenotype and inhibition of this pathway will promote and M1 phenotype.Part 3 The potential mechanism underlying the PD-1/PD-L1 axis modulating macrophage differentiation during early pregnancy[Purpose] To explore the potential mechanism underlying the regulatory effects of the PD-1/PD-L1 pathway on macrophage polarization during early pregnancy.[Methods] An in vitro model consisting of peripheral CD14+ monocytes isolated from women with NP(n=8)was used.With PD-1/PD-L1 axis blockade(anti-PD-1 m Ab(10 μg/m L)),CD14+ monocytes were treated with rh GM-CSF for 7d.The m RNA levels of genes potentially underlying macrophage polarization modulated by PD-1 signaling were determined by q RT-PCR.[Results] Compared with the control macrophages,PD-1 blockade increased m RNA expression of glucose transporter Glut-1(P < 0.01).However,anti-PD-1 m Ab administration decreased carnitine palmitoyltransferase(CPT)ⅠA m RNA expression(P < 0.01)and enhanced the m RNA expression of the glutamine transporters(P < 0.01),sodiumcoupled neutral amino acid transporter(SNAT)-1 and-2(P < 0.01).PD-1 blockade promoted the m RNA levels of the enzymes associated with glycolysis,such as hexokinase(HK)-2(P < 0.01)and pyruvate dehydrogenase kinase(PDK)-1(P < 0.01).PD-1 blockade also augmented the expression of fatthy acid synthase(FASN)m RNA(P < 0.05).PD-1 blockade enhanced the m RNA expression levels of Phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)and mammalian target of rapamycin(m-TOR)(P < 0.01).The levels of mitogenactivated protein kinase/extracellular signal-regulated kinase(MEK)/extracellular signal-regulated kinase(ERK)m RNA also increased with anti-PD-1 m Abs administration(P < 0.01).[Conclusions] PD-1 blockade profoundly reprogrammed metabolism during macrophage differentiation,characterized by enhanced glycolysis,which might be jointly regulated by the PI3K/AKT/m-TOR and MRK/ERK signaling.Part 4 The trophoblast derived soluble PD-L1 promotes the differentiation of M2 macrophages[Purpose] To test the expression of s PD-L1 in trophoblast cell line and human primary trophoblast cells,and the potential effect of s PD-L1 on macrophage differentiation.[Methods] Villous was collect from women with NP,then trophoblasts were isolated by enzyme digestion and cultured in vitro under low serum medium to obtain conditioned medium from human primary trophoblast cells.Swan 71 and 3A trophoblast cell lines were also cultured in low serum to obtain conditioned medium.Then s PD-L1 expression was analyzed by Simple Plex and m PD-L1 by Western Blot.CD14+ monocytes were sorted by immunomagnetic method from healthy women with normal fertility and educated with trophoblast conditioned medium(TCM)for 7 days with or without anti-PD-1 m Ab administration.The macrophage profile and cytokine profile were analyzed by flow cytometry and Simple Plex.[Results] Both m PD-L1 and s PD-L1 were expressed in Swan 71 cells.Compared with m PD-L1,s PD-L1 secretion level gradually increased with time(P < 0.01).The same pattern was also observed in 3A and human primary trophoblasts(P < 0.01).TCM induced M2-type macrophage differentiation,while the percentage of M2 macrophages in anti-PD-1 m Ab treatment group was significantly reduced;in addition,higher levels of pro-inflammatory cytokines such as IL-6 and TNF-α were produced in macrophage with anti-PD-1 m Ab treatment.[Conclusions] The trophoblasts constitutively secrete s PD-L1,block PD-1 receptor attenuates the ability of TCM to induce macrophage differentiation into M2 type;these data indicate that trophoblast-derived s PD-L1 is involved in M2 macrophages Differentiation.Part 5 The modulatory effects of PD-1 signaling on macrophage polarization in vivo[Purpose] To further investigate the role of the PD-1 signaling in regulating macrophage polarization in maternal-fetal tolerance and pregnancy maintenance.[Methods] We established an allogenic normal pregnancy model by mating CBA/J females with BALB/c males(CBA/J×BALB/c).Then,we challenged the normal pregnant mice(CBA/J×BALB/c)with PD-1 blocking antibodies on Day 4.5,Day 6.5 and Day 8.5.We used the abortion-prone pregnant model by mating CBA/J females with DBA/2 males(CBA/J×DBA/2).All the mice were sacrificed on Day 10.5,the embryo resorption rate was analyzed.The profile of uterine and spleen macrophages as well as PD-1 expression were determined by flow cytometry.[Results] Administration of anti-PD-1 m Abs to CBA/J×BALB/c pregnant mice induced significant fetal loss(P < 0.05),similar to the rate of fetal loss in the CBA/J×DBA/2 abortion prone mice(P < 0.05).Compared to the CBA/J×BALB/c normal pregnant mice,we observed a significant decrease in the percentage of M2 macrophages in the uterus of CBA/J×BALB/c mice treated with anti-PD-1 m Abs(P < 0.05).The CBA/J×DBA/2 abortion prone mice also showed a lower percentage of M2 macrophages compared to the normal pregnant group(P < 0.05).Treatment with anti-PD-1 m Ab increased the M1/M2 ratios in the uterus and spleen of CBA/J×BALB/c pregnant mice(P < 0.05).Similar high M1/M2 ratios were observed in the CBA/J×DBA/2 abortion prone mice(P < 0.05).In the CBA/J×DBA/2 abortion prone mice,PD-1+ uterine macrophages were significantly lower compared with the CBA/J×BALB/c normal pregnant mice(P < 0.05).This was also the case for the CBA/J×BALB/c mice receiving anti-PD-1 m Ab treatment,where we found lower PD-1+ uterine macrophages(P < 0.05).Anti-PD-1 m Ab administration had no effect on the expression of PD-1 in spleen macrophages(P > 0.05).Most of the M2 uterine macrophages were PD-1 positive cells in the CBA/J×BALB/c normal pregnant mice.However,much less M2 uterine macrophages were PD-1 positive in the CBA/J×DBA/2 abortion prone mice and PD-1 blockade CBA/J×BALB/c mice(P < 0.05).However,anti-PD-1 m Ab administration had no effect on the percentages of spleen PD-1+ M2 macrophages in the CBA/J×BALB/c mice(P > 0.05).[Conclusions] Our in vivo data from mice further demonstrate that macrophages polarization during early pregnancy is influenced by the PD-1/PD-L1 axis.PD-1 deficiency or blockade of PD-1 pathway induced monocytes/macrophages polarization into an M1 phenotype,which may be potentially responsible for the observed pregnancy loss.更多还原