The Role And Mechanism Of PD-1/PD-L1 Signaling Pathway In Macrophage Differentiation And Function During Early Pregnancy

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 ?g/m L))or blockade(anti-PD-L1 m Ab(10 ?g/m L)and anti-PD-1 m Ab(10 ?g/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)-1? m RNA and tumor necrosis factor(TNF)-? 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 ?g/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)?A 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-? 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.