Yonghong Zhang1,2, Lina Ma1, Xiaohui Hu1, Jinlu Ji1, Gil Mor2, Aihua Liao1. 1. Family Planning Research Institute, Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China. 2. Division of Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA.
Abstract
STUDY QUESTION: What is the role of the programmed cell death-1 (PD-1)/PD-1 ligand-1 (PD-L1) axis in macrophage polarization during early pregnancy? SUMMARY ANSWER: PD-1 signaling is a major regulator of macrophage differentiation and function, and it is critical for the success of a pregnancy. WHAT IS KNOWN ALREADY: The predominance of decidual macrophages (DMs) with an M2 phenotype is an important contributor to maternal-fetal tolerance during early pregnancy. STUDY DESIGN, SIZE, DURATION: Twenty-four women with recurrent miscarriage (RM) and 70 women undergoing elective termination of an early normal pregnancy (NP) were included. Twelve female CBA/J, four male DBA/2, and four male BALB/c mice were included and mating carried out. The 12 CBA/J pregnant mice were then categorized into three groups of four mice: healthy control group CBA/J×BALB/c, abortion-prone pregnant group CBA/J×DBA/2 and normal pregnancies CBA/J×BALB/c treated with anti-PD-1 monoclonal antibodies. PARTICIPANTS/MATERIALS, SETTING, METHODS: The profile of DMs, and the expression of PD-1 and PD-L1 in DMs from women with NP and RM were measured by flow cytometry. PD-L1 expression in human villi was determined by quantitative RT-PCR (qRT-PCR) and western blot. An in vitro model consisting of peripheral CD14+ monocytes isolated from women with NP was used. The profile of differentiated macrophages and their phagocytotic activity were then measured by flow cytometry. The mRNA levels of genes potentially underlying macrophage polarization modulated by PD-1 signaling were determined by qRT-PCR. Twelve pregnant mice were included in our in vivo model and underwent different treatment. The embryo resorption rate, and macrophage profile as well as PD-1 expression in murine spleens and uterus were analyzed by flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with NP, women with RM had elevated percentages of M1 DMs (P < 0.01), and reduced frequencies of M2 DMs (P < 0.05), as well as decreased PD-1 protein expression (P < 0.05) in the DMs. In addition, decreased mRNA and protein levels of PD-L1 expression in placental villi were observed in women with RM (P < 0.001). Using in vitro experiments, compared to the control group, we found that PD-1 activation by recombinant human (rh) PD-L1 Fc (human PD-L1 fused to the Fc region of human IgG1) drove the differentiation of macrophages with immuno-modulatory characteristics (P < 0.01). However, PD-1 blockade promoted dominance of the M1 phenotype (P < 0.01). PD-1 polarized macrophages showed enhanced phagocytic activity (P < 0.01), which was decreased with PD-1 blockade (P < 0.001). Furthermore, PD-1 blockade promoted the expression of pro-inflammatory cytokines and interferon regulatory factor (IRF) 5 (P < 0.05), while IRF4 expression was inhibited (P < 0.05). In addition, PD-1 blockade promoted macrophage glycolysis (P < 0.01) and inhibited fatty acid oxidation (P < 0.05). The mRNA expression levels of both phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin and mitogen-activated protein kinase/extracellular signal-regulated kinase/extracellular signal-regulated kinase were upregulated (P < 0.05) with PD-1 blockade during DM metabolic reprogramming. Moreover, in vivo mice data showed that PD-1 blockade or deficiency was associated with decreased M2 percentages at the maternal-fetal interface (P < 0.05) and embryo loss (P < 0.05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Whether the changes in DM polarization seen in miscarriage tissues are a cause or consequence of the demise of the pregnancy still requires further investigation. In addition, conducting metabolite analysis is required to further measure bioenergetic profiles. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study on the role of the PD-1/PD-L1 axis in macrophage polarization during early pregnancy; such exploration enhances our understanding of the physiology of early pregnancy. Our study also indicates that targeting the PD-1 pathway may represent a novel therapeutic strategy to prevent pregnancy loss. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Nature Science Foundation of China (No. 81671490) and Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, HUST (No. 5001519002). None of the authors has any conflict of interest to declare.
STUDY QUESTION: What is the role of the programmed cell death-1 (PD-1)/PD-1 ligand-1 (PD-L1) axis in macrophage polarization during early pregnancy? SUMMARY ANSWER: PD-1 signaling is a major regulator of macrophage differentiation and function, and it is critical for the success of a pregnancy. WHAT IS KNOWN ALREADY: The predominance of decidual macrophages (DMs) with an M2 phenotype is an important contributor to maternal-fetal tolerance during early pregnancy. STUDY DESIGN, SIZE, DURATION: Twenty-four women with recurrent miscarriage (RM) and 70 women undergoing elective termination of an early normal pregnancy (NP) were included. Twelve female CBA/J, four male DBA/2, and four male BALB/c mice were included and mating carried out. The 12 CBA/J pregnant mice were then categorized into three groups of four mice: healthy control group CBA/J×BALB/c, abortion-prone pregnant group CBA/J×DBA/2 and normal pregnancies CBA/J×BALB/c treated with anti-PD-1 monoclonal antibodies. PARTICIPANTS/MATERIALS, SETTING, METHODS: The profile of DMs, and the expression of PD-1 and PD-L1 in DMs from women with NP and RM were measured by flow cytometry. PD-L1 expression in human villi was determined by quantitative RT-PCR (qRT-PCR) and western blot. An in vitro model consisting of peripheral CD14+ monocytes isolated from women with NP was used. The profile of differentiated macrophages and their phagocytotic activity were then measured by flow cytometry. The mRNA levels of genes potentially underlying macrophage polarization modulated by PD-1 signaling were determined by qRT-PCR. Twelve pregnant mice were included in our in vivo model and underwent different treatment. The embryo resorption rate, and macrophage profile as well as PD-1 expression in murine spleens and uterus were analyzed by flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with NP, women with RM had elevated percentages of M1 DMs (P < 0.01), and reduced frequencies of M2 DMs (P < 0.05), as well as decreased PD-1 protein expression (P < 0.05) in the DMs. In addition, decreased mRNA and protein levels of PD-L1 expression in placental villi were observed in women with RM (P < 0.001). Using in vitro experiments, compared to the control group, we found that PD-1 activation by recombinant human (rh) PD-L1 Fc (human PD-L1 fused to the Fc region of human IgG1) drove the differentiation of macrophages with immuno-modulatory characteristics (P < 0.01). However, PD-1 blockade promoted dominance of the M1 phenotype (P < 0.01). PD-1 polarized macrophages showed enhanced phagocytic activity (P < 0.01), which was decreased with PD-1 blockade (P < 0.001). Furthermore, PD-1 blockade promoted the expression of pro-inflammatory cytokines and interferon regulatory factor (IRF) 5 (P < 0.05), while IRF4 expression was inhibited (P < 0.05). In addition, PD-1 blockade promoted macrophage glycolysis (P < 0.01) and inhibited fatty acid oxidation (P < 0.05). The mRNA expression levels of both phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin and mitogen-activated protein kinase/extracellular signal-regulated kinase/extracellular signal-regulated kinase were upregulated (P < 0.05) with PD-1 blockade during DM metabolic reprogramming. Moreover, in vivo mice data showed that PD-1 blockade or deficiency was associated with decreased M2 percentages at the maternal-fetal interface (P < 0.05) and embryo loss (P < 0.05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Whether the changes in DM polarization seen in miscarriage tissues are a cause or consequence of the demise of the pregnancy still requires further investigation. In addition, conducting metabolite analysis is required to further measure bioenergetic profiles. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study on the role of the PD-1/PD-L1 axis in macrophage polarization during early pregnancy; such exploration enhances our understanding of the physiology of early pregnancy. Our study also indicates that targeting the PD-1 pathway may represent a novel therapeutic strategy to prevent pregnancy loss. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Nature Science Foundation of China (No. 81671490) and Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, HUST (No. 5001519002). None of the authors has any conflict of interest to declare.
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