Mechanism of PD-1/PD-L1 signaling pathway regulating Treg/Th17 in the occurrence and development of preeclampsia

WANG Li, CHEN Xiao-ju , LIN Qing, ZHENG Lin-mei, KONG Jiao, MAO Dong-rui

Department of Obstetrics, Hainan Provincial People’s Hospital, Hainan Hospital Affiliated to Hainan Medical University, Haikou 570013, China

Keywords:PD-1/PD-L1 Treg/Th17 Preeclampsia

ABSTRACT Objective: To detect the expression levels of programmed cell death 1 (PD-1) and its ligand PD-L1, regulatory cell (Treg) and T helper cell 17 (Th17) specific nuclear transcription factors forkhead box protein 3 (Foxp3) and the retinoic acid-related orphan nuclear receptorgammat(RORγc) in the placenta of normal pregnant women and in preeclamptic (PE) women, and to study the relationship between their differential expression and the development of PE.Methods: Between March 2021 and March 2022, 40 patients with pre-eclampsia who were treated at the Obstetrical Department in Hainan Hospital were selected, including 20 cases of mild (PE) and 20 cases of severe (sPE), and 20 normal pregnant women with singleton cesarean section in the same period were selected to collect placental tissue. PD-1, PD-L1,Foxp3 and RORγc in placenta were detected by qRT-PCR and Western blot. Results: (1) The results of qRT-PCR assay showed that the mRNA expression of PD?1, PD-L1, Foxp3 protein decreased significantly compared with the control group. The changes in PD?1, PD?L1 and Foxp3 became more obvious as the disease progressed (P＜0.05); The mRNA expression of RORγc increased significantly compared with the control group. The changes of RORγc mRNA became more obviousas as the disease progressed (P＜0.05);(2)The results of Western blot assay show, compared with the normal group, the protein expression of PD?1, PD?L1, Foxp3 mRNA decreased significantly. With the aggravation of the disease, the changes of PD-1 and PD-L1 became more obvious, with significant difference (P＜0.05); Compared with the normal group, the protein expression of RORγc increased significantly. With the aggravation of the disease, the changes of RORγc protein became more obvious with significant difference(P＜0.05). Conclusion: PD-1/PD-Ll participates in the occurrence and development of preeclampsia by regulating Treg/Th17 immune balance.

1. Introduction

Preeclampsia (PE) is a disease unique to pregnancy, characterized by new onset of hypertension and multiple organ function involvement after 20 weeks of gestation, which is one of the causes of increased maternal and infant morbidity and mortality[1,2]. Its etiology is unclear, and its mechanisms are complex and diverse. At present, it is believed that the main pathophysiological changes causing PE are immune imbalance at the maternal-fetal interface, hypoxia, abnormal trophoblast invasion, and excessive systemic inflammatory reaction [3,4]. Among them, foreign antigens cause immunological imbalance at the maternal-fetal interface,and maternal immune tolerance induces chronic low-grade inflammation, causing research hotspots, but the specific mechanism is unclear [5].

PD-1 is peripheral tissue T cell-mediated immune response of the main immunosuppressive factor, to limit the effect of T cell response mediated peripheral tissue damage, not only involved in infectious disease caused by viruses and bacteria, the tumor immune escape,graft rejection and autoimmune [6-8], but with the maternal-fetal immune tolerance induction and pregnancy maintenance is closely related to the [9]. Studies have shown [10-13] that the expression level of PD-1 on peripheral blood Treg cells of PE patients is increased compared with that of normal pregnant women in the same period, but the expression level of PD-1 on Thl7 cells is decreased compared with that of normal pregnant women in the same period,and this difference is positively correlated with the severity of the disease, and this differential expression inhibits the production and differentiation of Treg cells. But abnormally activated Thl7 cells. Therefore, we speculate that the dysfunction of PD-1/PD-L1 pathway may be the cause of Treg/Th17 imbalance in PE patients. In this study, the expression levels of PD-1, PD-L1, Foxp3 and RORγc at the maternal-fetal interface of PE patients were observed, and their expression and interaction in PE were investigated. The summary is as follows:

2. Data and Methods

2. 1 General Information

Pregnant women with singleton pregnancy who were hospitalized and terminated in Hainan Hospital Affiliated to Hainan Medical College from March 2021 to March 2022 were selected, including 20 cases in PE group, 20 cases in sPE group and 20 cases in control group. The diagnostic criteria were referred to the eighth edition of Obstetrics and Gynecology. There were no significant differences in age, parity and parity among all groups (P＞0.05). Exclusion criteria: this pregnancy was a multiple pregnancy, had diabetes,hypertension, chronic kidney disease, hepatitis and other diseases before pregnancy, or had autoimmune diseases or blood diseases, or had received blood transfusion, transplantation, immunotherapy and other treatments, or had a history of alcohol, tobacco and drug abuse.This project was approved by the Medical Ethics Committee of Hainan Hospital, and the informed consent of research content was signed.

2.2 Research Methods

2.2.1 Specimen collection and processing

Placental tissues were obtained from subjects in PE group, sPE group and control group. After delivery of the placenta by cesarean section, aseptic operation was performed. About 100 mg placenta tissue was cut from the maternal surface of the placenta, and the calcified or bleeding part was not removed. The placenta was rinsed with normal saline three times, then placed in the EP tube of RNA enzyme and stored in a -80 ℃ refrigerator.

2.2.2 qRT?PCR was used to detect the expression levels of PD-1, PD-L1, Foxp3 and RORγc mRNA in placenta

The mRNA series of target genes were searched on NCBI, and primers were designed using Primer6.0 software. The primers for PD?1, PD?L1, Foxp3 and RORγc were synthesized by Guangdong Wenhuas Biological Co., LTD. The preserved placental tissue was removed, total RNA of the tissue was extracted and its purity was detected. CDNA was synthesized by reverse transcription mode of PCR (the operation was strictly carried out in the kit instruction).PCR amplification reaction was performed by using cDNA template and carefully following the operation steps of the kit (ABI Company,USA). The target genes PD?1, PD?L1, Foxp3 and RORγc were detected. The above steps were repeated 3 times, and the final value was the average of the three times to calculate the mRNA expression level. Relative gene expression levels were expressed as 2-ΔΔCT(GAPDH as internal reference).

2.2.3 Western blot analysis of the expression levels of PD-1,PD-L1, Foxp3 and RORγc proteins in placenta

The frozen placental tissue was removed, and the tissue protein was extracted with RIPA lysate (Solibo, Beijing). The protein concentration was quantified with BCA kit (Solibo, Beijing). After SDS-PAGE electrophoresis, the membrane was transferred, and the membrane was immersed in the blocking solution for 1 h at room temperature. Rabbit anti-human PD-1 and PD-L1, mouse anti-human Foxp3 and RORγC (all Abcam, USA), and mouse anti-GAPDH (CST, USA) were combined, and then placed in a 4 ℃refrigerator. The next day, goat anti-rabbit/anti-mouse anti-antibody was added and mixed with light blowing and shaking for 1 h at room temperature. The results were thoroughly washed, scanned by gel imaging system and analyzed by ImageJ software.

2.3 Statistical processing

All data were input into SPSS21.0 software for processing. Normal measurement data were represented as (±s), and differences were compared by ANOVA. If the distribution is not normal, it is represented by M (P25, P75), and the rank-sum test is used to compare the differences. Enumeration data were represented by example (%),and differences were compared by chi-square test. Test level α = 0.05(two-sided), correction for pairwise comparisons = 0.05 / number of comparisons. P＜0.05 was considered statistically significant.

3. Results

3.1 Analysis of clinical data

Clinical characteristics of PE, sPE and control groups. There were no significant differences in age, gestational age, parity and parity among the three groups (P＞0.05), while there were significant differences in systolic blood pressure and diastolic blood pressure(P＜0.01), as shown in Table 2.are shown in Table 3.

Tab 1 PCR primer sequence

3.2 mRNA expression levels of PD-1, PD-L1, Foxp3 and RORγC

The mRNA expression levels of PD?1, PD?L1, Foxp3 and RORγc in placenta were detected by qRT-PCR. The results showed that compared with the control group, the mRNA expression levels of PD?1 in PE group (0.19±0.01) and sPE group (0.08±0.01) were significantly decreased (see Figure 1A). The mRNA expression level of PD?L1 in PE group (0.21±0.011) and sPE group (0.09±0.01)also decreased (see Figure 1B). The mRNA expression levels of Foxp3 in PE group (0.49±0.04) and sPE group (0.22±0.02) were also significantly decreased (see Figure 1C). The mRNA levels of PD?1, PD?L1 and Foxp3 in sPE group decreased more significantly than those in PE group, and the differences between the two groups were statistically significant (P＜0.05). The mRNA expression of RORγc gene in PE group (1.29±0.0) and sPE group (1.89±0.05)was significantly higher than that in normal maternal placenta. The mRNA expression of RORγc gene in sPE group was significantly higher than that in PE group (Figure 1D), and the differences between groups were statistically significant (P＜0.05). The results

3.3 Expression levels of placental PD-1, PD-L1, Foxp3 and RORγc proteins in control, PE and sPE groups

Western blot showed that compared with the control group, the protein expression level of PD-1 in PE group (0.59±0.01) and sPE group (0.38±0.02) was significantly decreased (see Figure 2B). The expression of PD-L1 protein was decreased in PE group (0.62±0.02)and sPE group (0.33±0.01) (see Figure 2C). The expression levels of Foxp3 protein in PE group (0.20±0.02) and sPE group (0.09±0.01)were also significantly decreased (see Figure 2D). Compared with PE group, the protein expression levels of PD-1, PD-L1 and Foxp3 in sPE group decreased more significantly, and the differences between the three groups were statistically significant (P＜0.05).Compared with the control group, the protein expression level of RORγc in PE group (0.87±0.03) and sPE group (1.68±0.05) was significantly increased. Compared with PE group, RORγc protein expression in sPE group increased more significantly (Figure 2 E), and the differences between the two groups were statistically significant (P＜0.05). The results are shown in Table 4.

Tab 3 The mRNA expression level of PD?1, PD?L1, Foxp3 and RORγc of all groups (n=20, ±s)

Tab 3 The mRNA expression level of PD?1, PD?L1, Foxp3 and RORγc of all groups (n=20, ±s)

Note: Compared with the control group, **P＜0.01; Compared with PE group, ##P＜0.01.

Group PD?1 PD?L1 Foxp3 RORγc sPE 0.08±0.01**## 0.09±0.01**## 0.22±0.02**## 1.89±0.05**##PE 0.19±0.01** 0.21±0.01** 0.49±0.04** 1.29±0.04**Control group 0.58±0.02 0.62±0.02 0.87±0.05 0.33±0.02 F 8 884 9 173 1 226 7 811 P 0.000 0.000 0.000 0.000

Fig 1 The mRNA expression level of PD?1, PD?L1, Foxp3 and RORγc

Tab 4 Protein expression levels of PD-1, PD-L1, Foxp3 and RORγc of all groups(n=20,±s)

Tab 4 Protein expression levels of PD-1, PD-L1, Foxp3 and RORγc of all groups(n=20,±s)

Note: Compared with the control group, **P＜0.01; Compared with PE group, ##P＜0.01.

Group PD-1 PD-L1 Foxp3 RORγc sPE 0.38±0.02**## 0.33±0.01**## 0.09±0.01**## 1.68±0.05**##PE 0.59±0.01** 0.62±0.02** 0.20±0.02** 0.87±0.03**Control group 0.98±0.03 0.89±0.03 0.58±0.03 0.28±0.02 F 3 556 2 011 2 864 8 362 P 0.000 0.000 0.000 0.000

4. Discussion

The adaptive and innate immune systems play important roles in the development and pathogenesis of different types of pregnancy disorders, such as preeclampsia. Dysregulation of spiral arteries and insufficient trophoblast invasion lead to PE through the production of various inflammatory cytokines and antiangiogenic factors from the placenta. The role of T lymphocytes in immune tolerance and immune response in pregnancy has become one of the research hotspots in recent years.

Th1, Th2, Th17, and Treg cells are derived from CD4+T lymphocytes, and the normal association between these cells is necessary to prevent pregnancy disorders such as PE. Treg plays an inhibitory role by regulating antigen presentation, target cell lysis,secretion of inhibitory cytokines and other different mechanisms[14,15], and plays an important role in maintaining normal pregnancy and reducing maternal-fetal complications [16]. If the number of Treg cells is reduced, the immunosuppressive function of Treg cells is weakened accordingly, resulting in impaired immune tolerance at the maternal-fetal interface, which is an important cause of infertility, recurrent abortion, preeclampsia and other adverse pregnancy outcomes [17]. Previous studies have confirmed that the immunosuppressive and cellular regulatory functions of Tregs are dependent on Foxp3 expression [18,19]. RORγc molecule is a key transcription factor regulating Th17 cells, which is involved in the pathogenesis of autoimmune diseases and the body's rejection after transplantation [20]. Studies have shown that Foxp3 overexpression can further induce naive CD4+T precursors to transform into Tregs[21]; conversely, Foxp3 dysfunction will lead to congenital Treg cell defects and severe systemic immune disorders in humans [22]. The results of this study showed that Foxp3 expression in placenta of preeclampsia patients was low, and decreased with the progression of the disease, and the difference was statistically significant (P＜0.01).These results suggest that the low expression of Foxp3 can lead to the decrease of immune suppression, resulting in the imbalance of maternal and fetal immune tolerance, and induce PE. The nuclear transcription factor RORγc of Th17 cells increased significantly with the aggravation of the disease (P＜0.01), suggesting that there is a lack of Treg number and excessive activation of Th17 cells at the maternal-fetal interface in PE patients, which is consistent with the conclusions of domestic and foreign researchers [23-27]. The imbalance of Treg and Th17 cells can induce chronic inflammation in PE. However, the main factors leading to the imbalance of cellular immunity have not been fully elucidated.

PD-1 is commonly found in activated T cells, B cells, macrophages and other effector cells, and PD-L1 is expressed not only on tumor cells and epithelial cells, but also on trophoblast cells of the placenta[27]. The interaction between PD-1 and PD-LI has become a key player in regulating immune response and peripheral tolerance. PD-1 regulates the immune system's response to the body by negatively regulating the secretion of proinflammatory factors by T cells[28-30]. Studies have confirmed that maternal-fetal tolerance and normal pregnancy maintenance are achieved by inducing Treg cell differentiation through PD-1/PD-L1 pathway, preventing Th17 cell activation, and promoting Treg/Th17 cell balance during pregnancy[31-34]. Research found that intraperitoneal injection of anti-PD-L1 into pregnant mice caused PD-1/PD-L1 pathway block, inhibited the development of Treg cells, and abnormally activated Thl7 cells,eventually leading to a decrease in the litter size and weight of the offspring mice, and an increase in the rate of embryonic abortion [35].The PD-1 / PD-L1 pathway defends against potentially pathogenic effector T cells by simultaneously utilizing two peripheral tolerance mechanisms :(1) promoting Treg development and function, and (2)directly inhibiting pathogenic effector T cells. In this study, compared with the normal control group, the expressions of PD-1 and PDL1 at the maternal-fetal interface were decreased in PE and sPE groups. The low expression of PD-L1 inhibited the differentiation of T helper cells into Treg cells and promoted the apoptosis of Treg cells, which was manifested by the decreased expression of Foxp3 at the maternal-fetal interface. The low expression of PD-1 promoted the differentiation and proliferation of Th17 cells, as indicated by the significantly increased expression level of RORγc at the maternalfetal interface. This result is consistent with the findings[35,27].Therefore, the alteration of PD-1 / PD-L1 pathway may be related to Treg/Th17 imbalance in human pregnancy.

In conclusion, the expression of PD-1/PD-L1 may be a key factor in balancing Tregs and Th17 cells and maintaining tolerance at the fetomaternal interface. Therefore, we speculate that CD4+T cells differentiate into Thl7 cells in the inflammatory environment of PE, resulting in Treg/Thl7 imbalance, and PD-1/PD-L1 signaling pathway plays an important regulatory role in this process.

Author’s contribution:

Wang Li participated in the topic selection, design, part of the experiment and the writing and modification of the paper; Chen Xiao-ju was responsible for specimen collection and clinical data statistics.

All authors declare no conflict of interest.