Expression of GRIM-19 in unexplained recurrent spontaneous abortion and possible pathogenesis

Expression of GRIM-19 in unexplained recurrent spontaneous abortion and possible pathogenesis Abstract STUDY QUESTION Is aberrant expression of gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) associated with unexplained recurrent spontaneous abortion (URSA)? SUMMARY ANSWER GRIM-19 deficiency may regulate regulatory T cell/T helper 17 cell (Treg/Th17) balance partly through reactive oxygen species (ROS)–mammalian target of rapamycin (mTOR) signaling axis in URSA. WHAT IS KNOWN ALREADY Immunological disorders may cause impaired maternal immune tolerance to the fetus and result in fetal rejection. The differentiation of Treg and Th17 cells is controlled by phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling pathway. GRIM-19 participates in the immune response, but its role in URSA is largely unknown. STUDY DESIGN, SIZE, DURATION The current study included 28 URSA patients and 30 non-pregnant healthy women. PARTICIPANTS/MATERIALS, SETTING, METHODS The proportion of Treg and Th17 cells in peripheral blood of URSA patients and control subjects were assessed with flow cytometry. The expression of GRIM-19 in peripheral blood lymphocytes (PBLs) was measured with quantitative real-time PCR and western blot analysis. Furthermore, the ROS level in the PBLs of URSA patients and control subjects were assessed by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Then, Akt/mTOR expression in the PBLs was measured. Downregulation of GRIM-19 in Jurkat cells was performed by specific siRNA. Then, intracellular ROS production and the expression of p-mTOR, which is known to enhance Th17 differentiation and decrease Treg cell differentiation, were detected. Finally, N-acetylcysteine (NAC) was used to decrease the intracellular ROS level, and the expression of p-mTOR was measured. MAIN RESULTS AND THE ROLE OF CHANCE The proportion of Treg cells was reduced in URSA patients, whereas the proportion of Th17 cells was increased. The expression of GRIM-19 was significantly lower in PBLs of URSA patients. Furthermore, there is a considerable increase in intracellular ROS production and a high level of p-Akt and p-mTOR expression in the PBLs of URSA patients compared with the control subjects. In parallel to this, downregulation of GRIM-19 in the Jurkat cells by siRNA results in an increased ROS production and an increased expression of p-mTOR. Importantly, the upregulation of p-mTOR resulting from GRIM-19 loss was significantly reversed in the cells treatment with ROS inhibitor N-acetyl-l-cysteine (NAC), indicating that ROS was indeed required for GRIM-19 depletion induced p-mTOR expression. LARGE SCALE DATA None. LIMITATIONS, REASONS FOR CAUTION A large number of researches have confirmed that the differentiation of Treg and Th17 cells is controlled by PI3K/Akt/mTOR signaling pathway. We have not shown the regulatory role of ROS and PI3K/Akt/mTOR in Treg and Th17 differentiation in this study. WIDER IMPLICATIONS OF THE FINDINGS Our study has demonstrated that GRIM-19 deficiency may play a role in regulating Treg/Th17 balance partly through ROS–mTOR signaling axis in URSA. The present study offers a new perspective to the roles of GRIM-19 in immunoregulation. STUDY FUNDING AND COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (Grant numbers 81571511, 81701528, 81370711 and 30901603), the Shandong Provincial Natural Science Foundation (Grant numbers ZR2017PH052 and ZR2013HM090) and the Science Foundation of Qilu Hospital of Shandong University, Fundamental Research Funds of Shandong University (Grant numbers 2015QLQN50 and 2015QLMS24). The authors declare that there is no conflict of interest that could prejudice the impartiality of the present research. GRIM-19, unexplained recurrent spontaneous abortion, regulatory T cells, T helper 17 cells, reactive oxygen species Introduction Recurrent spontaneous abortion (RSA) is a common problem among couples of reproductive age and is defined as three or more consecutive pregnancy losses before the 20th week of gestation (Patriarca et al., 2000; Poole and Claman, 2004; Yang et al., 2008). Unexplained RSA (URSA) still affects ~50% of RSA patients, although the causes of RSA include chromosomal aberrations, hematological problems, hormonal disorders, uterine anatomical defects, infectious and immunological abnormalities (Christiansen et al., 2005). Gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) was isolated by using antisense gene knockout as a death-associated protein (Angell et al., 2000), official full name NADH:ubiquinone oxidoreductase subunit A13 (NDUFA13). Further study in mice indicated that GRIM-19 plays an essential role in complex I assembly and electron transfer activity, whereas disruption of mitochondrial membrane potential (MMP) by GRIM-19 mutants increases the cells’ sensitivity to apoptotic stimuli (Lu and Cao, 2008). More important, GRIM-19 also participates in the immune response. A recent report in murine autoimmune arthritis has shown that GRIM-19 augmented regulatory T (Treg) cell differentiation, whereas it suppressed T helper 17 (Th17) cell differentiation, by inhibiting signal transducer and activator of transcription 3 (STAT3) phosphorylation (Moon et al., 2014). Another group reported that GRIM-19 heterozygous mice are prone to spontaneous urinary tract infection with reduced bacterial killing ability and pro-inflammatory cytokine production (Chen et al., 2012). Although our previous study has demonstrated that there is an association between missed abortion and low levels of GRIM-19, the potential role of GRIM-19 in URSA is largely unknown (Chen et al., 2015). GRIM-19 plays an essential role in the assembly and enzymatic activity of mitochondrial complex I, and mitochondria are the main source of intracellular reactive oxygen species (ROS). Aberrant expression of GRIM-19 may lead to changes in intracellular level of ROS. Safranova et al. (2003) reported increase ROS in peripheral blood granulocytes in women with habitual abortions. Meanwhile, ROS were critical for activation of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling pathway, in a manner that was dependent on mitochondrial respiratory chain (Silva et al., 2011). In addition, differentiation of Treg and Th17 cells is controlled by PI3K/Akt/mTOR signaling pathway (Kim et al., 2015). Studies have indicated that URSA is largely associated with the failure of fetomaternal immunologic tolerance (Wu et al., 2014). One of the most important immunological factors that plays a primary role in controlling the fetomaternal tolerance and results in a successful pregnancy are Treg cells. Treg cells suppress excessive immune response of other cells and maintain tolerance to self-antigens (Aluvihare et al., 2004; Somerset et al., 2004). The frequency of Treg cells was downregulated during human miscarriage (Hosseini et al., 2016). Th17 cells play an important role in induction of inflammation by producing pro-inflammatory cytokines. Recent data have shown a pathogenic effect of these cells in autoimmunity, transplant rejection and other diseases (Tesmer et al., 2008; Hirota et al., 2010; Sereshki et al., 2014). In URSA patients, it has been reported that the proportion of Th17 cells and the expression of IL-23, the Th17-inducing cytokine, are higher in the peripheral blood than in normal pregnancies (Saifi et al., 2014). Treg and Th17 cells are two distinct subsets of CD4+ T cells, and the balance between the two cell types could have an impact on the control of inflammation and autoimmunity. There is clear evidence that Treg/Th17 imbalance plays a potential role in the pathogenesis of URSA (Sereshki et al., 2014; Zhu et al., 2017). A decrease in Treg/Th17 ratio was observed in URSA patients due to the increased differentiation of Th17 cells and decreased differentiation of Treg cells from naive T cells through heightened circulating IL-6 levels (Zhu et al., 2017). In this study, we report that the proportion of Treg cells was reduced in URSA patients, whereas the proportion of Th17 cells was increased. Low level of GRIM-19 in peripheral blood lymphocytes (PBLs) of URSA patients was observed. Furthermore, there is a considerable increase in intracellular ROS production and a high level of p-Akt and p-mTOR, which is known to enhance Th17 differentiation and decrease Treg cells differentiation. Furthermore, the molecular mechanisms were investigated in Jurkat cells in vitro. Our study offers a new perspective to the roles of GRIM-19 in immunoregulation. Materials and Methods Ethical approval The study has been reviewed and approved by the Institutional Review Boards of Qilu Hospital of Shandong University. Written informed consent was obtained from all human subjects. Subjects All study participants were recruited at the department of gynecology and obstetrics, Qilu Hospital of Shandong University. Twenty-eight URSA patients with a mean (± standard deviation) age of 30.0 ± 4.1 (range: 23–40) years, who had at least three consecutive first trimester abortions (7–12 weeks of gestation) were recruited. The mean number of abortions was 3.9 ± 1.1 (range: 3–7). The diagnosis of URSA was made after excluding any verifiable causes: chromosomal abnormality, abnormalities of the uterus or cervix, endocrine and metabolic diseases, infection, congenital thrombophilias and autoimmune disease. Karyotype analysis of all abortion couples and abortuses was performed to exclude any chromosome problems. Pelvic examination and ultrasound were used to rule out abnormalities of uterus and cervix, and if necessary, a hysterosalpingography had to be performed for further confirmation. Serum samples were analyzed for FSH, LH, estradiol (E2), prolactin (PRL) and testosterone (T). Progesterone was analyzed at the seventh day after the LH surge in the same cycle. Free triiodothyronine, free thyroxin, thyrotropic stimulating hormone, thyroid peroxidase antibody and thyroglobulin antibody were analyzed. Endocrine diseases (hyperprolactinemia, luteal function defect, polycystic ovary syndrome and hyperandrogenemia) were excluded. Metabolic diseases (hyperthyroidism, insulin resistance, diabetes and hypothyroidism) were excluded. Chlamydia and ureaplasma infection were rule out by cervical mucus culture. Congenital thrombophilias was excluded by measurement of protein C, protein S and antithrombin III activity. The activated partial thrombin time was used to test the lupus anticoagulant. Systemic lupus erythematosus was excluded by measurement of the anticardiolipin antibodies, extracted nuclear antigen antibodies, and antinuclear antibodies. All male partners of URSA patients should have normal semen status, according to World Health Organization criteria. The control group comprised 30 non-pregnant healthy women, with a mean age of 30.3 ± 4.8 (range: 22–41) years (Table I). Control group women had at least one successful pregnancy without any disease and had no history of ectopic pregnancy, spontaneous abortion, preterm delivery, still birth, pre-eclampsia and abnormal pregnancy. Table I Clinical characteristics of subjects. Clinical characteristics URSA Control n 28 30 Age (y) 30.0 ± 4.1 30.3 ± 4.8 Number of abortions 3.9 ± 1.1 0.0 ± 0.0 Clinical characteristics URSA Control n 28 30 Age (y) 30.0 ± 4.1 30.3 ± 4.8 Number of abortions 3.9 ± 1.1 0.0 ± 0.0 Table I Clinical characteristics of subjects. Clinical characteristics URSA Control n 28 30 Age (y) 30.0 ± 4.1 30.3 ± 4.8 Number of abortions 3.9 ± 1.1 0.0 ± 0.0 Clinical characteristics URSA Control n 28 30 Age (y) 30.0 ± 4.1 30.3 ± 4.8 Number of abortions 3.9 ± 1.1 0.0 ± 0.0 All participants included in the control and URSA groups were not pregnant, as confirmed by a negative blood hCG test, and were nonsmokers with normal body mass index who took no medications. All participants were at least three months from their last abortion or pregnancy. Blood samples were taken from both groups during the luteal phase (at Days 19–23 of the menstrual cycle, particularly on Day 21). Isolation of PBLs A total of 5 ml heparinized venous blood was taken from the case and control groups. Human peripheral blood mononuclear cells (PBMCs) were separated from freshly isolated peripheral blood of subjects by gradient centrifugation on Ficoll-Hypaque (Lymphoprep, AXIS-SHIELD PoCAs, Oslo, Norway) at 800g for 30 min at room temperature. Cells at the interface were harvested, washed twice, and resuspended in Roswell Park Memorial Institute (RPMI)−1640. The cells were then suspended in complete culture medium (RPMI-1640, 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 50 g/ml of streptomycin) and were incubated for 2 h at 37°C in 5% CO2 to allow adherent cells to attach to the plastic. The supernatant containing lymphocytes was then collected. Lymphocyte viability was determined by trypan blue exclusion. Only samples with >90% viable lymphocytes were used. Quantitative real-time PCR Total RNA was extracted from PBLs (5 × 105 cells) using Trizol reagent (Invitrogen, California, USA), according to the manufacturer’s instructions. Purity and quantification assessment were performed by optical density measurements at 260 and 280 nm. cDNA was synthesized by using a Quantscript RT Kit (Roche, Clare, Ireland). Real-time quantification of target mRNA was performed using a SYBR PrimescriptTM RT-PCR kit (TAKARA, Dalian, China), normalized to the expression level of GAPDH. The real-time PCR conditions were as follows: initial denaturation at 95°C for 10 s, 40 cycles of 15 s at 95°C (denaturation) and 30 s at 56°C (annealing and extension) (Applied Biosystems 7500, Life Technologies Corporation, California, USA). The following primer sequences were used: GRIM-19, Forward: 5′-TCGGGGACTGTCGGGGTAC-3′, and Reverse: 5′-AGGGTCCTCCGGTCCTTCT-3′; GAPDH, Forward: 5′-GCCTTCCGTGTTCCTACCC-3′ and Reverse: 5′-CGAAGTCGCAGGAGACAACC-3′. All experiments were performed three times for each sample. Relative gene expression levels were analyzed by 2−∆∆CT method. Western blot analysis Protein was extracted from the PBLs and Jurkat cells in lysis buffer (Beyotime, China). The protein concentration was evaluated with the bicinchoninic acid (BCA) Protein Assay kit (Beyotime, China). Proteins (30 μg/sample) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%), and transferred to polyvinylidene difluoride (PVDF) membranes (BioRad, USA). The membrane was blocked with 5% skimmed milk for 2 h at room temperature, and subsequently incubated with primary antibodies against following proteins: anti-GRIM-19 (Abcam, UK, 1:1000), anti-AKT (Cell Signaling Technology, USA, 1:500), anti-p-AKT (Cell Signaling Technology, USA, 1:500), anti-mTOR (Cell Signaling Technology, USA, 1:500), anti-p-mTOR (Cell Signaling Technology, USA, 1:500), anti-GAPDH (Abcam, UK, 1:2000) overnight at 4°C. After washing with Tris-buffered saline Tween-20 (TBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h (Zhongshan Golden Bridge Biotechnology Co., Ltd, China). Immunoreactivity was detected by enhanced chemiluminescence (ECL) reagents (Merck Millipore, USA), and quantified by densitometry using Quantity One software (BioRad, USA). Intracellular ROS measurement The level of intracellular ROS was quantified by an oxidation-sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). PBLs were washed twice with serum-free medium and then incubated in the dark with 10 μM DCFH-DA solution for 30 min at 37°C. The fluorescence was analyzed quantitatively by fluorescence microscope and flow cytometry after being washed three times with phosphate-buffered saline (PBS). Quantification of relative fluorescence intensity was performed using ZEN2011 software (ZEISS, Germany). A primary gate based on physical parameters (forward and side light scatter) was set to exclude dead cells and debris. The excitation and emission wavelengths were set at 488 and 525 nm. Data are expressed as mean fluorescent signal intensity (MFI). Antioxidant enzyme activity assays Superoxide dismutase (SOD) activity in PBLs was measured by using a commercially available kit (Nanjing Jiancheng Bioengineering Institute, China) based on the auto-oxidation of hydroxylamine. The developed blue color was measured at 550 nm. Glutathione peroxidase (GSH-Px) activity was assessed using a GSH-Px kit (Nanjing Jiancheng Bioengineering Institute, China) by the velocity method. The reaction was initiated by the addition of H2O2. A series of enzymatic reactions was activated by GSH-Px in the homogenate which subsequently led to the conversion of GSH (reduced glutathione) to oxidized glutathione (GSSG). The change in absorbance during the conversion of GSH to GSSG was recorded spectrophotometrically at 412 nm. Flow cytometric detection of Treg/Th17 cell percentages A total of 5 ml heparinized venous blood samples were taken from both groups, and PBMCs were separated. Then PBMCs were suspended at a density of 2 × 106 cells/ml in complete culture medium (RPMI-1640, 10% FBS, 100 U/ml of penicillin, 50 g/ml of streptomycin and 2 mM glutamine). Then, cultures were stimulated with phorbol myristate acetate (PMA, 50 ng/ml, Sigma, USA) plus ionomycin (1 μg/ml, Sigma, USA) in the presence of monensin (500 ng/ml, Sigma, USA) for 4.5 h in a 37°C incubator at 5% CO2. As PMA rapidly induced cell membrane surface CD4 endocytosis, we applied CD3+CD8− gates to represent the CD4+T cells according to several previous studies (Cedeno-Laurent et al., 2010; Zhu et al., 2017). For Treg analysis, the cells were incubated with anti-human CD3-PE (eBioscience, USA), anti-human CD8−FITC (eBioscience, USA) and anti-human CD25-PerCP-Cy5.5 (eBioscience, USA) at 4°C for 30 min. After the surface staining, the cells were fixed and permeabilized with Perm/Fix solution (BioLegend, USA) and were then stained with anti-human Foxp3-APC (eBioscience, USA). For Th17 analysis, the cells were incubated with anti-human CD3-PE (eBioscience, USA) and anti-human CD8−FITC (eBioscience, USA) at 4°C for 30 min. After the surface staining, the cells were fixed and permeabilized with Perm/Fix solution (BioLegend, USA) and were then stained with anti-human IL-17 A-APC (eBioscience, USA). Stained cells were washed twice with PBS and analyzed on a FACS Calibur flow cytometer. For the detection of Treg cells, we first gate on lymphocyte (gate P1) using forward scatter (FSC) and side scatter (SSC), then gate on CD3+CD8− T cells (gate P2), and analyzed CD25+Foxp3+ T cells in a CD3+CD8− gate. For the detection of Th17 cells, we first gate on lymphocyte (gate P1) using FSC and SSC, then gate on CD3+ T cells (gate P2), and analyzed CD8−IL-17 A+ T cells in a CD3+ gate. All staining was performed according to manufacturer’s protocols. Isotype controls were used to confirm antibody specificity. Single color stain controls were used to enable correct compensation. Cell lines and culture Jurkat E6-1 human CD4+ T-lymphocytes obtained from the American Type Culture Collection (ATCC). The cells were maintained routinely in RPMI-1640 medium supplemented with 10% FBS and 2 mM glutamine at 37°C in a humidified 5% CO2 incubator. Knockdown GRIM-19 by siRNA (GenePharma Co., Ltd, China) was performed by Lipofectamine 2000 (Invitrogen, USA) in accordance with the manufacturer’s instructions. The following sequences were used: GRIM-19, 5′-GGAUUGGAACCCUGAUCUATT-3′, and scramble, 5′-UUCUCCGAACGUGUCACGUTT-3′. The culture medium was replaced after 6 h of incubation. Then, 48 h after transfection, the cells were collected. In antioxidant group, cells were cultured with 2 mM N-acetylcysteine (NAC, Sigma, USA) for 24 h. Statistical analysis The SPSS 18.0 statistical software program was used for statistical analysis. Statistical analyses of more than two groups were performed with one-way ANOVA followed by Tukey’s post hoc test or an independent samples t test. P < 0.05 were considered to be statistically significant. Results Aberrant expression of GRIM-19 in the PBLs of patients with URSA To determine whether GRIM-19 is associated with URSA, we examined the expression of GRIM-19 in the PBLs of patients with URSA and non-pregnant healthy women. The mRNA and protein expression of GRIM-19 was significantly lower in URSA patients (n = 28) than that in control group (n = 30) (protein expression GRIM-19/GAPDH: 0.45 ± 0.21 vs 0.79 ± 0.21, **P < 0.01) (Fig. 1). Figure 1 View largeDownload slide Aberrant expression of GRIM-19 in the PBLs of patients with URSA. (A) The mRNA level of GRIM-19 in the peripheral blood lymphocytes (PBLs) of patients with unexplained recurrent spontaneous abortion (URSA) (n = 28) and the control subjects (n = 30) was detected by quantitative real-time PCR. (B) A western blot analysis was performed to assess the protein levels of GRIM-19 in the PBLs of URSA patients (n = 28) and the control subjects (n = 30) (GRIM-19/GAPDH: 0.45 ± 0.21 vs 0.79 ± 0.21). The representative examples are shown. GAPDH served as a loading control. All data are expressed as mean ± SD. *P < 0.05, **P < 0.01, Student’s t test. Figure 1 View largeDownload slide Aberrant expression of GRIM-19 in the PBLs of patients with URSA. (A) The mRNA level of GRIM-19 in the peripheral blood lymphocytes (PBLs) of patients with unexplained recurrent spontaneous abortion (URSA) (n = 28) and the control subjects (n = 30) was detected by quantitative real-time PCR. (B) A western blot analysis was performed to assess the protein levels of GRIM-19 in the PBLs of URSA patients (n = 28) and the control subjects (n = 30) (GRIM-19/GAPDH: 0.45 ± 0.21 vs 0.79 ± 0.21). The representative examples are shown. GAPDH served as a loading control. All data are expressed as mean ± SD. *P < 0.05, **P < 0.01, Student’s t test. Intracellular level of ROS in the PBLs of patients with URSA Mitochondria are the main source of intracellular ROS, notably via the formation of superoxide in the electron transport chain (ETC) (Angelova and Abramov, 2016). Previous study showed that deficiency of GRIM-19 may lead to a collapse in MMP (Chen et al., 2015), so we evaluated the intracellular level of ROS in the PBLs of patients with URSA. Obvious increases in ROS levels were seen in PBLs of URSA group (Fig. 2A). Relative fluorescence intensity for images of each group is shown in Fig. 2B. Flow cytometric analysis confirmed that the intracellular ROS levels of PBLs in URSA group (n = 20, MFI: 42.17 ± 10.91) were increased to those of the control group (n = 20, MFI: 29.87 ± 8.13, **P < 0.01) (Fig. 2C). As such, the roles of GRIM-19 in URSA may be linked to intracellular ROS. Figure 2 View largeDownload slide Intracellular level of ROS in the PBLs of patients with URSA. (A) ROS accumulation in the PBLs of URSA patients (n = 20) and control subjects (n = 20) was visualized with the fluorescent dye DCFH-DA, representative example is shown. (B) Relative fluorescence intensity for images of URSA group (n = 20) and control group (n = 20). (C) Flow cytometric analysis confirmed that the intracellular ROS levels of PBLs in URSA group (n = 20, MFI: 42.17 ± 10.91) were increased to those of control group (n = 20, MFI: 29.87 ± 8.13). Representative examples are shown. (D) The activities of total SOD and GSH-Px in PBLs of URSA patients and control subjects (n = 20, SOD: 36.86 ± 9.44 vs 52.19 ± 8.41, GSH-Px: 96.87 ± 8.41 vs 112.64 ± 7.83). All data are expressed as mean ± SD. **P < 0.01, Student’s t test. Figure 2 View largeDownload slide Intracellular level of ROS in the PBLs of patients with URSA. (A) ROS accumulation in the PBLs of URSA patients (n = 20) and control subjects (n = 20) was visualized with the fluorescent dye DCFH-DA, representative example is shown. (B) Relative fluorescence intensity for images of URSA group (n = 20) and control group (n = 20). (C) Flow cytometric analysis confirmed that the intracellular ROS levels of PBLs in URSA group (n = 20, MFI: 42.17 ± 10.91) were increased to those of control group (n = 20, MFI: 29.87 ± 8.13). Representative examples are shown. (D) The activities of total SOD and GSH-Px in PBLs of URSA patients and control subjects (n = 20, SOD: 36.86 ± 9.44 vs 52.19 ± 8.41, GSH-Px: 96.87 ± 8.41 vs 112.64 ± 7.83). All data are expressed as mean ± SD. **P < 0.01, Student’s t test. The change of oxidative stress related factors SOD and GSH-Px, as free radical scavengers, mainly scavenge hydrogen peroxide and hydroxyl free radicals, finally reduce cell injury from oxidative stress (Li et al., 2016). Therefore, we subsequently evaluated the activities of total SOD and GSH-Px in PBLs of URSA patients. The activity of SOD and GSH-Px were decreased, respectively in PBLs of URSA group compared to control group, as shown in Fig. 2D (n = 20, SOD: 36.86 ± 9.44 vs 52.19 ± 8.41, **P < 0.01. GSH-Px: 96.87 ± 8.41 vs 112.64 ± 7.83, **P < 0.01). Expression of PI3K/Akt/mTOR pathway in the PBLs of patients with URSA In a previous study, it was confirmed that ROS were critical for IL-7-mediated activation of PI3K/Akt/mTOR signaling pathway, in a manner that was dependent on mitochondrial respiratory chain (Silva et al., 2011). In addition, evidence for an involvement of the PI3K/Akt/mTOR network in Treg and Th17 differentiation and function has been accumulating (Kim et al., 2015). Thus, we evaluated the activation of PI3K/Akt/mTOR pathway in the PBLs of patients with URSA. The protein levels of p-Akt and p-mTOR in PBLs from patients with URSA were both significantly higher than those from control group. The overall expression of Akt and mTOR, however, was not significantly different in any of the groups (Fig. 3A). Figure 3 View largeDownload slide Expression of PI3K/Akt/mTOR pathway and proportional change of Treg and Th17 cells in URSA patients. (A) Western blot analysis was performed to assess the protein levels of PI3K/Akt/mTOR pathway in the PBLs of URSA patients (n = 20) and the control subjects (n = 20). The representative examples are shown. GAPDH served as a loading control. All data are expressed as mean ± SD. **P < 0.01, Student’s t test. (B) Flow cytometric identification of Treg cells in the PBMCs of a patient with URSA (n = 20, 4.32 ± 1.52%) and control subjects (n = 20, 6.23 ± 1.41%). Lymphocytes were identified and gated on a forward/side-scatter plot. Percentages of CD3+CD8−CD25+Foxp3+ Treg cells were identified. The representative examples are shown. All data are expressed as mean ± SD. **P < 0.01, Student’s t test. (C) Flow cytometric identification of Th17 cells in the PBMCs of a patient with URSA (n = 20, 3.21 ± 0.68%) and control subjects (n = 20, 1.76 ± 0.46%). Lymphocytes were identified and gated on a forward/side-scatter plot. Percentages of CD3+CD8−IL-17+ Th17 cells were identified. The representative examples are shown. All data are expressed as mean ± SD. **P < 0.01, Student’s t test. Figure 3 View largeDownload slide Expression of PI3K/Akt/mTOR pathway and proportional change of Treg and Th17 cells in URSA patients. (A) Western blot analysis was performed to assess the protein levels of PI3K/Akt/mTOR pathway in the PBLs of URSA patients (n = 20) and the control subjects (n = 20). The representative examples are shown. GAPDH served as a loading control. All data are expressed as mean ± SD. **P < 0.01, Student’s t test. (B) Flow cytometric identification of Treg cells in the PBMCs of a patient with URSA (n = 20, 4.32 ± 1.52%) and control subjects (n = 20, 6.23 ± 1.41%). Lymphocytes were identified and gated on a forward/side-scatter plot. Percentages of CD3+CD8−CD25+Foxp3+ Treg cells were identified. The representative examples are shown. All data are expressed as mean ± SD. **P < 0.01, Student’s t test. (C) Flow cytometric identification of Th17 cells in the PBMCs of a patient with URSA (n = 20, 3.21 ± 0.68%) and control subjects (n = 20, 1.76 ± 0.46%). Lymphocytes were identified and gated on a forward/side-scatter plot. Percentages of CD3+CD8−IL-17+ Th17 cells were identified. The representative examples are shown. All data are expressed as mean ± SD. **P < 0.01, Student’s t test. Proportional change of Treg and Th17 cells in PBMCs of URSA patients Treg and Th17 cells play a major role in tolerating conceptus antigens and therefore may contribute to the maintenance of pregnancy, so we determined the proportion of Treg and Th17 cells in URSA patients compared to healthy non-pregnant women. The proportion of Treg cells in PBMCs in URSA patients (n = 20, 4.32 ± 1.52%) was statistically significantly lower than those in control women (n = 20, 6.23 ± 1.41%, **P < 0.01) (Fig. 3B). Meanwhile, the percentage of Th17 cells in PBMCs in URSA patients (n = 20, 3.21 ± 0.68%) was significantly higher than that in control women (n = 20, 1.76 ± 0.46%, **P < 0.01) (Fig. 3C). Considering the broad involvement of mTOR in Treg and Th17 differentiation and function, these data suggested a possible role for GRIM-19 in the Treg/Th17 balance by regulating the activation of mTOR. Downregulation of GRIM-19 and its effect in intracellular ROS production and the level of p-mTOR in Jurkat cells To determine the effect of the low level of GRIM-19 in PBLs of URSA patients, we downregulated the expression of GRIM-19 by specific siRNA in the Jurkat E6-1 human CD4+ T-lymphocytes. Immunoblotting indicated notable depletion of GRIM-19 in the GRIM-19 siRNA group (Fig. 4A). Then, the intracellular ROS production and the protein level of p-mTOR in the GRIM-19 siRNA group and the scramble control group were analyzed. The cells in the GRIM-19 siRNA group had higher levels of ROS than those in the control group (Fig. 4B). More strikingly, the protein level of p-mTOR increased significantly in the GRIM-19 siRNA group, and the expression level of total mTOR did not differ (Fig. 4A). Collectively, all these results strongly suggested that depletion of GRIM-19 would affect intracellular ROS production and PI3K/Akt/mTOR signaling pathway in URSA. Figure 4 View largeDownload slide Regulation of p-mTOR by GRIM-19–ROS–mTOR axis in Jurkat cells. (A) The Jurkat cells were transfected with GRIM-19 siRNA and treated with ROS inhibitor NAC. The expression levels of GRIM-19, mTOR and p-mTOR in the control, GRIM-19 siRNA, GRIM-19 siRNA + NAC and NAC group were evaluated by immunoblot. GAPDH was used as a loading control. Three independent experiments were carried out. The representative examples are shown. *P < 0.05, **P < 0.01, one-way ANOVA test. (B) Flow cytometric analysis was applied to detect the ROS generation in the control, GRIM-19 siRNA, GRIM-19 siRNA + NAC and NAC group by staining with DCFH-DA. Three independent experiments were carried out. The representative examples are shown. All data are expressed as mean fluorescent signal intensity (MFI) ± SD. **P < 0.01, one-way ANOVA test. G19: GRIM-19. Figure 4 View largeDownload slide Regulation of p-mTOR by GRIM-19–ROS–mTOR axis in Jurkat cells. (A) The Jurkat cells were transfected with GRIM-19 siRNA and treated with ROS inhibitor NAC. The expression levels of GRIM-19, mTOR and p-mTOR in the control, GRIM-19 siRNA, GRIM-19 siRNA + NAC and NAC group were evaluated by immunoblot. GAPDH was used as a loading control. Three independent experiments were carried out. The representative examples are shown. *P < 0.05, **P < 0.01, one-way ANOVA test. (B) Flow cytometric analysis was applied to detect the ROS generation in the control, GRIM-19 siRNA, GRIM-19 siRNA + NAC and NAC group by staining with DCFH-DA. Three independent experiments were carried out. The representative examples are shown. All data are expressed as mean fluorescent signal intensity (MFI) ± SD. **P < 0.01, one-way ANOVA test. G19: GRIM-19. Regulation of p-mTOR by GRIM-19–ROS–mTOR axis in Jurkat cells When the cells were transfected with GRIM-19 siRNA, protein level of p-mTOR and level of intracellular ROS ascended accordingly. To confirm ROS as a linker bridging GRIM-19 depletion and mTOR activation, GRIM-19 knockdown cells were treated with antioxidant NAC. Notable depletion of intracellular ROS was observed in the cells treated with NAC (Fig. 4B). As depicted in Fig. 4A, the upregulation of p-mTOR resulted from GRIM-19 loss was significantly reversed in the cells treatment with NAC, indicating that ROS was indeed required for GRIM-19 depletion induced p-mTOR expression. These results suggested that GRIM-19 may be involved in URSA partly through ROS–mTOR–Treg/Th17 axis. Discussion To date, no reports have been made about the role of GRIM-19 in URSA, although it has been demonstrated previously to be essential to the immunoregulation. In this study, we revealed a new molecular mechanism underlying the association between GRIM-19 deficiency and URSA. One of the major outcomes of this study showed that the expression of GRIM-19 decreased significantly in the PBLs of patients with URSA, demonstrating the relevance between a low level of GRIM-19 and URSA. Our study showed a reducing number of Treg cells and an increasing number of Th17 cells in PBMCs of URSA women compared to control subjects that agreed with other studies. Further studies revealed that there is a considerable increase in intracellular ROS production and a high level of PI3K/Akt/mTOR activation in the PBLs of URSA patients compared with the control subjects. In addition, our study using Jurkat cells demonstrated that downregulation of GRIM-19 leads to an increased ROS production and an elevated level of p-mTOR. Meanwhile, treatment of Jurkat cells with antioxidant NAC decreased its p-mTOR expression. Involvement of the PI3K/Akt/mTOR network in Treg and Th17 differentiation and function has been demonstrated previously. Therefore, we conclude that GRIM-19 deficiency may play a role in regulating Treg/Th17 balance partly through ROS–mTOR signaling axis in URSA. GRIM-19 is a functional subunit of mitochondrial respiratory chain complex I and plays an essential role in the assembly and enzymatic activity of complex I. A lack of GRIM-19 leads to a collapse in MMP, and abnormality of the mitochondrion structure, morphology and cellular distribution (Huang et al., 2004; Chen et al., 2015). Mitochondria produce ROS as a by-product of a number of enzymatic reactions and the ETC (Angelova and Abramov, 2016). Emerging evidence suggests that dysregulated ROS signaling may contribute to a development of processes which lead to human diseases (Silva et al., 2016). A previous study showed that inhibition of respiration and decrease of MMP can stimulate superoxide production (Nohl et al., 1993; Angelova and Abramov, 2016). In our study, a considerable increase in the intracellular ROS production in the PBLs of URSA patients was observed. To study the correlation between GRIM-19 and the intracellular ROS production, the ROS level in Jurkat cells was examined after GRIM-19 knockdown. We found that the ROS level in the GRIM-19 siRNA group to be significantly higher than in the control group. These results indicated that a deficiency of GRIM-19 in URSA is related to intracellular ROS production. ROS activates many adaptation signaling pathways in cells, in which mTOR plays an important role in autophagy, apoptosis, metabolism, growth, survival and immunity (Wullschleger et al., 2006; Roy et al., 2014; Wang et al., 2016). Increasing evidence strongly suggests that mTOR is regulated by many upstream regulators, such as PI3K, AKT and AMPK, and ROS is the initial regulator (Hay and Sonenberg, 2004; Wang et al., 2016). ROS-AKT-mTOR axis plays an important role in autophagy of cancer cells (Fiorini et al., 2015). Safranova et al. (2003) reported increase ROS in peripheral blood granulocytes in women with habitual abortions. In our study, the upregulation of p-mTOR resulted from GRIM-19 loss was significantly reversed in the cells treatment with NAC, indicating that ROS was indeed required for GRIM-19 depletion induced p-mTOR expression. These results indicated that mTOR is mediated by ROS generation, and GRIM-19 may be involved in URSA via affecting intracellular ROS production and downstream PI3K/Akt/mTOR signaling pathway. Dysfunctions in immune system regulation may result in pregnancy abnormalities such as URSA. Immunological disorders may cause impaired maternal immune tolerance to the fetus and result in fetal rejection (Sereshki et al., 2014). Several types of T cells have been associated with the pathogenesis of URSA, including Treg and Th17 cell. Treg cells are one of the most important immunological factors that play a primary role in suppressing excessive immune response of other cells, maintaining tolerance to self-antigens, and results in a successful pregnancy (Zenclussen, 2006). Th17 cells play a major role in induction of inflammation by producing pro-inflammatory cytokines and matrix metalloproteinase (Sereshki et al., 2014). Several studies have shown the balance between Treg cells and Th17 cells under normal and pathologic conditions (Schaub et al., 2008; Rong et al., 2009; Kimura and Kishimoto, 2010). A recent study indicated that in patients with URSA, immunotherapy with mononuclear cells derived from the baby’s father could affect Treg/Th17 balance, and they found that the Treg bias would be beneficial for pregnancy (Wu et al., 2014). Another study indicated that the imbalance between Th17 and Treg cells during the proliferative phase of menstrual cycles in the URSA group may be considered a cause for spontaneous abortion (Sereshki et al., 2014). In our study, we found a reducing number of Treg cells and an increasing number of Th17 cells in PBMCs of URSA women compared to control subjects. Previous studies have found that the differentiation of Th17 cells is controlled by PI3K/Akt/mTOR signaling pathway. It was reported recently that the PI3K-AKT-mTORC1-S6K axis positively regulates Th17 differentiation by promoting the nuclear translocation of RAR-related orphan receptor (ROR)γt (Kurebayashi et al., 2012; Kim et al., 2013; Koga et al., 2014). In contrast, the inhibition of PI3K and mTOR increases Treg cell differentiation (Okkenhaug et al., 2006; Wan et al., 2016). A study in follicular thyroid cancer cells found that the ratio of Treg/Th17 was increased significantly after rapamycin treatment, which is consistent with the previous studies (Zhou et al., 2016). Thus, our study indicated that the imbalance between Th17 and Treg cells in URSA is largely associated with the high level of PI3K/Akt/mTOR signaling pathway. In conclusion, we have provided evidence demonstrating that there may be an association between URSA and low levels of GRIM-19. GRIM-19 deficiency may play a role in regulating Treg/Th17 balance partly through ROS–mTOR signaling axis in URSA. Authors’ roles L.C. and Y.Y. conceived the study. Y.Y., L.Y.C., X.H.D., H.L.Y. performed experiments. L.C., Y.Y., L.Y.C., X,H.D. and H.L.Y. performed data analysis. L.C., L.Y.C. and Y.Y. wrote, reviewed and edited the article. All authors edited and approved the final draft of the article. Funding National Natural Science Foundation of China (Grant numbers 81571511, 81701528, 81370711 and 30901603), the Shandong Provincial Natural Science Foundation (Grant numbers ZR2017PH052 and ZR2013HM090) and the Science Foundation of Qilu Hospital of Shandong University, Fundamental Research Funds of Shandong University (Grant numbers 2015QLQN50 and 2015QLMS24). Conflict of interest The authors declare that there is no conflict of interest that could prejudice the impartiality of the present research. References Aluvihare VR , Kallikourdis M , Betz AG . Regulatory T cells mediate maternal tolerance to the fetus . Nat Immunol 2004 ; 5 : 266 – 271 . Angell JE , Lindner DJ , Shapiro PS , Hofmann ER , Kalvakolanu DV . Identification of GRIM-19, a novel cell death-regulatory gene induced by the interferon-beta and retinoic acid combination, using a genetic approach . J Biol Chem 2000 ; 275 : 33416 – 33426 . Angelova PR , Abramov AY . 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For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Human Reproduction Oxford University Press

Expression of GRIM-19 in unexplained recurrent spontaneous abortion and possible pathogenesis

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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10.1093/molehr/gay020
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Abstract

Abstract STUDY QUESTION Is aberrant expression of gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) associated with unexplained recurrent spontaneous abortion (URSA)? SUMMARY ANSWER GRIM-19 deficiency may regulate regulatory T cell/T helper 17 cell (Treg/Th17) balance partly through reactive oxygen species (ROS)–mammalian target of rapamycin (mTOR) signaling axis in URSA. WHAT IS KNOWN ALREADY Immunological disorders may cause impaired maternal immune tolerance to the fetus and result in fetal rejection. The differentiation of Treg and Th17 cells is controlled by phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling pathway. GRIM-19 participates in the immune response, but its role in URSA is largely unknown. STUDY DESIGN, SIZE, DURATION The current study included 28 URSA patients and 30 non-pregnant healthy women. PARTICIPANTS/MATERIALS, SETTING, METHODS The proportion of Treg and Th17 cells in peripheral blood of URSA patients and control subjects were assessed with flow cytometry. The expression of GRIM-19 in peripheral blood lymphocytes (PBLs) was measured with quantitative real-time PCR and western blot analysis. Furthermore, the ROS level in the PBLs of URSA patients and control subjects were assessed by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Then, Akt/mTOR expression in the PBLs was measured. Downregulation of GRIM-19 in Jurkat cells was performed by specific siRNA. Then, intracellular ROS production and the expression of p-mTOR, which is known to enhance Th17 differentiation and decrease Treg cell differentiation, were detected. Finally, N-acetylcysteine (NAC) was used to decrease the intracellular ROS level, and the expression of p-mTOR was measured. MAIN RESULTS AND THE ROLE OF CHANCE The proportion of Treg cells was reduced in URSA patients, whereas the proportion of Th17 cells was increased. The expression of GRIM-19 was significantly lower in PBLs of URSA patients. Furthermore, there is a considerable increase in intracellular ROS production and a high level of p-Akt and p-mTOR expression in the PBLs of URSA patients compared with the control subjects. In parallel to this, downregulation of GRIM-19 in the Jurkat cells by siRNA results in an increased ROS production and an increased expression of p-mTOR. Importantly, the upregulation of p-mTOR resulting from GRIM-19 loss was significantly reversed in the cells treatment with ROS inhibitor N-acetyl-l-cysteine (NAC), indicating that ROS was indeed required for GRIM-19 depletion induced p-mTOR expression. LARGE SCALE DATA None. LIMITATIONS, REASONS FOR CAUTION A large number of researches have confirmed that the differentiation of Treg and Th17 cells is controlled by PI3K/Akt/mTOR signaling pathway. We have not shown the regulatory role of ROS and PI3K/Akt/mTOR in Treg and Th17 differentiation in this study. WIDER IMPLICATIONS OF THE FINDINGS Our study has demonstrated that GRIM-19 deficiency may play a role in regulating Treg/Th17 balance partly through ROS–mTOR signaling axis in URSA. The present study offers a new perspective to the roles of GRIM-19 in immunoregulation. STUDY FUNDING AND COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (Grant numbers 81571511, 81701528, 81370711 and 30901603), the Shandong Provincial Natural Science Foundation (Grant numbers ZR2017PH052 and ZR2013HM090) and the Science Foundation of Qilu Hospital of Shandong University, Fundamental Research Funds of Shandong University (Grant numbers 2015QLQN50 and 2015QLMS24). The authors declare that there is no conflict of interest that could prejudice the impartiality of the present research. GRIM-19, unexplained recurrent spontaneous abortion, regulatory T cells, T helper 17 cells, reactive oxygen species Introduction Recurrent spontaneous abortion (RSA) is a common problem among couples of reproductive age and is defined as three or more consecutive pregnancy losses before the 20th week of gestation (Patriarca et al., 2000; Poole and Claman, 2004; Yang et al., 2008). Unexplained RSA (URSA) still affects ~50% of RSA patients, although the causes of RSA include chromosomal aberrations, hematological problems, hormonal disorders, uterine anatomical defects, infectious and immunological abnormalities (Christiansen et al., 2005). Gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) was isolated by using antisense gene knockout as a death-associated protein (Angell et al., 2000), official full name NADH:ubiquinone oxidoreductase subunit A13 (NDUFA13). Further study in mice indicated that GRIM-19 plays an essential role in complex I assembly and electron transfer activity, whereas disruption of mitochondrial membrane potential (MMP) by GRIM-19 mutants increases the cells’ sensitivity to apoptotic stimuli (Lu and Cao, 2008). More important, GRIM-19 also participates in the immune response. A recent report in murine autoimmune arthritis has shown that GRIM-19 augmented regulatory T (Treg) cell differentiation, whereas it suppressed T helper 17 (Th17) cell differentiation, by inhibiting signal transducer and activator of transcription 3 (STAT3) phosphorylation (Moon et al., 2014). Another group reported that GRIM-19 heterozygous mice are prone to spontaneous urinary tract infection with reduced bacterial killing ability and pro-inflammatory cytokine production (Chen et al., 2012). Although our previous study has demonstrated that there is an association between missed abortion and low levels of GRIM-19, the potential role of GRIM-19 in URSA is largely unknown (Chen et al., 2015). GRIM-19 plays an essential role in the assembly and enzymatic activity of mitochondrial complex I, and mitochondria are the main source of intracellular reactive oxygen species (ROS). Aberrant expression of GRIM-19 may lead to changes in intracellular level of ROS. Safranova et al. (2003) reported increase ROS in peripheral blood granulocytes in women with habitual abortions. Meanwhile, ROS were critical for activation of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling pathway, in a manner that was dependent on mitochondrial respiratory chain (Silva et al., 2011). In addition, differentiation of Treg and Th17 cells is controlled by PI3K/Akt/mTOR signaling pathway (Kim et al., 2015). Studies have indicated that URSA is largely associated with the failure of fetomaternal immunologic tolerance (Wu et al., 2014). One of the most important immunological factors that plays a primary role in controlling the fetomaternal tolerance and results in a successful pregnancy are Treg cells. Treg cells suppress excessive immune response of other cells and maintain tolerance to self-antigens (Aluvihare et al., 2004; Somerset et al., 2004). The frequency of Treg cells was downregulated during human miscarriage (Hosseini et al., 2016). Th17 cells play an important role in induction of inflammation by producing pro-inflammatory cytokines. Recent data have shown a pathogenic effect of these cells in autoimmunity, transplant rejection and other diseases (Tesmer et al., 2008; Hirota et al., 2010; Sereshki et al., 2014). In URSA patients, it has been reported that the proportion of Th17 cells and the expression of IL-23, the Th17-inducing cytokine, are higher in the peripheral blood than in normal pregnancies (Saifi et al., 2014). Treg and Th17 cells are two distinct subsets of CD4+ T cells, and the balance between the two cell types could have an impact on the control of inflammation and autoimmunity. There is clear evidence that Treg/Th17 imbalance plays a potential role in the pathogenesis of URSA (Sereshki et al., 2014; Zhu et al., 2017). A decrease in Treg/Th17 ratio was observed in URSA patients due to the increased differentiation of Th17 cells and decreased differentiation of Treg cells from naive T cells through heightened circulating IL-6 levels (Zhu et al., 2017). In this study, we report that the proportion of Treg cells was reduced in URSA patients, whereas the proportion of Th17 cells was increased. Low level of GRIM-19 in peripheral blood lymphocytes (PBLs) of URSA patients was observed. Furthermore, there is a considerable increase in intracellular ROS production and a high level of p-Akt and p-mTOR, which is known to enhance Th17 differentiation and decrease Treg cells differentiation. Furthermore, the molecular mechanisms were investigated in Jurkat cells in vitro. Our study offers a new perspective to the roles of GRIM-19 in immunoregulation. Materials and Methods Ethical approval The study has been reviewed and approved by the Institutional Review Boards of Qilu Hospital of Shandong University. Written informed consent was obtained from all human subjects. Subjects All study participants were recruited at the department of gynecology and obstetrics, Qilu Hospital of Shandong University. Twenty-eight URSA patients with a mean (± standard deviation) age of 30.0 ± 4.1 (range: 23–40) years, who had at least three consecutive first trimester abortions (7–12 weeks of gestation) were recruited. The mean number of abortions was 3.9 ± 1.1 (range: 3–7). The diagnosis of URSA was made after excluding any verifiable causes: chromosomal abnormality, abnormalities of the uterus or cervix, endocrine and metabolic diseases, infection, congenital thrombophilias and autoimmune disease. Karyotype analysis of all abortion couples and abortuses was performed to exclude any chromosome problems. Pelvic examination and ultrasound were used to rule out abnormalities of uterus and cervix, and if necessary, a hysterosalpingography had to be performed for further confirmation. Serum samples were analyzed for FSH, LH, estradiol (E2), prolactin (PRL) and testosterone (T). Progesterone was analyzed at the seventh day after the LH surge in the same cycle. Free triiodothyronine, free thyroxin, thyrotropic stimulating hormone, thyroid peroxidase antibody and thyroglobulin antibody were analyzed. Endocrine diseases (hyperprolactinemia, luteal function defect, polycystic ovary syndrome and hyperandrogenemia) were excluded. Metabolic diseases (hyperthyroidism, insulin resistance, diabetes and hypothyroidism) were excluded. Chlamydia and ureaplasma infection were rule out by cervical mucus culture. Congenital thrombophilias was excluded by measurement of protein C, protein S and antithrombin III activity. The activated partial thrombin time was used to test the lupus anticoagulant. Systemic lupus erythematosus was excluded by measurement of the anticardiolipin antibodies, extracted nuclear antigen antibodies, and antinuclear antibodies. All male partners of URSA patients should have normal semen status, according to World Health Organization criteria. The control group comprised 30 non-pregnant healthy women, with a mean age of 30.3 ± 4.8 (range: 22–41) years (Table I). Control group women had at least one successful pregnancy without any disease and had no history of ectopic pregnancy, spontaneous abortion, preterm delivery, still birth, pre-eclampsia and abnormal pregnancy. Table I Clinical characteristics of subjects. Clinical characteristics URSA Control n 28 30 Age (y) 30.0 ± 4.1 30.3 ± 4.8 Number of abortions 3.9 ± 1.1 0.0 ± 0.0 Clinical characteristics URSA Control n 28 30 Age (y) 30.0 ± 4.1 30.3 ± 4.8 Number of abortions 3.9 ± 1.1 0.0 ± 0.0 Table I Clinical characteristics of subjects. Clinical characteristics URSA Control n 28 30 Age (y) 30.0 ± 4.1 30.3 ± 4.8 Number of abortions 3.9 ± 1.1 0.0 ± 0.0 Clinical characteristics URSA Control n 28 30 Age (y) 30.0 ± 4.1 30.3 ± 4.8 Number of abortions 3.9 ± 1.1 0.0 ± 0.0 All participants included in the control and URSA groups were not pregnant, as confirmed by a negative blood hCG test, and were nonsmokers with normal body mass index who took no medications. All participants were at least three months from their last abortion or pregnancy. Blood samples were taken from both groups during the luteal phase (at Days 19–23 of the menstrual cycle, particularly on Day 21). Isolation of PBLs A total of 5 ml heparinized venous blood was taken from the case and control groups. Human peripheral blood mononuclear cells (PBMCs) were separated from freshly isolated peripheral blood of subjects by gradient centrifugation on Ficoll-Hypaque (Lymphoprep, AXIS-SHIELD PoCAs, Oslo, Norway) at 800g for 30 min at room temperature. Cells at the interface were harvested, washed twice, and resuspended in Roswell Park Memorial Institute (RPMI)−1640. The cells were then suspended in complete culture medium (RPMI-1640, 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 50 g/ml of streptomycin) and were incubated for 2 h at 37°C in 5% CO2 to allow adherent cells to attach to the plastic. The supernatant containing lymphocytes was then collected. Lymphocyte viability was determined by trypan blue exclusion. Only samples with >90% viable lymphocytes were used. Quantitative real-time PCR Total RNA was extracted from PBLs (5 × 105 cells) using Trizol reagent (Invitrogen, California, USA), according to the manufacturer’s instructions. Purity and quantification assessment were performed by optical density measurements at 260 and 280 nm. cDNA was synthesized by using a Quantscript RT Kit (Roche, Clare, Ireland). Real-time quantification of target mRNA was performed using a SYBR PrimescriptTM RT-PCR kit (TAKARA, Dalian, China), normalized to the expression level of GAPDH. The real-time PCR conditions were as follows: initial denaturation at 95°C for 10 s, 40 cycles of 15 s at 95°C (denaturation) and 30 s at 56°C (annealing and extension) (Applied Biosystems 7500, Life Technologies Corporation, California, USA). The following primer sequences were used: GRIM-19, Forward: 5′-TCGGGGACTGTCGGGGTAC-3′, and Reverse: 5′-AGGGTCCTCCGGTCCTTCT-3′; GAPDH, Forward: 5′-GCCTTCCGTGTTCCTACCC-3′ and Reverse: 5′-CGAAGTCGCAGGAGACAACC-3′. All experiments were performed three times for each sample. Relative gene expression levels were analyzed by 2−∆∆CT method. Western blot analysis Protein was extracted from the PBLs and Jurkat cells in lysis buffer (Beyotime, China). The protein concentration was evaluated with the bicinchoninic acid (BCA) Protein Assay kit (Beyotime, China). Proteins (30 μg/sample) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%), and transferred to polyvinylidene difluoride (PVDF) membranes (BioRad, USA). The membrane was blocked with 5% skimmed milk for 2 h at room temperature, and subsequently incubated with primary antibodies against following proteins: anti-GRIM-19 (Abcam, UK, 1:1000), anti-AKT (Cell Signaling Technology, USA, 1:500), anti-p-AKT (Cell Signaling Technology, USA, 1:500), anti-mTOR (Cell Signaling Technology, USA, 1:500), anti-p-mTOR (Cell Signaling Technology, USA, 1:500), anti-GAPDH (Abcam, UK, 1:2000) overnight at 4°C. After washing with Tris-buffered saline Tween-20 (TBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h (Zhongshan Golden Bridge Biotechnology Co., Ltd, China). Immunoreactivity was detected by enhanced chemiluminescence (ECL) reagents (Merck Millipore, USA), and quantified by densitometry using Quantity One software (BioRad, USA). Intracellular ROS measurement The level of intracellular ROS was quantified by an oxidation-sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). PBLs were washed twice with serum-free medium and then incubated in the dark with 10 μM DCFH-DA solution for 30 min at 37°C. The fluorescence was analyzed quantitatively by fluorescence microscope and flow cytometry after being washed three times with phosphate-buffered saline (PBS). Quantification of relative fluorescence intensity was performed using ZEN2011 software (ZEISS, Germany). A primary gate based on physical parameters (forward and side light scatter) was set to exclude dead cells and debris. The excitation and emission wavelengths were set at 488 and 525 nm. Data are expressed as mean fluorescent signal intensity (MFI). Antioxidant enzyme activity assays Superoxide dismutase (SOD) activity in PBLs was measured by using a commercially available kit (Nanjing Jiancheng Bioengineering Institute, China) based on the auto-oxidation of hydroxylamine. The developed blue color was measured at 550 nm. Glutathione peroxidase (GSH-Px) activity was assessed using a GSH-Px kit (Nanjing Jiancheng Bioengineering Institute, China) by the velocity method. The reaction was initiated by the addition of H2O2. A series of enzymatic reactions was activated by GSH-Px in the homogenate which subsequently led to the conversion of GSH (reduced glutathione) to oxidized glutathione (GSSG). The change in absorbance during the conversion of GSH to GSSG was recorded spectrophotometrically at 412 nm. Flow cytometric detection of Treg/Th17 cell percentages A total of 5 ml heparinized venous blood samples were taken from both groups, and PBMCs were separated. Then PBMCs were suspended at a density of 2 × 106 cells/ml in complete culture medium (RPMI-1640, 10% FBS, 100 U/ml of penicillin, 50 g/ml of streptomycin and 2 mM glutamine). Then, cultures were stimulated with phorbol myristate acetate (PMA, 50 ng/ml, Sigma, USA) plus ionomycin (1 μg/ml, Sigma, USA) in the presence of monensin (500 ng/ml, Sigma, USA) for 4.5 h in a 37°C incubator at 5% CO2. As PMA rapidly induced cell membrane surface CD4 endocytosis, we applied CD3+CD8− gates to represent the CD4+T cells according to several previous studies (Cedeno-Laurent et al., 2010; Zhu et al., 2017). For Treg analysis, the cells were incubated with anti-human CD3-PE (eBioscience, USA), anti-human CD8−FITC (eBioscience, USA) and anti-human CD25-PerCP-Cy5.5 (eBioscience, USA) at 4°C for 30 min. After the surface staining, the cells were fixed and permeabilized with Perm/Fix solution (BioLegend, USA) and were then stained with anti-human Foxp3-APC (eBioscience, USA). For Th17 analysis, the cells were incubated with anti-human CD3-PE (eBioscience, USA) and anti-human CD8−FITC (eBioscience, USA) at 4°C for 30 min. After the surface staining, the cells were fixed and permeabilized with Perm/Fix solution (BioLegend, USA) and were then stained with anti-human IL-17 A-APC (eBioscience, USA). Stained cells were washed twice with PBS and analyzed on a FACS Calibur flow cytometer. For the detection of Treg cells, we first gate on lymphocyte (gate P1) using forward scatter (FSC) and side scatter (SSC), then gate on CD3+CD8− T cells (gate P2), and analyzed CD25+Foxp3+ T cells in a CD3+CD8− gate. For the detection of Th17 cells, we first gate on lymphocyte (gate P1) using FSC and SSC, then gate on CD3+ T cells (gate P2), and analyzed CD8−IL-17 A+ T cells in a CD3+ gate. All staining was performed according to manufacturer’s protocols. Isotype controls were used to confirm antibody specificity. Single color stain controls were used to enable correct compensation. Cell lines and culture Jurkat E6-1 human CD4+ T-lymphocytes obtained from the American Type Culture Collection (ATCC). The cells were maintained routinely in RPMI-1640 medium supplemented with 10% FBS and 2 mM glutamine at 37°C in a humidified 5% CO2 incubator. Knockdown GRIM-19 by siRNA (GenePharma Co., Ltd, China) was performed by Lipofectamine 2000 (Invitrogen, USA) in accordance with the manufacturer’s instructions. The following sequences were used: GRIM-19, 5′-GGAUUGGAACCCUGAUCUATT-3′, and scramble, 5′-UUCUCCGAACGUGUCACGUTT-3′. The culture medium was replaced after 6 h of incubation. Then, 48 h after transfection, the cells were collected. In antioxidant group, cells were cultured with 2 mM N-acetylcysteine (NAC, Sigma, USA) for 24 h. Statistical analysis The SPSS 18.0 statistical software program was used for statistical analysis. Statistical analyses of more than two groups were performed with one-way ANOVA followed by Tukey’s post hoc test or an independent samples t test. P < 0.05 were considered to be statistically significant. Results Aberrant expression of GRIM-19 in the PBLs of patients with URSA To determine whether GRIM-19 is associated with URSA, we examined the expression of GRIM-19 in the PBLs of patients with URSA and non-pregnant healthy women. The mRNA and protein expression of GRIM-19 was significantly lower in URSA patients (n = 28) than that in control group (n = 30) (protein expression GRIM-19/GAPDH: 0.45 ± 0.21 vs 0.79 ± 0.21, **P < 0.01) (Fig. 1). Figure 1 View largeDownload slide Aberrant expression of GRIM-19 in the PBLs of patients with URSA. (A) The mRNA level of GRIM-19 in the peripheral blood lymphocytes (PBLs) of patients with unexplained recurrent spontaneous abortion (URSA) (n = 28) and the control subjects (n = 30) was detected by quantitative real-time PCR. (B) A western blot analysis was performed to assess the protein levels of GRIM-19 in the PBLs of URSA patients (n = 28) and the control subjects (n = 30) (GRIM-19/GAPDH: 0.45 ± 0.21 vs 0.79 ± 0.21). The representative examples are shown. GAPDH served as a loading control. All data are expressed as mean ± SD. *P < 0.05, **P < 0.01, Student’s t test. Figure 1 View largeDownload slide Aberrant expression of GRIM-19 in the PBLs of patients with URSA. (A) The mRNA level of GRIM-19 in the peripheral blood lymphocytes (PBLs) of patients with unexplained recurrent spontaneous abortion (URSA) (n = 28) and the control subjects (n = 30) was detected by quantitative real-time PCR. (B) A western blot analysis was performed to assess the protein levels of GRIM-19 in the PBLs of URSA patients (n = 28) and the control subjects (n = 30) (GRIM-19/GAPDH: 0.45 ± 0.21 vs 0.79 ± 0.21). The representative examples are shown. GAPDH served as a loading control. All data are expressed as mean ± SD. *P < 0.05, **P < 0.01, Student’s t test. Intracellular level of ROS in the PBLs of patients with URSA Mitochondria are the main source of intracellular ROS, notably via the formation of superoxide in the electron transport chain (ETC) (Angelova and Abramov, 2016). Previous study showed that deficiency of GRIM-19 may lead to a collapse in MMP (Chen et al., 2015), so we evaluated the intracellular level of ROS in the PBLs of patients with URSA. Obvious increases in ROS levels were seen in PBLs of URSA group (Fig. 2A). Relative fluorescence intensity for images of each group is shown in Fig. 2B. Flow cytometric analysis confirmed that the intracellular ROS levels of PBLs in URSA group (n = 20, MFI: 42.17 ± 10.91) were increased to those of the control group (n = 20, MFI: 29.87 ± 8.13, **P < 0.01) (Fig. 2C). As such, the roles of GRIM-19 in URSA may be linked to intracellular ROS. Figure 2 View largeDownload slide Intracellular level of ROS in the PBLs of patients with URSA. (A) ROS accumulation in the PBLs of URSA patients (n = 20) and control subjects (n = 20) was visualized with the fluorescent dye DCFH-DA, representative example is shown. (B) Relative fluorescence intensity for images of URSA group (n = 20) and control group (n = 20). (C) Flow cytometric analysis confirmed that the intracellular ROS levels of PBLs in URSA group (n = 20, MFI: 42.17 ± 10.91) were increased to those of control group (n = 20, MFI: 29.87 ± 8.13). Representative examples are shown. (D) The activities of total SOD and GSH-Px in PBLs of URSA patients and control subjects (n = 20, SOD: 36.86 ± 9.44 vs 52.19 ± 8.41, GSH-Px: 96.87 ± 8.41 vs 112.64 ± 7.83). All data are expressed as mean ± SD. **P < 0.01, Student’s t test. Figure 2 View largeDownload slide Intracellular level of ROS in the PBLs of patients with URSA. (A) ROS accumulation in the PBLs of URSA patients (n = 20) and control subjects (n = 20) was visualized with the fluorescent dye DCFH-DA, representative example is shown. (B) Relative fluorescence intensity for images of URSA group (n = 20) and control group (n = 20). (C) Flow cytometric analysis confirmed that the intracellular ROS levels of PBLs in URSA group (n = 20, MFI: 42.17 ± 10.91) were increased to those of control group (n = 20, MFI: 29.87 ± 8.13). Representative examples are shown. (D) The activities of total SOD and GSH-Px in PBLs of URSA patients and control subjects (n = 20, SOD: 36.86 ± 9.44 vs 52.19 ± 8.41, GSH-Px: 96.87 ± 8.41 vs 112.64 ± 7.83). All data are expressed as mean ± SD. **P < 0.01, Student’s t test. The change of oxidative stress related factors SOD and GSH-Px, as free radical scavengers, mainly scavenge hydrogen peroxide and hydroxyl free radicals, finally reduce cell injury from oxidative stress (Li et al., 2016). Therefore, we subsequently evaluated the activities of total SOD and GSH-Px in PBLs of URSA patients. The activity of SOD and GSH-Px were decreased, respectively in PBLs of URSA group compared to control group, as shown in Fig. 2D (n = 20, SOD: 36.86 ± 9.44 vs 52.19 ± 8.41, **P < 0.01. GSH-Px: 96.87 ± 8.41 vs 112.64 ± 7.83, **P < 0.01). Expression of PI3K/Akt/mTOR pathway in the PBLs of patients with URSA In a previous study, it was confirmed that ROS were critical for IL-7-mediated activation of PI3K/Akt/mTOR signaling pathway, in a manner that was dependent on mitochondrial respiratory chain (Silva et al., 2011). In addition, evidence for an involvement of the PI3K/Akt/mTOR network in Treg and Th17 differentiation and function has been accumulating (Kim et al., 2015). Thus, we evaluated the activation of PI3K/Akt/mTOR pathway in the PBLs of patients with URSA. The protein levels of p-Akt and p-mTOR in PBLs from patients with URSA were both significantly higher than those from control group. The overall expression of Akt and mTOR, however, was not significantly different in any of the groups (Fig. 3A). Figure 3 View largeDownload slide Expression of PI3K/Akt/mTOR pathway and proportional change of Treg and Th17 cells in URSA patients. (A) Western blot analysis was performed to assess the protein levels of PI3K/Akt/mTOR pathway in the PBLs of URSA patients (n = 20) and the control subjects (n = 20). The representative examples are shown. GAPDH served as a loading control. All data are expressed as mean ± SD. **P < 0.01, Student’s t test. (B) Flow cytometric identification of Treg cells in the PBMCs of a patient with URSA (n = 20, 4.32 ± 1.52%) and control subjects (n = 20, 6.23 ± 1.41%). Lymphocytes were identified and gated on a forward/side-scatter plot. Percentages of CD3+CD8−CD25+Foxp3+ Treg cells were identified. The representative examples are shown. All data are expressed as mean ± SD. **P < 0.01, Student’s t test. (C) Flow cytometric identification of Th17 cells in the PBMCs of a patient with URSA (n = 20, 3.21 ± 0.68%) and control subjects (n = 20, 1.76 ± 0.46%). Lymphocytes were identified and gated on a forward/side-scatter plot. Percentages of CD3+CD8−IL-17+ Th17 cells were identified. The representative examples are shown. All data are expressed as mean ± SD. **P < 0.01, Student’s t test. Figure 3 View largeDownload slide Expression of PI3K/Akt/mTOR pathway and proportional change of Treg and Th17 cells in URSA patients. (A) Western blot analysis was performed to assess the protein levels of PI3K/Akt/mTOR pathway in the PBLs of URSA patients (n = 20) and the control subjects (n = 20). The representative examples are shown. GAPDH served as a loading control. All data are expressed as mean ± SD. **P < 0.01, Student’s t test. (B) Flow cytometric identification of Treg cells in the PBMCs of a patient with URSA (n = 20, 4.32 ± 1.52%) and control subjects (n = 20, 6.23 ± 1.41%). Lymphocytes were identified and gated on a forward/side-scatter plot. Percentages of CD3+CD8−CD25+Foxp3+ Treg cells were identified. The representative examples are shown. All data are expressed as mean ± SD. **P < 0.01, Student’s t test. (C) Flow cytometric identification of Th17 cells in the PBMCs of a patient with URSA (n = 20, 3.21 ± 0.68%) and control subjects (n = 20, 1.76 ± 0.46%). Lymphocytes were identified and gated on a forward/side-scatter plot. Percentages of CD3+CD8−IL-17+ Th17 cells were identified. The representative examples are shown. All data are expressed as mean ± SD. **P < 0.01, Student’s t test. Proportional change of Treg and Th17 cells in PBMCs of URSA patients Treg and Th17 cells play a major role in tolerating conceptus antigens and therefore may contribute to the maintenance of pregnancy, so we determined the proportion of Treg and Th17 cells in URSA patients compared to healthy non-pregnant women. The proportion of Treg cells in PBMCs in URSA patients (n = 20, 4.32 ± 1.52%) was statistically significantly lower than those in control women (n = 20, 6.23 ± 1.41%, **P < 0.01) (Fig. 3B). Meanwhile, the percentage of Th17 cells in PBMCs in URSA patients (n = 20, 3.21 ± 0.68%) was significantly higher than that in control women (n = 20, 1.76 ± 0.46%, **P < 0.01) (Fig. 3C). Considering the broad involvement of mTOR in Treg and Th17 differentiation and function, these data suggested a possible role for GRIM-19 in the Treg/Th17 balance by regulating the activation of mTOR. Downregulation of GRIM-19 and its effect in intracellular ROS production and the level of p-mTOR in Jurkat cells To determine the effect of the low level of GRIM-19 in PBLs of URSA patients, we downregulated the expression of GRIM-19 by specific siRNA in the Jurkat E6-1 human CD4+ T-lymphocytes. Immunoblotting indicated notable depletion of GRIM-19 in the GRIM-19 siRNA group (Fig. 4A). Then, the intracellular ROS production and the protein level of p-mTOR in the GRIM-19 siRNA group and the scramble control group were analyzed. The cells in the GRIM-19 siRNA group had higher levels of ROS than those in the control group (Fig. 4B). More strikingly, the protein level of p-mTOR increased significantly in the GRIM-19 siRNA group, and the expression level of total mTOR did not differ (Fig. 4A). Collectively, all these results strongly suggested that depletion of GRIM-19 would affect intracellular ROS production and PI3K/Akt/mTOR signaling pathway in URSA. Figure 4 View largeDownload slide Regulation of p-mTOR by GRIM-19–ROS–mTOR axis in Jurkat cells. (A) The Jurkat cells were transfected with GRIM-19 siRNA and treated with ROS inhibitor NAC. The expression levels of GRIM-19, mTOR and p-mTOR in the control, GRIM-19 siRNA, GRIM-19 siRNA + NAC and NAC group were evaluated by immunoblot. GAPDH was used as a loading control. Three independent experiments were carried out. The representative examples are shown. *P < 0.05, **P < 0.01, one-way ANOVA test. (B) Flow cytometric analysis was applied to detect the ROS generation in the control, GRIM-19 siRNA, GRIM-19 siRNA + NAC and NAC group by staining with DCFH-DA. Three independent experiments were carried out. The representative examples are shown. All data are expressed as mean fluorescent signal intensity (MFI) ± SD. **P < 0.01, one-way ANOVA test. G19: GRIM-19. Figure 4 View largeDownload slide Regulation of p-mTOR by GRIM-19–ROS–mTOR axis in Jurkat cells. (A) The Jurkat cells were transfected with GRIM-19 siRNA and treated with ROS inhibitor NAC. The expression levels of GRIM-19, mTOR and p-mTOR in the control, GRIM-19 siRNA, GRIM-19 siRNA + NAC and NAC group were evaluated by immunoblot. GAPDH was used as a loading control. Three independent experiments were carried out. The representative examples are shown. *P < 0.05, **P < 0.01, one-way ANOVA test. (B) Flow cytometric analysis was applied to detect the ROS generation in the control, GRIM-19 siRNA, GRIM-19 siRNA + NAC and NAC group by staining with DCFH-DA. Three independent experiments were carried out. The representative examples are shown. All data are expressed as mean fluorescent signal intensity (MFI) ± SD. **P < 0.01, one-way ANOVA test. G19: GRIM-19. Regulation of p-mTOR by GRIM-19–ROS–mTOR axis in Jurkat cells When the cells were transfected with GRIM-19 siRNA, protein level of p-mTOR and level of intracellular ROS ascended accordingly. To confirm ROS as a linker bridging GRIM-19 depletion and mTOR activation, GRIM-19 knockdown cells were treated with antioxidant NAC. Notable depletion of intracellular ROS was observed in the cells treated with NAC (Fig. 4B). As depicted in Fig. 4A, the upregulation of p-mTOR resulted from GRIM-19 loss was significantly reversed in the cells treatment with NAC, indicating that ROS was indeed required for GRIM-19 depletion induced p-mTOR expression. These results suggested that GRIM-19 may be involved in URSA partly through ROS–mTOR–Treg/Th17 axis. Discussion To date, no reports have been made about the role of GRIM-19 in URSA, although it has been demonstrated previously to be essential to the immunoregulation. In this study, we revealed a new molecular mechanism underlying the association between GRIM-19 deficiency and URSA. One of the major outcomes of this study showed that the expression of GRIM-19 decreased significantly in the PBLs of patients with URSA, demonstrating the relevance between a low level of GRIM-19 and URSA. Our study showed a reducing number of Treg cells and an increasing number of Th17 cells in PBMCs of URSA women compared to control subjects that agreed with other studies. Further studies revealed that there is a considerable increase in intracellular ROS production and a high level of PI3K/Akt/mTOR activation in the PBLs of URSA patients compared with the control subjects. In addition, our study using Jurkat cells demonstrated that downregulation of GRIM-19 leads to an increased ROS production and an elevated level of p-mTOR. Meanwhile, treatment of Jurkat cells with antioxidant NAC decreased its p-mTOR expression. Involvement of the PI3K/Akt/mTOR network in Treg and Th17 differentiation and function has been demonstrated previously. Therefore, we conclude that GRIM-19 deficiency may play a role in regulating Treg/Th17 balance partly through ROS–mTOR signaling axis in URSA. GRIM-19 is a functional subunit of mitochondrial respiratory chain complex I and plays an essential role in the assembly and enzymatic activity of complex I. A lack of GRIM-19 leads to a collapse in MMP, and abnormality of the mitochondrion structure, morphology and cellular distribution (Huang et al., 2004; Chen et al., 2015). Mitochondria produce ROS as a by-product of a number of enzymatic reactions and the ETC (Angelova and Abramov, 2016). Emerging evidence suggests that dysregulated ROS signaling may contribute to a development of processes which lead to human diseases (Silva et al., 2016). A previous study showed that inhibition of respiration and decrease of MMP can stimulate superoxide production (Nohl et al., 1993; Angelova and Abramov, 2016). In our study, a considerable increase in the intracellular ROS production in the PBLs of URSA patients was observed. To study the correlation between GRIM-19 and the intracellular ROS production, the ROS level in Jurkat cells was examined after GRIM-19 knockdown. We found that the ROS level in the GRIM-19 siRNA group to be significantly higher than in the control group. These results indicated that a deficiency of GRIM-19 in URSA is related to intracellular ROS production. ROS activates many adaptation signaling pathways in cells, in which mTOR plays an important role in autophagy, apoptosis, metabolism, growth, survival and immunity (Wullschleger et al., 2006; Roy et al., 2014; Wang et al., 2016). Increasing evidence strongly suggests that mTOR is regulated by many upstream regulators, such as PI3K, AKT and AMPK, and ROS is the initial regulator (Hay and Sonenberg, 2004; Wang et al., 2016). ROS-AKT-mTOR axis plays an important role in autophagy of cancer cells (Fiorini et al., 2015). Safranova et al. (2003) reported increase ROS in peripheral blood granulocytes in women with habitual abortions. In our study, the upregulation of p-mTOR resulted from GRIM-19 loss was significantly reversed in the cells treatment with NAC, indicating that ROS was indeed required for GRIM-19 depletion induced p-mTOR expression. These results indicated that mTOR is mediated by ROS generation, and GRIM-19 may be involved in URSA via affecting intracellular ROS production and downstream PI3K/Akt/mTOR signaling pathway. Dysfunctions in immune system regulation may result in pregnancy abnormalities such as URSA. Immunological disorders may cause impaired maternal immune tolerance to the fetus and result in fetal rejection (Sereshki et al., 2014). Several types of T cells have been associated with the pathogenesis of URSA, including Treg and Th17 cell. Treg cells are one of the most important immunological factors that play a primary role in suppressing excessive immune response of other cells, maintaining tolerance to self-antigens, and results in a successful pregnancy (Zenclussen, 2006). Th17 cells play a major role in induction of inflammation by producing pro-inflammatory cytokines and matrix metalloproteinase (Sereshki et al., 2014). Several studies have shown the balance between Treg cells and Th17 cells under normal and pathologic conditions (Schaub et al., 2008; Rong et al., 2009; Kimura and Kishimoto, 2010). A recent study indicated that in patients with URSA, immunotherapy with mononuclear cells derived from the baby’s father could affect Treg/Th17 balance, and they found that the Treg bias would be beneficial for pregnancy (Wu et al., 2014). Another study indicated that the imbalance between Th17 and Treg cells during the proliferative phase of menstrual cycles in the URSA group may be considered a cause for spontaneous abortion (Sereshki et al., 2014). In our study, we found a reducing number of Treg cells and an increasing number of Th17 cells in PBMCs of URSA women compared to control subjects. Previous studies have found that the differentiation of Th17 cells is controlled by PI3K/Akt/mTOR signaling pathway. It was reported recently that the PI3K-AKT-mTORC1-S6K axis positively regulates Th17 differentiation by promoting the nuclear translocation of RAR-related orphan receptor (ROR)γt (Kurebayashi et al., 2012; Kim et al., 2013; Koga et al., 2014). In contrast, the inhibition of PI3K and mTOR increases Treg cell differentiation (Okkenhaug et al., 2006; Wan et al., 2016). A study in follicular thyroid cancer cells found that the ratio of Treg/Th17 was increased significantly after rapamycin treatment, which is consistent with the previous studies (Zhou et al., 2016). Thus, our study indicated that the imbalance between Th17 and Treg cells in URSA is largely associated with the high level of PI3K/Akt/mTOR signaling pathway. In conclusion, we have provided evidence demonstrating that there may be an association between URSA and low levels of GRIM-19. GRIM-19 deficiency may play a role in regulating Treg/Th17 balance partly through ROS–mTOR signaling axis in URSA. Authors’ roles L.C. and Y.Y. conceived the study. Y.Y., L.Y.C., X.H.D., H.L.Y. performed experiments. L.C., Y.Y., L.Y.C., X,H.D. and H.L.Y. performed data analysis. L.C., L.Y.C. and Y.Y. wrote, reviewed and edited the article. All authors edited and approved the final draft of the article. Funding National Natural Science Foundation of China (Grant numbers 81571511, 81701528, 81370711 and 30901603), the Shandong Provincial Natural Science Foundation (Grant numbers ZR2017PH052 and ZR2013HM090) and the Science Foundation of Qilu Hospital of Shandong University, Fundamental Research Funds of Shandong University (Grant numbers 2015QLQN50 and 2015QLMS24). Conflict of interest The authors declare that there is no conflict of interest that could prejudice the impartiality of the present research. References Aluvihare VR , Kallikourdis M , Betz AG . Regulatory T cells mediate maternal tolerance to the fetus . Nat Immunol 2004 ; 5 : 266 – 271 . Angell JE , Lindner DJ , Shapiro PS , Hofmann ER , Kalvakolanu DV . Identification of GRIM-19, a novel cell death-regulatory gene induced by the interferon-beta and retinoic acid combination, using a genetic approach . J Biol Chem 2000 ; 275 : 33416 – 33426 . Angelova PR , Abramov AY . 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For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Molecular Human ReproductionOxford University Press

Published: May 11, 2018

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