Human Wharton's jelly‐derived mesenchymal stem cells prevent pregnancy loss in a rat by JAK/STAT‐mediated immunomodulation

Spontaneous abortion (SA) is a multiple‐original syndrome with immune imbalance as one of its major risk factors. As Wharton's jelly‐mesenchymal stem cells (WJ‐MSCs) are considered to be able to prevent abortion, this study aims to explore the currently poorly understood underlining molecular signaling pathways and regulatory mechanisms of WJ‐MSCs in pregnancy maintenance.


INTRODUCTION
Spontaneous abortion (SA) is a common clinical disease that seriously affects the physical and mental health of women of childbearing age.Many risk factors, including genetic factors, endocrine disorders, infectious agents, immune dysfunction, and genital anatomical abnormalities, contribute to this complex pathology. 1,2While ≥50% of its causes are not clear so far, the immune imbalance has been reported to be an important factor associated with SA and infertility. 3During pregnancy, the maternal immune system must be modified to accept the semiallogeneic fetus.The immune relationship between embryonic and maternal tissue is extremely complex involving human leukocyte antigens, the complement pathway, cytokines, blood group antigens, trophoblast cell membrane antigen, and intercellular adhesion molecules. 4As a result, the role of T helper cells (Th) and the balance of secreted cytokines in SA is an active area of research.
Helper T cells are divided into Th1, Th2, Th3, and Th17 subtypes based on their cytokine production.Th1 cells secrete Interleukin-2 (IL-2), Interferon-γ (IFN-γ), Tumor necrosis factor-α (TNF-α), Tumor necrosis factor-β (TNF-β) and play a role in cellular immune responses.Th2 cells secrete IL-4, IL-5, IL-6, IL-10, and IL-13, and are involved in humoral immunity.Th3 cells produce transforming growth factor-β (TGF-β) which inhibits the expression of Th1 cytokines.A normal pregnancy exhibits a shift toward Th2 cytokine production and inhibition of Th1 responses and disturbance of this balance may result in unexplained recurrent spontaneous abortion (URSA). 5Additionally, Th17 cells produce IL-17 which can cause an inflammatory response at the maternal interface that jeopardizes pregnancy. 5,6he importance of T cells subtypes and the cytokine environment at the maternal-fetal interface suggests that immunotherapy may be a viable method of treating URSA in the clinic. 7,8urrent treatments for URSA include active immunotherapy, but their effectiveness is limited. 9,10With progress in the understanding of reproductive immunology, the immune mechanism of SA and its immunological treatment has become an important topic of research. 11,12t has been reported that the expression of related factors from Th1/Th2/Th3/Th17 (IL-2, IL-3, IL-5, IL-6, IL-10, IL-17) mainly plays a biological function through JAK/STAT pathway, and these interleukins can activate JAK/STAT signaling pathway under specific conditions. 13There are other pathways involved in interleukin mediating immune response, such as inhibiting the ability of nuclear factor-κB (NF-κB) to bind to DNA or inhibiting the activity of IκB kinase reversely to inhibit NF-κB activation.In order to study the JAK/STAT or NF Nf-κb signaling pathway, many researchers [13][14][15] cocultured signaling pathway inhibitors to with cells, so as to inhibit the effects of related signaling pathways and factors in cells.One study 16 confirmed that MSCs could regulate the ratio of Th1/Th2 and Th17/Treg cells and the expression of related factors in lymphocytes through the JAK/STAT signaling pathway by co-culturing lymphocytes with JAK/STAT inhibitors.
While mesenchymal stem cells (MSCs), as pluripotent stem cells, have been studied in tissue engineering, hematopoietic stem cell transplantation, and gene therapy, MSCs have been widely studied in the treatment of immune diseases because of their role in immune regulation. 17It has been shown that Wharton's jelly (WJ) in the human umbilical cord is an important source of MSCs which secrets a large number of cytokines and possesses no tumorigenicity, lower infection rate of pathogenic microorganisms, fewer ethical problems, multidirectional differentiation potential and lower immunogenicity than bone marrow. 18,19It has been suggested 20 that the expression of IFN-γ secreted by Th1 decreased significantly, while the expression of IL-4, IL-13, and other factors secreted by Th2 increased after the co-culture of WJ-MSCs and immune cells.It has been also found that WJ-MSCs affect the differentiation and function of Th17 cells in the peripheral lymphocyte system and promote the proliferation and differentiation of Tregs cells to maintain the balance between Th17 cells and Tregs cells and improve the immune microenvironment of the body.The above results suggest the importance of WJ-MSCs for local immune tolerance.
Bromocriptine is a prolactin inhibitor to trigger abortion by disrupting the endocrine environment, 21 such as low progesterone (P), low prolactin (PRL), and abnormal Th1 immune response.The uterine decidua is one of the main sources of PRL synthesis and secretion during pregnancy.Animal experiments have revealed that it plays an important role in regulating ovarian function and promoting the development of fetal organ tissues and appropriate physiological high blood PRL is an important factor in maintaining pregnancy. 22Bromocriptine can inhibit the secretion of PRL by the pituitary gland and reduce the progesterone accordingly, disrupting the balance of Th1/Th2 cytokines and leading to miscarriage. 23Human WJ-MSCs have previously been shown to prevent SA in this rat model by decreasing the expression of IFN-γ and IL-17 and up-regulating the expression of IL-10, 21 but the detailed mechanism of the immunomodulatory effect remains elusive.Here we utilized bromocriptine-induced abortion in rats to investigate the therapeutic effects of WJ-MSCs on Th1/Th2/Th3 cytokines and to examine the roles of JAK/STAT and NF-κB signaling in immunomodulation of WJ-MSCs.

Isolation, culture, and characterization of WJ-MSCs
Human umbilical cords were obtained from normal fullterm deliveries with the informed consent of the mother.The preparation of WJ-MSCs was approved by the Research Ethics Committee at the Affiliated Hospital of Nantong University.WJ-MSCs were isolated, cultured, and characterized using the methods described in a previous study. 21-MSCs treated with JAK/STAT or NF-κB inhibitors P3 WJ-MSCs were cultured with DMEM/F12 medium (USA, Hyclone) containing 10% fetal bovine serum (FBS; USA, Hyclone) with/without the inhibitors of JAK/STAT (Germany, MERCK, AG490, 10 μmol/L) or NF-κB (Solarbio, BAY-11-7085, 10 μmol/L).The medium was removed 5 h later, WJ-MSCs were washed twice with PBS and then cultured with DMEM/F12 medium containing 10% FBS.The morphological changes of WJ-MSCs were observed under an optical microscope.Cells were measured in terms of their viability or injected into rats through the tail vein immediately after these operations.We selected STAT3 as a star molecule to detect Western blotting (WB) protein in WJ-MSCs to verify the effectiveness of the inhibitor experiment.Primary antibodies: anti-STAT3 (Sigma-Aldrich, SAB2104416), anti-p-STAT3 (Sigma-Aldrich, SAB4300033).The specific experimental method is the same as the WB experiment in the following text.

Cell viability test
The viability of WJ-MSCs treated with JAK/STAT inhibitor or NF-κB inhibitor was measured by a Cell Counting Kit-8 (CCK-8) kit (Dojindo, Kumamoto, Japan).All experimental procedures were performed according to the manufacturer's instructions.

Rat model of spontaneous abortion
The protocol was approved by the Care and Use Committee of the Laboratory Animal Research Center of Nantong University.Age-matched virgin female (200-250 g; n = 48) and male (200-250 g; n = 48) Sprague-Dawley rats were obtained from the Laboratory Animal Center of Nantong University.Day 1 of pregnancy was defined by the presence of a vaginal plug.Pregnant rats were randomly divided into six groups.Group A served as a normal pregnancy group and received a vehicle injection of 0.2 mL of PBS by subcutaneous injection on gestational days 6-8 and 1 mL of DMEM/F12 by tail vein injection on day 9 of gestation.Group B received a subcutaneous injection with 0.8 mL/kg of 75% ethanol on gestational days 6-8 and 1 mL of DMEM/F12 via tail vein injection on day 9 of gestation.Group C served as the abortion model and received subcutaneous injections of 0.8 mL/kg of bromocriptine (0.5 mg/mL) in 75% ethanol on gestational days 6-8 and 1 mL of DMEM/F12 by tail vein injection on day 9 of gestation.Group D was similar to group C but with an additional injection of 1 Â 10 7 WJ-MSCs 21 dispersed in 1 mL of DMEM/F12.WJ-MSCs were treated with JAK/STAT or NF-kB signal pathway inhibitors for 5 h and then injected into rats.The rats injected with 1 Â 10 7 treated WJ-MSCs dispersed in 1 mL DMEM/F12 served as group E or F.

Tissues and serum collection
Pregnant rats in each group were euthanized on day 14 of gestation and embryo resorption was assessed.The fetal/ placental unit and decidua were removed from the uterus and rinsed with saline.A portion of the fresh tissues was stored at À80 C for real-time polymerase chain reaction (RT-PCR) or WB.Whole blood was collected from the orbit and serum was obtained by centrifuging blood in serum separation tubes.Serum was stored at À80 C before being subjected to enzyme-linked immunosorbent assays (ELISA).

RT-PCR analysis of Th-related cytokines
Total RNAs were isolated from tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) in RNA-free centrifuge tubes.According to the instructions of the cDNA synthesis Kit (Roche, Switzerland), 1 μg total RNA was reverse transcribed in 5 Â Reaction Buffer, 10 mmol/L dNTPs, 1 μmol/L oligo-dT primer, 20 U RNase inhibitor, 200 U/μL RT enzyme, and an appropriate amount of DEPC-treated water.Quantitative RT-PCR (qRT-PCR) was performed in a total volume of 20 μL reactions containing 2 μL cDNA, 10 μL SYBR Green (Roche, Switzerland), indicated primer pairs, and 6 μL ddH 2 O. Primer pairs used are shown in Table S1.

Statistical methods
Results were analyzed with SPSS 22.0 data analysis software.Data are expressed as mean ± standard error of the mean (mean ± SEM).p < 0.05 is considered to be statistically significant.

Isolation, culture, and characterization of WJ-MSCs
The recent development of stem cell technology has revolutionized the treatment of various diseases and laid the foundation for immunomodulation. 24To investigate WJ-MSCs' function, we first cultured MSCs obtained from human umbilical cords. 21Our results showed that a small number of WJ-MSCs appeared around the implanted blocks after seeding for 6-7 days (Figure 1a).After 9-10 days of culture, cells were gathered in pieces (Figure 1b).After 12-14 days of culture, cells formed a single layer as a result of rapid proliferation and exhibited a uniform spindle-shaped reminiscent of fibroblasts (Figure 1c).Microscopic results showed that JAK/STAT or NF-ĸB inhibition did not cause a discernible difference in cell (Figure 1d-f).However, the inhibition altered the growth and viability of WJ-MSC as measured by a CCK8 assay (Figure 1g).To verify the effectiveness of the inhibitor experiment, we selected STAT3 as a star molecule to detect WB protein in cells.Results showed that p-STAT3 was significantly inhibited compared to the control groups (Figure 1h, i).
Flow cytometry of passage three cells showed that WJ-derived cells expressed the MSC markers CD105, CD29, CD73, CD166, and CD44, and were negative for the markers CD34, CD11b, CD14, CD45, and HLA-DR (Figure S1a), which was consistent with human MSCs. 25,26Studies 27,28 have shown that MSCs have multiple differentiation potentials, including muscle cells, hepatocytes, osteoblasts, adipocytes, chondrocytes, and stromal cells.They have also been successfully induced to be endometrial cells. 29In this study, the differentiation potentials of MSCs into osteoblasts and adipocytes were confirmed (Figure S1b).would improve abortion and the potential mechanism, MSCs prepared from a human umbilical cord pretreated with or without JAK/STAT or NF-ĸB inhibitors were injected into a pregnant rat in the presence of bromocriptine.The results showed that absorbing embryos were rarely detected in normal or ethanol pregnancies (Figure 2a, a1, b1), but were frequently detected in the bromocriptine-induced abortion model (Figure 2a, c1).Treatment with WJ-MSC significantly reduced the visible occurrence of embryo absorption (Figure 2a, d1).While JAK/STAT inhibitor but not NF-κB inhibitor primed WJ-MSC obviously compromised the effect of abortion prevention of WJ-MSC (Figure 2a, e1, f1).Quantification revealed that embryo absorption was significantly increased in the bromocriptine-treated group compared to the normal pregnancy or ethanol injection group (Figure 2b).WJ-MSC treatment significantly reduced the abortion rate in bromocriptinetreated pregnancy (Figure 2b).There is actually no statistical difference between normal pregnancy group versus WJ-MSC treatment group ( p > 0.05, p = 0.0723) and ethanol control group versus WJ-MSC treatment group ( p > 0.05, p = 0.0685) (Figure 2b).JAK/STAT inhibition abolished pregnancy-preserving effects of WJ-MSC but NF-κB inhibition did not (Figure 2b).

Cytokine profiles during the abortion and the effects of WJ-MSC transfusion serum cytokines by ELISA
To further evaluate the local environmental immune profile changed by WJ-MSCs, the cytokines secreted from Th1, Th2, and Th3 cells were investigated in different groups.ELISA analysis showed that the levels of Th1-related cytokines IL-2, TNF-α, and TNF-β in the bromocriptine-induced abortion group were significantly higher than in the normal pregnancy group or ethanol injection group (Figure 3a), while the levels of the Th2-related cytokines IL-4, IL-5, and IL-13 (Figure 3b), and Th3 cell-cytokine TGF-β were significantly decreased (Figure 3c).These results suggested that WJ-MSCs transfused into the abortion rat restored cytokine profiles to comparable levels of normal pregnancy group or ethanol injection group (Figure 3).JAK/STAT pretreated WJ-MSC prevented their rescues effect on cytokine profiles, but not NF-κB inhibition (Figure 3).

Cytokines in decidual and placental tissues by Western blot
The results above showed that the mechanism of WJ-MSCs regulating abortion primarily depended on JAK/STAT signaling pathway.The expression of cytokines was detected in both decidua and placenta, the two components of the maternal-fetus interface.Western blot results showed that Th2 cytokines (IL-4, IL-5, IL-13) were significantly reduced in the bromocriptineinduced abortion group compared to the normal pregnancy group or ethanol group (Figure 4b), while the Th1 cytokines TNF-α and TNF-β significantly increased except IL-2 (Figure 4a).WJ-MSC injection significantly reversed these abortion-related effects on cytokine (Figure 4).JAK/STAT inhibition of WJ-MSC abolished their ability to reverse Th2-related cytokine changes (Figure 4).

Cytokine mRNA in decidual and placental tissues
To further validate our results, we extracted the mRNA from decidual and placental tissues.Our results suggested that Th2 (IL-4, IL-5, and IL-13) and Th3 (TGF-β) cytokine mRNA levels were significantly reduced in the bromocriptine-induced abortion group compared to the normal pregnancy or ethanol group (Figure 5b, c), while the Th1 (IL-2, TNF-α, and TNF-β) cytokine mRNA levels were significantly increased (Figure 5a).WJ-MSC treatment of the abortion group significantly reversed these abortion-related effects on cytokine mRNA.JAK/STAT inhibition of WJ-MSC prevented them from reversing abortion-related cytokine mRNA changes (Figure 5).

DISCUSSION
The bromocriptine-induced abortion model examined here involved a shift from a Th2-dominated immune response that supports pregnancy to a detrimental Th1-dominated response.Human WJ-MSCs had immunomodulatory properties that prevented this shift and rescued pregnancy.There was a decrease of IL-2 in the WJ-MSCs group by ELISA and PCR in different groups but its expression detected by the Western Blot experiment showed less discrepancy which might be due to the secretory nature of IL-2 or the discrepancies between the post-translational protein and mRNA.Combined with the ELISA and PCR results, we could believe that there is some persuasive evidence to suggest that there is a difference in IL-2.Human WJ-MSCs decreased the Th1 (IL-2, TNF-α, and TNF-β) cytokines and increased the Th2 (IL-4, IL-5, and IL-13) cytokines in serum and decidual/placental tissue.Additionally, there was also evidence that WJ-MSCs promoted the expression of TGF-β, an anti-inflammatory cytokine produced by Th3 and regulatory T cells.Our study demonstrated that the immunomodulatory and pregnancy-preserving effects of WJ-MSCs were dependent on JAK/STAT signaling in WJ-MSCs, but not NF-κB signaling.
The T cell cytokines profile presented here expanded our knowledge of the effects of WJ-MSCs in the rat bromocriptine-induced abortion group because in our previous study 21   WJ-MSCs injection were detected in both peripheral blood and decidual/placental tissue which suggested that both systemic and local immune responses at the maternal/fetal interface contributed to this hemostasis maintenance and pregnancy success.Our results are consistent with reports that human endometrial MSCs prevented abortion in a mouse incompatible pregnancy abortion model, 30 and that mouse bone marrow-derived MSCs prevented abortion in LPS-mediated and incompatible mating mouse models. 31Our observation that WJ-MSCs could tip T cell cytokine responses from Th1 to Th2 was similar to the findings from the therapeutic application of MSCs to other models including mouse diabetes, 32 rat corneal transplant, 33 rat autoimmune uveoretinitis, 34 rat renal injury, 35 and mouse colitis. 36ur results suggested that JAK/STAT signaling, an important mediator of responses to injury and inflammation, was required for WJ-MSCs to modify T-cell responses.JAK/STAT signaling in human bone marrow MSCs had been implicated in the production of indoleamine 2,3-dioxygenase, a suppressor of T cell proliferation. 37Other studies of human bone marrow MSCs indicated the requirement of JAK/STAT signaling for the release of modulators of immunity 38,39 and homing of MSCs to sites of injury/inflammation. 40 In order to investigate the signaling pathways through which stem cells inhibit the occurrence of miscarriage, we preprocessed the stem cells and treated them with relevant signaling pathway inhibitors before transplantation.Zanders et al. 41 treated C2C12 cells (mouse myoblasts) with AG490, which attenuated STAT3 phosphorylation.Plus, in vivo experiments showed that treatment with AG490 attenuated sepsis-induced STAT3 phosphorylation. 41][43][44][45] To verify the effectiveness of the inhibitor experiment, we selected STAT3 as a star molecule to detect WB protein in cells.7][48] The inhibition of JAK-STAT decreased the cell viability of WJ-MSCs, which may be a reason why the treatment effect for miscarriage is reduced.Of course, we have also conducted repeated verification.Although the addition of AG490 and BAY-11-7085 stem cell viability was affected, the therapeutic effect of AG490 on stem cells decreased while BAY-11-708 did not show any significant changes, indicating that the weakened efficacy of our stem cell therapy for miscarriage is related to the inhibition of the JAK/STAT signaling pathway, rather than a decrease in cell viability.Taken together, these findings indicate the broad importance of JAK/STAT signaling in the therapeutic properties of MSC.Manipulating JAK/STAT signaling may allow the therapeutic effects of WJ-MSCs to be optimized.
The immunomodulatory properties of MSCs from various tissues make MSCs potentially useful for treating a range of diseases and injuries.WJ-MSCs are an attractive source for therapy because they can be obtained in large numbers from umbilical cords that would otherwise be discarded.The utilization of MSCs to prevent human recurrent SA might be practicable due to the pregnancypreserving immunomodulation of these allogeneic cells rather than long-term engraftment.Our results presented here further corroborated that the administration of xenogeneic human MSCs has the desired effect of modulating a rat immune response.WJ-MSCs have been shown to be beneficial for graft versus host disease in human patients undergoing hematopoietic stem cell transplantation, and clinical trials were underway for a range of other conditions. 49Collectively, our study blazes a new trail to combat recurrent abortion and provides a promising strategy for clinical application.
It was unclear how WJ-MSCs inhibited immunity in immune regulation.To determine whether WJ-MSCs F I G U R E 1 Isolated and cultured human WJ-MSCs.(a) 6-7 days after seeding, a small number of WJ-MSCs appeared around the implanted blocks.(b) After 9-10 days of culture, cells are gathered in pieces.(c) After 12-14 days of culture, the majority of cells exhibited a uniform spindle shape similar to fibroblasts.(d)-(f) JAK/STAT or NF-ĸB inhibition did not alter the appearance of cultured WJ-MSC.(g) JAK/STAT or NF-ĸB inhibition significantly alters the growth and viability of WJ-MSC as measured by a CCK8 assay.(h)-(i) STAT3 and p-STAT3 protein expression level in WJ-MSCs detected by Western Blot.(n = 3, **p < 0.01).(a)-(f) were taken using 100Â magnification (n = 8, *p < 0.05, **p < 0.01).