| The mechanism of immune tolerance during pregnancy is one of the basic problems in life sciences, and more and more domestic and overseas scholars are throwing themselves to the researchs on the mechanism. Many cells and molecules are involved in the establishment and maintenance of maternal-fetal immune tolerance, and the process is extremely delicate and complex. Both T cells and NK cells are important immune cells in the pregnant uterus. They can regulate maternal-fetal immune tolerance by secrete a variety of cytokines. When the body is attacked by virus, the immune system will not only cause an effective immune response to clear the external microorganisms, but also ensure that the maternal-fetal immune balance is not destroyed, so that the pregnancy will go smoothly. This is modulated by profound mechanisms. At present, the mechanism by which maternal-fetal immune tolerance is established has not been fully elucidated. The study of virus affects on maternal-fetal immune tolerance will help us to further reveal the mechanism.Innate recognition receptors are thought to be involved in the recognition of viral components and activation of antiviral responses in immune and non-immune cells. Toll-like receptors (TLRs) is a family of innate pattern recognition receptors that can recognize virus-associated molecules and activate antiviral responses. Both TLR3 and TLR7/8 are TLR family members, and involved in the process of antiviral immune. However, the effect of TLR3 and TLR7/8 activation on immune cells of maternal-fetal interface and its mechanism have not been elucidated. In the present study, non-obese diabetic (NOD) mouse mould, which was characterized by a deficiency of NK cells, was used to further explore the roles of T cells and NK cells in the process of maternal-fetal immune tolerance through comparative study with non-immune deficient mice. In addition, the effect of TLR3 and TLR7/8 activation on immune cells at maternal-fetal interface and its mechanism would be initially elucidated.Objectives1. To investigate the effect of specific TLR3 and TLR7 agonists, poly (I:C) and R837, individually and in combination, on uterine immune cell function and their subsequent effects on pregnancy outcome by comparing NOD mice and BALB/c mice.2. To observe the signaling pathways of immune responses caused by activation of TLRs by respectively inhibit JNK MAPK and ERK MAPK signaling pathway.Materials and Methods1. Female BALB/c or NOD mice were co-caged with C57BL/6 males, generating naturally established allogeneic pregnant BALB/c×C57BL/6 and NODxC57BL/6 models. The pregnant mice could be divided into two groups, the group of NOD mice and BALB/c mice, and 10 pregnant mice were obtained in each group. According to the different treatments, they were further divided into groups of poly (I:C), R837, both poly (I:C) and R837 (the group of combined stimulation), PBS group, DMSO, respectively. In total, there were 10 groups. Pregnant mice were intraperitoneally injected totally three times with poly (I:C) (200ng/ml, in a volume of 200μl, dissolved in PBS), with R837(20 ng/ml; 200μl dissolved in DMSO), with a combination of poly (I:C) and R837 (40 ng poly (I:C) in 100μl PBS and 4 ng R837 in 100μl DMSO), or with an equal volume of PBS or DMSO as a solvent control, at gestational days 2.5,4.5, and 6.5 respectively. Embryo-depleted placentas and decidual tissue including decidua basalis, were harvested at day 12.5. Then we calculate the percentage of embryo resorption. Lymphocytes were purified from placentas and decidual tissue, and the status of cytokine expression in CD45+ cell was detected by an FACS Calibur flow cytometer.2. The allogeneic pregnant models were established as above, and pregnant mice were obtained in NOD group and BALB/c group respectively. Then they were further divided into CD49b+ group and CD3+ group, which were further divided into groups of poly (I:C), R837, both of poly (I:C) and R837 (the group of combined stimulation), PBS, DMSO, respectively. In total, there were 20 groups. Under sterile conditions, magnetic-affinity cell sorting (MACS) was used to purify placental CD3+ and CD49b+ cells from non-stimulated BALB/c and NOD female mices on gestational day 12.5. The purity and viability of the purified cells was determined by flow cytometry and propidium iodide staining. Purified CD3+ and CD49b+ cells were seeded onto 24 well plates. Poly (I:C) (final concentration 50μg/ml), R837(final concentration 10μg/ml), or a combination of both was added into the culture medium. After 6 days of cultivation, the cells were harvested and the expression pattern of TNF-a in CD3+ cells or CD49b+ cells was detected by flow cytometry.3. The allogeneic pregnant models were established as above, and pregnant mice were obtained in NOD group and BALB/c group respectively. They were further divided into spleen group and placenta group, which were further divided into groups of poly (I:C), R837, both of poly (I:C) and R837 (the group of combined stimulation), PBS DMSO, respectively. In total, there were 20 groups. Then CD49b+ NK cells were purified under sterile condition from spleen or pooled placentas and decidua basalis at day 12.5 of gestation by MACS. Cells were cultured in poly(I:C) (final concentration,200 ng/ml), R837(100 ng/ml), a combination of poly (I:C) and R837 (final concentrations,200 and 100 ng/ml, respectively) or an equal volume of PBS or DMSO. After 72 h cultivation, the cells were harvested. Standard 51Cr release assay was used to detect NK cell cytotoxicity.4. The allogeneic pregnant models were established as above, and pregnant mices were obtained in NOD group and BALB/c group respectively. They were further divided into SP600125 group, PD98059 group and control group, which were further divided into groups of poly (I:C), R837, both of poly (I:C) and R837(the group of combined stimulation). In total, there were 18 groups. Under sterile conditions, placental CD3+ cells from NOD mice on gestational day 12.5 were seeded onto 24 well plates and cultivated for 24h. Cells were treated with SP600125, an inhibitor of c-Jun N terminal kinase (JNK) MAPK, PD98059, an inhibitor of extracellular signal-regulated kinase (ERK) MAPK or the same volume of DMSO and cultivated for 24h. Cells were then treated with poly (I:C) (50μg/ml), R837(10μg/ml) or a combination of both and cultured for 72h. The expression pattern of TNF-αin CD3+ cell was detected by flow cytometry.Results1. An additive increase in the embryo resorption rate in NOD×C57BL/6 mice and BALB/c×C57BL/6 mice was observed after injection of both poly (I:C) and R837. Both were significantly higher than the corresponding solvent control groups (P<0.01 for both). In comparison, the resorption rate in the poly (I:C) and R837 combination group was significantly higher than that in either the poly (I:C) group or the R837 group (P<0.01 for both). In addition, the resorption rates in NOD groups were considerably higher than the corresponding groups in BALB/c mice (P<0.05 for PBS groups and P<0.01 for DMSO, poly (I:C), R837, and poly (I:C) plus R837 groups). The TNF-α+ and IFN-γ+ percentages in CD45+ cells were markedly increased upon induction with poly (I:C) or R837 (P<0.05 or P<0.01). The percentages were further increased upon treatment with both poly (I:C) and R837 (.P<0.01). In contrast, no such trends were observed in the percentages of IL-4+, TGF-β+, or IL-10+ cells. However, the percentages in some BALB/c groups were significantly higher than the corresponding NOD groups.2. In BALB/c mice, the percentage of CD3+ and CD49b+ cells positive for TNF-α+ was increased significantly upon individual stimulation with poly (I:C) or R837 and was increased even further in response to combined stimulation of these TLR agonists (P<0.01 for all). In NOD mice, the percentage of CD3+ cells positive for TNF-α+ was significantly increased upon stimulation with poly (I:C) or R837 and this percentage was further increased by combination of poly (I:C) and R837 (P<0.01 for all). However, the NOD CD49b+ TNF-α+ cell percentage was not increased when stimulated by either poly (I:C), or R837, or a combination of both (P>0.05 for all). The purity and viability of the purified cells routinely exceeded 97% and 95% respectively, as determined by flow cytometry and propidium iodide staining.3. The cytotoxicity of splenic NK cells and uterine NK (uNK) cells derived from BALB/c mice was significantly increased by single-used agonist as well as by combined stimulation of agonists. The cytotoxicity level of uNK cells was significantly lower than that observed in the cells derived from spleens in BALB/c. In contrast, there was lower cytotoxicity in NOD splenic NK cells and uNK cells. The cytotoxicity was slightly increased upon single agonist stimulation and increased further upon combined stimulation in NOD splenic NK cells, and was not increased in response to stimulation of either single or combined agonists in NOD uNK cells.4. The increase in the proportion of CD3+TNF-α+ cells by poly (I:C), R837 or a combination of both poly (I:C) and R837 was partially abrogated by SP600125 and almost completely abrogated by PD98059. This trend was observed in both BALB/c and NOD mice (P<0.01 or P<0.05). The percentage level of CD3+ TNF-α+ cells induced by both poly (I:C) and R837 was still significantly higher than those of cells induced by either poly (I:C) or R837 alone (P<0.05 or P<0.01).ConclusionsThe TLR3 and TLR7 signals are transmitted via both Thl-type T cells and NK1-type NK cells, which express more cytokines (such as TNF-αand IFN-γ), cause an effective immune response, and induce an additive increase in the embryo resorption rate. However, in NOD mice, the TLRs signals are mainly transmitted via Thl-type T cells, rather than NK cells. Furthermore, the ERK MAPK pathway may be critical in TLR3 and TLR7 signaling. Combined TLR3 and TLR7/8 agonist stimulation appeared to have an additive effect in triggering Th1-type T cells.
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