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IL-10 regulate decidual Tregs apoptosis contributing to the abnormal pregnancy with Toxoplasma gondii infection
Microbial Pathogenesis 89 (2015) 210e216
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
IL-10 regulate decidual Tregs apoptosis contributing to the abnormal pregnancy with Toxoplasma gondii infection Kaixue Lao c, 1, Mingdong Zhao b, 1, Zhidan Li a, 1, Xianbing Liu a, Haixia Zhang a, Yuzhu Jiang a, Yanlin Wang c, **, Xuemei Hu a, * a b c
Department of Immunology, Binzhou Medical University, Yantai, Shandong, 264003, PR China Department of Radiology, Affiliated Hospital of Binzhou Medical University, Binzhou, Shandong, 256603, PR China Department of Gynecology and Obstetrics, Binzhou Affiliated Hospital of Binzhou Medical University, Binzhou, Shandong, 256603, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 July 2015 Received in revised form 29 September 2015 Accepted 4 October 2015 Available online 2 November 2015
This study aims to investigate whether IL-10 regulate decidual Treg cells apoptosis to reverse the abnormal pregnancy outcomes with Toxoplasma gondii (T. gondii) infection. Recombinant mouse IL-10 (rIL-10) treatment and IL-10 deficiency (IL-10/) abnormal pregnancy animal models with T. gondii infection were established. Apoptosis related molecules cleaved Caspase-3 and Caspase-8 in decidual Treg cells were examined using flow cytometry. The levels of cleaved Caspase-3 and Caspase-8 in decidual Treg cells were up-regulated with T. gondii infection. Compared to infected group, the expressions of cleaved Caspase-3 and Caspase-8 in decidual Treg cells were down-regulated in rIL-10treated group, while up-regulated in infected IL-10/ group. In addition, pregnant outcomes were improved in rIL-10-treated group, while worse in IL-10/ group compared to infected group. These findings revealed that IL-10 reduced the decidual Treg cells apoptosis contributing to improving adverse pregnant outcomes following T. gondii infection. © 2015 Elsevier Ltd. All rights reserved.
Keywords: IL-10 Regulatory T cells Apoptosis Abnormal pregnancy outcomes Toxoplasma gondii
1. Introduction Toxoplasma gondii (T. gondii), an opportunistic parasite, can infect an extensive range of warm-blooded animals, including humans [1]. Acute toxoplasmosis, especially during pregnancy, may give rise to the fetus infection, with a result of severe developmental disorders, or even worse, like fetal death [2e4]. Nevertheless, the mechanisms underlying T. gondii infection caused adverse pregnancy outcomes remain poorly understand. Also effective prevent and treatment methods to cure toxoplasmosis need to be established. IL-10, known as a typical Th2 cytokine, was increased in normal pregnancy with a beneficial effect [5]. Some result showed that an 80% increase in resorption rate of CBA/J DBA/2 pregnancies was observed after anti-IL-10 treatment [6]. And IL-10/ females
* Corresponding author. Department of Immunology, Binzhou Medical University, No. 346 Guan-Hai Road, Lai-shan, Yantai, Shandong, 264003, PR China. ** Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (X. Hu). 1 Kaixue Lao, Mingdong Zhao and Zhidan Li contributed equally to this work. http://dx.doi.org/10.1016/j.micpath.2015.10.002 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
mated with IL-10/ males had 27% more viable fetuses than pregnancy wile-type mice on day 18 of gestation [7]. These results emphasized the importance of IL-10 in maintaining normal pregnancy outcomes. In our previous studies, we found that IL-10 was decreased in placenta, accompanied by adverse pregnancy outcomes with T. gondii infection. While supplement of rIL-10 could reverse the abnormal pregnancy outcomes induced by T. gondii infection [8]. These results demonstrated that IL-10 involved in abnormal pregnancy outcomes caused by T. gondii infection. However, the mechanism of IL-10 in improving abnormal pregnancy outcomes caused by T. gondii infection remains to need further explored. Many evidences suggest that regulatory T (Treg) cells, act as a critical role of immune suppression, allow the maternal immune system to tolerate the fetal allograft [9e14]. And lower frequencies of Treg cells were observed in adverse pregnancy [15,16]. The depletion of Treg cells could lead to abortion owing to immunologic rejection [9]. These results declared that the enough number of Treg cells was needed in keeping normal pregnancy. In our previous research, we have found that T. gondii infection could induce Treg cells apoptosis and then result in abnormal pregnancy outcomes, while adoptive transfer of Treg cells could improve adverse
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pregnancy [17]. Our results suggested that the decreased number of Treg cells resulted from apoptosis may be an important mechanism in serious pregnancy outcomes induced by T. gondii infection. However the mechanism of Treg cells apoptosis lead to abnormal pregnancy outcomes with T. gondii infection was still unclear. Some research reported that IL-10 could modulate the expressions of different apoptosis molecules to prevent cells apoptosis [18e23]. Caspase-8, as a key signaling molecule in extrinsic pathway, could transmit the death signal generated by the apoptotic stimulus to caspase-3 via direct cleavage and activation of Caspase-3, with a result of rapidly dismantling the cell [24e26]. Interestingly, IL-10 could prevent Treg cells from apoptosis via extrinsic pathway [26]. Yet, IL-10 decrease the apoptosis of Treg cells to improve the abnormal pregnant outcomes caused by T. gondii infection was need further confirmed. In this study, we focus on declaring that IL-10 reducing the apoptosis of decidual Treg cells was the reason of improving the abnormal pregnancy outcomes induced by T. gondii infection. 2. Materials and methods 2.1. Animals C57BL/6 wild type (WT) mice were purchased from Beijing Weitong Lihua Experimental Animal Technical Co., Ltd. IL-10 deficiency (IL-10/) C57BL/6 mice were obtained from Model Animal Research Center of Nanjing University. All mice were used at 6e8week old (females) and 8e10-week old (males). Mice were housed five per cage at 25 C with air humidity of 50e60% and a 12 h light 12 h dark cycle, with abundant sterilized water and food (purchased from Jiangsu Biological Engineering Co. Ltd.) supported. After being cohabited with males at a ratio of 2:1, females next day with a vaginal plug, considered as gestational day (gd) 0, were segregated and randomized into four groups: the uninfected (WT) group, the infected group (IG), the infected mice with recombinant mouse IL-10 (rIL-10) treated group (rIL-10treated group), the infected IL-10/ group (IL-10/ group). At least 10 mice were tested in each group.
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2.4. Maintenance of T. gondii RH trophozoites T. gondii tachyzoite RH strain was kept in our laboratory. We infected each Kunming mouse with 2 106 T. gondii RH trophozoites via an intraperitoneal (i.p.) injection. T. gondii tachyzoite were removed 54e72 h later under sterile condition and injected into another healthy mouse at an appropriate concentration. All the manipulations were obeyed the aseptic principles. 2.5. Establishment of infection and treatment model of pregnancy Pregnancy mice in infected group were inoculated i.p. with 400 tachyzoites in 200 ml sterile phosphate buffer solution (PBS) on gd 7, and 200 ml sterile PBS was inoculated in WT group at the same time. rIL-10 was injected via the tail vein at a dose of 1 mg/mouse on gd 6 and gd 8 respectively in rIL-10-treated group. 2.6. Pregnant outcomes Both mice were sacrificed at 7 days post-infection (dpi). We took the total numbers of implantations and resorption sites into account. The resorption sites were identified by their size, their necrotic and hemorrhagic appearance of embryos and placenta. The abortion percentage was calculated as the ratio of resorption sites to total implantation sites. 2.7. Cell preparation and flow cytometry
All experiments were performed in terms of the ethical standards formulated by the Institutional Animal Experimental Ethics Committee of Binzhou Medical University.
Animals were euthanized on gd 14. Single-cell suspensions were separately prepared from placenta and uterine by scissoring and digesting the tissues, then filtered through a 48 mm sterile nets and lysed the erythrocytes. Cells were stained with Percp-cy5.5conjugated anti-CD4 mAb (BD Pharmingen, USA) in the dark for 30 min at 4 C. For intracellular staining, cells were fixated and permeabilized in 1 Fix/Perm buffer (eBioscience, USA) for 30 min according to the manufacturer's instructions, and then intracellular molecules were stained with APC-conjugated anti-Foxp3 mAb (eBioscience, USA), PE-conjugated anti-cleaved Caspase-3 mAb (BD Pharmingen, USA) and PE-conjugated anti-cleaved Caspase-8 mAb (Cell Signaling Technology, USA) in the dark for 30 min at 4 C, respectively. All cells were washed 3 times with PBS and analyzed by BD FACSAria Flow cytometry (Becton Dickinson, USA) and BD FACSDiva software (Becton Dickinson).
2.3. Genotyping the IL-10/ mice
2.8. Data analysis and statistics
IL-10 deficiency (IL-10/) C57BL/6 mice used in this experiment were obtained from Model Animal Research Center of Nanjing University. And the genotype of IL-10/ mice was confirmed by extracting genomic DNA from tail tissue. cDNA was synthesized using the Reverse Transcribe kit (Fermentas, Canada). After initial denaturation (5 min at 95 C), 40 amplification cycles were performed (denaturation for 30 s at 95 C, annealing for 30 s at 60 C, and extension for 30 s at 72 C), followed by a final extension for 5 min at 72 C. The PCR products were separated by electrophoresis in a 1.5% agarose gel with a 100-bp DNA marker (Transgene, France) for size estimation. The gel was stained with GelStain (Transgene, china) to visualize the DNA. Primers used for PCR amplification were oIMR7376 (mutant reverse) 50 -CCA CAC GCG TCA CCT TAA TA30 ; oIMR9573 (common forward) 50 -CTT GCA CTA CCA AAG CCA CA30 ; and oIMR9574 (wild-type reverse) 50 -GTT ATT GTC TTC CCG GCT GT-30 . The expected sizes of the PCR products were 312 bp (mutant), 312 and 137 bp (heterozygote), and 137 bp (wild-type) [27].
Data were presented as means ± SEM. Data were analyzed by the SPSS 17.0 statistical software package. Unpaired t-tests were used to compare two independent groups. An associated probability (p values) of <0.05 or <0.01 was considered significant or very significant respectively.
2.2. Ethics statement
3. Results 3.1. rIL-10 treatment model of adverse pregnancy outcomes caused by T. gondii infection was established successfully Compared to infected group (Fig. 1AeC), the mice mental state (Fig. 1D), the conditions of placenta hemorrhage and necrosis (Fig. 1E and F) were all improved in rIL-10-treated group. Additionally, the abortion rate decreased (Fig.1G), the mean weight of placenta and fetal were all increased (Fig. 1H and I) in rIL-10-treated group. So the rIL-10 treatment animal model was established successfully accompanying with adverse pregnancy outcomes caused by T. gondii infection.
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Fig. 1. The effect of rIL-10 treatment on pregnancy outcomes of mice exposed to T. gondii. Hemorrhagic, necrotic, and resorbed fetal phenotype were markedly improved in rIL-10treated group than that of infected group (AeF). The rate of fetal loss was significantly less, and the mean weight of placenta and fetus were higher in rIL-10-treated group compared with those from infected group (GeI, *p < 0.05).
3.2. IL-10/ mice genotyping According to materials and methods, homozygote, heterozygote, and wild-type animals should produce bands 312 bp, 312 bp and 137 bp, and 137 bp, respectively, in size. In the present study, a 312bp band alone was observed (Fig. 2). The IL-10/ mice in this experiment were homozygotic.
3.3. Worse pregnancy outcomes were induced by T. gondii infection in IL-10/ mice In order to further confirm the role of IL-10 in abnormal pregnancy outcomes caused by T. gondii infection, IL-10/ mice were
used. The mice exhibited more serious mental and behavioral disorders, such as flagging, shambling, extrados and moving less in IL10/ group (Fig. 3D) than in infected group (Fig. 3A). More necrosis and resorbed fetuses phenotype were observed in IL-10/ group (Fig. 3E and F) compared to infected group (Fig. 3B and C). In addition, the resorption rate was significantly higher, and the mean weight of placenta and fetal were less in IL-10/ group in contrast to those from infected group (Fig. 3GeI, *p < 0.05). 3.4. T. gondii infection caused more apoptosis of decidual Treg cells via up-regulating cleaved Caspase-3 and Caspase-8 expressions Levels of cleaved Caspase-3 and Caspase-8 in decidual Treg cells were detected by FACS analysis. Results showed that cleaved Caspase-3 expression in decidual Treg cells was obviously increased in infected group compared to uninfected WT group (Fig. 4A, **p < 0.01). And as shown in Fig. 4B, the level of cleaved Caspase-8 in decidual Treg cells was also significantly increased after T. gondii infection (**p < 0.01). 3.5. rIL-10 treatment reduced the levels of cleaved Caspase-3 and Caspase-8 in decidual Treg cells
Fig. 2. Genotyping of IL-10/ mice. Wild-type control mice (C57BL/6) produced a 137bp band alone and heterozygote generated both 312 and 137 bp, while a 312-bp alone was observed for the homozygote mouse.
To explore whether IL-10 has effect on regulating the apoptosis of decidual Treg cells, cleaved Caspase-3 and Caspase-8 expressions were detected from mice with rIL-10 treatment. As shown in Fig. 5A and B, the levels of cleaved Caspase-3 and Caspase-8 in decidual Treg cells were lower in rIL-10-treated group than that of infected group (*p < 0.05).
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Fig. 3. Fetal resorption in infected group and IL-10/ group in response to T. gondii. More serious status and an increase in placental hemorrhage were detected in IL-10/ group than in infected group (AeF). The resorption rate of fetal loss was markedly higher in IL-10/ group in contrast to that of infected group (G, *p < 0.05). The mean weight of placenta and fetus were less in IL-10/ group compared to those from infected group (HeI, *p < 0.05).
Fig. 4. The effect of T. gondii infection on cleaved Caspase-3 and Caspase-8 expressions in decidual Treg cells. The levels of cleaved Caspase-3 and Caspase-8 in decidual Treg cells were markedly up-regulated in T. gondii infected group than uninfected WT group (A and B, **p < 0.01).
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Fig. 5. Effect of rIL-10 treatment on expressions of cleaved Caspase-3 and Caspase-8 in decidual Treg cells in response to T. gondii. The levels of cleaved Caspase-3 and Caspase-8 in Treg cells were lower in IL-10-treated group than that of infected group (A and B, *p < 0.05).
3.6. Cleaved Caspase-3 and Caspase-8 expressions in decidual Treg cells were up-regulated in IL-10/ mice The expressions of cleaved Caspase-3 and Caspase-8 in decidual Treg cells of the IL-10/ mice were analyzed to further validate the effect of IL-10 on decidual Treg cells apoptosis. Results showed that higher cleaved Caspase-3 and Caspase-8 expressions were detected in IL-10/ group compared with that of infected group (Fig. 6A and B, *p < 0.05, **p < 0.01).
4. Discussion T. gondii infection could induce maternal immune deregulation and adverse consequences such as resorption, fetal loss during gestation, especially in the first trimester pregnancy [28]. IL-10 was a pregnancy-compatible cytokine [29] and in the absence of IL-10, mice resulted in fetal resorption in response to very low doses of LPS which unaffected the pregnancy in wild-type mice [30]. IL-10/ mice succumbed within the first 2 wk of the T. gondii infection
Fig. 6. The levels of cleaved Caspase-3 and Caspase-8 in decidual Treg cells in infected group and IL-10/ group with T. gondii infection. Higher cleaved Caspase-3 and Caspase-8 expressions in decidual Treg cells were detected respectively in IL-10/ group compared to that of infected group (A and B, *p < 0.05, **p < 0.01).
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suggested IL-10 was required for resisting to T. gondii [31,32]. In our previous study, result showed that IL-10 was decreased in placenta, and supplement of IL-10 could improve abnormal pregnancy outcomes following T. gondii infection [8]. In present study, lower resorption ratio, higher mean weight of placenta and fetus were observed in rIL-10-treated group compared to infected group. These results suggested that rIL-10 treatment animal model was established successfully accompanying with abnormal pregnancy with T. gondii Infection. IL-10/ mice were used to further confirm the role of IL-10 in improving adverse pregnancy outcomes caused by T. gondii infection. Worse pregnancy outcomes were observed in IL-10/ group than infected group, which further demonstrated that lack of IL-10 may be one of the most important factors in abnormal pregnancy outcomes induced by T. gondii infection. Moreover, our previous studies have declared that adding rIL-10 could improve adverse pregnancy with T. gondii infection through regulating the imbalance of Th1/Th2 and reducing trophoblasts apoptosis induced by T. gondii infection [8,18]. However, other underlining mechanism of IL-10 in improving abnormal pregnancy outcomes caused by T. gondii infection was still to be further investigated. Sufficient quantity of the Treg cells is necessary to the maintaining of normal pregnancy [33,34]. While apoptosis of Treg cells at the maternal-fetal interface was increased with T. gondii infection [17,35]. One study showed that T. gondii infection could cause Treg cells apoptosis by intrinsic apoptosis pathway [35]. In this study, we observed cleaved Caspase-3 and Caspase-8 expressions were increased significantly with T. gondii infection, suggesting that T. gondii infection could trigger the decidual Treg cells apoptosis via extrinsic apoptosis pathway as well, leading to adverse pregnancy outcomes. Some results showed that IL-10 have regulation functions to apoptosis of Treg cells via down-regulating cleaved Caspase-3 and Caspase-8 expressions [26]. However, whether the IL-10 could regulate the apoptosis of decidual Treg cells induced by T. gondii infection through extrinsic pathway is still unknown. In this study, the levels of cleaved Caspase-3 and Caspase-8 were lower in rIL-10treated group while higher in IL-10/ group than in infected group. These results revealed that IL-10 could obviously decrease the apoptosis of decidual Treg cells induced by T. gondii infection through extrinsic apoptosis pathway, and thus improve the abnormal pregnancy outcomes with T. gondii infection. All these results revealed that IL-10 may play an immunoprotective role in improving abnormal pregnancy outcomes induced by T. gondii infection through reducing the decidual Treg cells apoptosis. It may provide a new insight into the mechanism of IL-10 in improving the adverse pregnancy outcomes caused by T. gondii infection. Conflict of interest All authors approved submission of this manuscript to Microbial Pathogenesis and have no conflicts of interest. Acknowledgments This study was supported in part by funds from the National Natural Science Foundation of China 81171591, 81273243 and 81201307, Natural Science Foundation of Shandong Province ZR2013HQ023, ZR2014HP010 and Taishan Scholar Foundation. References [1] L.C. Gavrilescu, E.Y. Denkers, IFN-gamma overproduction and high level apoptosis are associated with high but not low virulence Toxoplasma gondii
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Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
IL-10 regulate decidual Tregs apoptosis contributing to the abnormal pregnancy with Toxoplasma gondii infection Kaixue Lao c, 1, Mingdong Zhao b, 1, Zhidan Li a, 1, Xianbing Liu a, Haixia Zhang a, Yuzhu Jiang a, Yanlin Wang c, **, Xuemei Hu a, * a b c
Department of Immunology, Binzhou Medical University, Yantai, Shandong, 264003, PR China Department of Radiology, Affiliated Hospital of Binzhou Medical University, Binzhou, Shandong, 256603, PR China Department of Gynecology and Obstetrics, Binzhou Affiliated Hospital of Binzhou Medical University, Binzhou, Shandong, 256603, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 July 2015 Received in revised form 29 September 2015 Accepted 4 October 2015 Available online 2 November 2015
This study aims to investigate whether IL-10 regulate decidual Treg cells apoptosis to reverse the abnormal pregnancy outcomes with Toxoplasma gondii (T. gondii) infection. Recombinant mouse IL-10 (rIL-10) treatment and IL-10 deficiency (IL-10/) abnormal pregnancy animal models with T. gondii infection were established. Apoptosis related molecules cleaved Caspase-3 and Caspase-8 in decidual Treg cells were examined using flow cytometry. The levels of cleaved Caspase-3 and Caspase-8 in decidual Treg cells were up-regulated with T. gondii infection. Compared to infected group, the expressions of cleaved Caspase-3 and Caspase-8 in decidual Treg cells were down-regulated in rIL-10treated group, while up-regulated in infected IL-10/ group. In addition, pregnant outcomes were improved in rIL-10-treated group, while worse in IL-10/ group compared to infected group. These findings revealed that IL-10 reduced the decidual Treg cells apoptosis contributing to improving adverse pregnant outcomes following T. gondii infection. © 2015 Elsevier Ltd. All rights reserved.
Keywords: IL-10 Regulatory T cells Apoptosis Abnormal pregnancy outcomes Toxoplasma gondii
1. Introduction Toxoplasma gondii (T. gondii), an opportunistic parasite, can infect an extensive range of warm-blooded animals, including humans [1]. Acute toxoplasmosis, especially during pregnancy, may give rise to the fetus infection, with a result of severe developmental disorders, or even worse, like fetal death [2e4]. Nevertheless, the mechanisms underlying T. gondii infection caused adverse pregnancy outcomes remain poorly understand. Also effective prevent and treatment methods to cure toxoplasmosis need to be established. IL-10, known as a typical Th2 cytokine, was increased in normal pregnancy with a beneficial effect [5]. Some result showed that an 80% increase in resorption rate of CBA/J DBA/2 pregnancies was observed after anti-IL-10 treatment [6]. And IL-10/ females
* Corresponding author. Department of Immunology, Binzhou Medical University, No. 346 Guan-Hai Road, Lai-shan, Yantai, Shandong, 264003, PR China. ** Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (X. Hu). 1 Kaixue Lao, Mingdong Zhao and Zhidan Li contributed equally to this work. http://dx.doi.org/10.1016/j.micpath.2015.10.002 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
mated with IL-10/ males had 27% more viable fetuses than pregnancy wile-type mice on day 18 of gestation [7]. These results emphasized the importance of IL-10 in maintaining normal pregnancy outcomes. In our previous studies, we found that IL-10 was decreased in placenta, accompanied by adverse pregnancy outcomes with T. gondii infection. While supplement of rIL-10 could reverse the abnormal pregnancy outcomes induced by T. gondii infection [8]. These results demonstrated that IL-10 involved in abnormal pregnancy outcomes caused by T. gondii infection. However, the mechanism of IL-10 in improving abnormal pregnancy outcomes caused by T. gondii infection remains to need further explored. Many evidences suggest that regulatory T (Treg) cells, act as a critical role of immune suppression, allow the maternal immune system to tolerate the fetal allograft [9e14]. And lower frequencies of Treg cells were observed in adverse pregnancy [15,16]. The depletion of Treg cells could lead to abortion owing to immunologic rejection [9]. These results declared that the enough number of Treg cells was needed in keeping normal pregnancy. In our previous research, we have found that T. gondii infection could induce Treg cells apoptosis and then result in abnormal pregnancy outcomes, while adoptive transfer of Treg cells could improve adverse
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pregnancy [17]. Our results suggested that the decreased number of Treg cells resulted from apoptosis may be an important mechanism in serious pregnancy outcomes induced by T. gondii infection. However the mechanism of Treg cells apoptosis lead to abnormal pregnancy outcomes with T. gondii infection was still unclear. Some research reported that IL-10 could modulate the expressions of different apoptosis molecules to prevent cells apoptosis [18e23]. Caspase-8, as a key signaling molecule in extrinsic pathway, could transmit the death signal generated by the apoptotic stimulus to caspase-3 via direct cleavage and activation of Caspase-3, with a result of rapidly dismantling the cell [24e26]. Interestingly, IL-10 could prevent Treg cells from apoptosis via extrinsic pathway [26]. Yet, IL-10 decrease the apoptosis of Treg cells to improve the abnormal pregnant outcomes caused by T. gondii infection was need further confirmed. In this study, we focus on declaring that IL-10 reducing the apoptosis of decidual Treg cells was the reason of improving the abnormal pregnancy outcomes induced by T. gondii infection. 2. Materials and methods 2.1. Animals C57BL/6 wild type (WT) mice were purchased from Beijing Weitong Lihua Experimental Animal Technical Co., Ltd. IL-10 deficiency (IL-10/) C57BL/6 mice were obtained from Model Animal Research Center of Nanjing University. All mice were used at 6e8week old (females) and 8e10-week old (males). Mice were housed five per cage at 25 C with air humidity of 50e60% and a 12 h light 12 h dark cycle, with abundant sterilized water and food (purchased from Jiangsu Biological Engineering Co. Ltd.) supported. After being cohabited with males at a ratio of 2:1, females next day with a vaginal plug, considered as gestational day (gd) 0, were segregated and randomized into four groups: the uninfected (WT) group, the infected group (IG), the infected mice with recombinant mouse IL-10 (rIL-10) treated group (rIL-10treated group), the infected IL-10/ group (IL-10/ group). At least 10 mice were tested in each group.
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2.4. Maintenance of T. gondii RH trophozoites T. gondii tachyzoite RH strain was kept in our laboratory. We infected each Kunming mouse with 2 106 T. gondii RH trophozoites via an intraperitoneal (i.p.) injection. T. gondii tachyzoite were removed 54e72 h later under sterile condition and injected into another healthy mouse at an appropriate concentration. All the manipulations were obeyed the aseptic principles. 2.5. Establishment of infection and treatment model of pregnancy Pregnancy mice in infected group were inoculated i.p. with 400 tachyzoites in 200 ml sterile phosphate buffer solution (PBS) on gd 7, and 200 ml sterile PBS was inoculated in WT group at the same time. rIL-10 was injected via the tail vein at a dose of 1 mg/mouse on gd 6 and gd 8 respectively in rIL-10-treated group. 2.6. Pregnant outcomes Both mice were sacrificed at 7 days post-infection (dpi). We took the total numbers of implantations and resorption sites into account. The resorption sites were identified by their size, their necrotic and hemorrhagic appearance of embryos and placenta. The abortion percentage was calculated as the ratio of resorption sites to total implantation sites. 2.7. Cell preparation and flow cytometry
All experiments were performed in terms of the ethical standards formulated by the Institutional Animal Experimental Ethics Committee of Binzhou Medical University.
Animals were euthanized on gd 14. Single-cell suspensions were separately prepared from placenta and uterine by scissoring and digesting the tissues, then filtered through a 48 mm sterile nets and lysed the erythrocytes. Cells were stained with Percp-cy5.5conjugated anti-CD4 mAb (BD Pharmingen, USA) in the dark for 30 min at 4 C. For intracellular staining, cells were fixated and permeabilized in 1 Fix/Perm buffer (eBioscience, USA) for 30 min according to the manufacturer's instructions, and then intracellular molecules were stained with APC-conjugated anti-Foxp3 mAb (eBioscience, USA), PE-conjugated anti-cleaved Caspase-3 mAb (BD Pharmingen, USA) and PE-conjugated anti-cleaved Caspase-8 mAb (Cell Signaling Technology, USA) in the dark for 30 min at 4 C, respectively. All cells were washed 3 times with PBS and analyzed by BD FACSAria Flow cytometry (Becton Dickinson, USA) and BD FACSDiva software (Becton Dickinson).
2.3. Genotyping the IL-10/ mice
2.8. Data analysis and statistics
IL-10 deficiency (IL-10/) C57BL/6 mice used in this experiment were obtained from Model Animal Research Center of Nanjing University. And the genotype of IL-10/ mice was confirmed by extracting genomic DNA from tail tissue. cDNA was synthesized using the Reverse Transcribe kit (Fermentas, Canada). After initial denaturation (5 min at 95 C), 40 amplification cycles were performed (denaturation for 30 s at 95 C, annealing for 30 s at 60 C, and extension for 30 s at 72 C), followed by a final extension for 5 min at 72 C. The PCR products were separated by electrophoresis in a 1.5% agarose gel with a 100-bp DNA marker (Transgene, France) for size estimation. The gel was stained with GelStain (Transgene, china) to visualize the DNA. Primers used for PCR amplification were oIMR7376 (mutant reverse) 50 -CCA CAC GCG TCA CCT TAA TA30 ; oIMR9573 (common forward) 50 -CTT GCA CTA CCA AAG CCA CA30 ; and oIMR9574 (wild-type reverse) 50 -GTT ATT GTC TTC CCG GCT GT-30 . The expected sizes of the PCR products were 312 bp (mutant), 312 and 137 bp (heterozygote), and 137 bp (wild-type) [27].
Data were presented as means ± SEM. Data were analyzed by the SPSS 17.0 statistical software package. Unpaired t-tests were used to compare two independent groups. An associated probability (p values) of <0.05 or <0.01 was considered significant or very significant respectively.
2.2. Ethics statement
3. Results 3.1. rIL-10 treatment model of adverse pregnancy outcomes caused by T. gondii infection was established successfully Compared to infected group (Fig. 1AeC), the mice mental state (Fig. 1D), the conditions of placenta hemorrhage and necrosis (Fig. 1E and F) were all improved in rIL-10-treated group. Additionally, the abortion rate decreased (Fig.1G), the mean weight of placenta and fetal were all increased (Fig. 1H and I) in rIL-10-treated group. So the rIL-10 treatment animal model was established successfully accompanying with adverse pregnancy outcomes caused by T. gondii infection.
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Fig. 1. The effect of rIL-10 treatment on pregnancy outcomes of mice exposed to T. gondii. Hemorrhagic, necrotic, and resorbed fetal phenotype were markedly improved in rIL-10treated group than that of infected group (AeF). The rate of fetal loss was significantly less, and the mean weight of placenta and fetus were higher in rIL-10-treated group compared with those from infected group (GeI, *p < 0.05).
3.2. IL-10/ mice genotyping According to materials and methods, homozygote, heterozygote, and wild-type animals should produce bands 312 bp, 312 bp and 137 bp, and 137 bp, respectively, in size. In the present study, a 312bp band alone was observed (Fig. 2). The IL-10/ mice in this experiment were homozygotic.
3.3. Worse pregnancy outcomes were induced by T. gondii infection in IL-10/ mice In order to further confirm the role of IL-10 in abnormal pregnancy outcomes caused by T. gondii infection, IL-10/ mice were
used. The mice exhibited more serious mental and behavioral disorders, such as flagging, shambling, extrados and moving less in IL10/ group (Fig. 3D) than in infected group (Fig. 3A). More necrosis and resorbed fetuses phenotype were observed in IL-10/ group (Fig. 3E and F) compared to infected group (Fig. 3B and C). In addition, the resorption rate was significantly higher, and the mean weight of placenta and fetal were less in IL-10/ group in contrast to those from infected group (Fig. 3GeI, *p < 0.05). 3.4. T. gondii infection caused more apoptosis of decidual Treg cells via up-regulating cleaved Caspase-3 and Caspase-8 expressions Levels of cleaved Caspase-3 and Caspase-8 in decidual Treg cells were detected by FACS analysis. Results showed that cleaved Caspase-3 expression in decidual Treg cells was obviously increased in infected group compared to uninfected WT group (Fig. 4A, **p < 0.01). And as shown in Fig. 4B, the level of cleaved Caspase-8 in decidual Treg cells was also significantly increased after T. gondii infection (**p < 0.01). 3.5. rIL-10 treatment reduced the levels of cleaved Caspase-3 and Caspase-8 in decidual Treg cells
Fig. 2. Genotyping of IL-10/ mice. Wild-type control mice (C57BL/6) produced a 137bp band alone and heterozygote generated both 312 and 137 bp, while a 312-bp alone was observed for the homozygote mouse.
To explore whether IL-10 has effect on regulating the apoptosis of decidual Treg cells, cleaved Caspase-3 and Caspase-8 expressions were detected from mice with rIL-10 treatment. As shown in Fig. 5A and B, the levels of cleaved Caspase-3 and Caspase-8 in decidual Treg cells were lower in rIL-10-treated group than that of infected group (*p < 0.05).
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Fig. 3. Fetal resorption in infected group and IL-10/ group in response to T. gondii. More serious status and an increase in placental hemorrhage were detected in IL-10/ group than in infected group (AeF). The resorption rate of fetal loss was markedly higher in IL-10/ group in contrast to that of infected group (G, *p < 0.05). The mean weight of placenta and fetus were less in IL-10/ group compared to those from infected group (HeI, *p < 0.05).
Fig. 4. The effect of T. gondii infection on cleaved Caspase-3 and Caspase-8 expressions in decidual Treg cells. The levels of cleaved Caspase-3 and Caspase-8 in decidual Treg cells were markedly up-regulated in T. gondii infected group than uninfected WT group (A and B, **p < 0.01).
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Fig. 5. Effect of rIL-10 treatment on expressions of cleaved Caspase-3 and Caspase-8 in decidual Treg cells in response to T. gondii. The levels of cleaved Caspase-3 and Caspase-8 in Treg cells were lower in IL-10-treated group than that of infected group (A and B, *p < 0.05).
3.6. Cleaved Caspase-3 and Caspase-8 expressions in decidual Treg cells were up-regulated in IL-10/ mice The expressions of cleaved Caspase-3 and Caspase-8 in decidual Treg cells of the IL-10/ mice were analyzed to further validate the effect of IL-10 on decidual Treg cells apoptosis. Results showed that higher cleaved Caspase-3 and Caspase-8 expressions were detected in IL-10/ group compared with that of infected group (Fig. 6A and B, *p < 0.05, **p < 0.01).
4. Discussion T. gondii infection could induce maternal immune deregulation and adverse consequences such as resorption, fetal loss during gestation, especially in the first trimester pregnancy [28]. IL-10 was a pregnancy-compatible cytokine [29] and in the absence of IL-10, mice resulted in fetal resorption in response to very low doses of LPS which unaffected the pregnancy in wild-type mice [30]. IL-10/ mice succumbed within the first 2 wk of the T. gondii infection
Fig. 6. The levels of cleaved Caspase-3 and Caspase-8 in decidual Treg cells in infected group and IL-10/ group with T. gondii infection. Higher cleaved Caspase-3 and Caspase-8 expressions in decidual Treg cells were detected respectively in IL-10/ group compared to that of infected group (A and B, *p < 0.05, **p < 0.01).
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suggested IL-10 was required for resisting to T. gondii [31,32]. In our previous study, result showed that IL-10 was decreased in placenta, and supplement of IL-10 could improve abnormal pregnancy outcomes following T. gondii infection [8]. In present study, lower resorption ratio, higher mean weight of placenta and fetus were observed in rIL-10-treated group compared to infected group. These results suggested that rIL-10 treatment animal model was established successfully accompanying with abnormal pregnancy with T. gondii Infection. IL-10/ mice were used to further confirm the role of IL-10 in improving adverse pregnancy outcomes caused by T. gondii infection. Worse pregnancy outcomes were observed in IL-10/ group than infected group, which further demonstrated that lack of IL-10 may be one of the most important factors in abnormal pregnancy outcomes induced by T. gondii infection. Moreover, our previous studies have declared that adding rIL-10 could improve adverse pregnancy with T. gondii infection through regulating the imbalance of Th1/Th2 and reducing trophoblasts apoptosis induced by T. gondii infection [8,18]. However, other underlining mechanism of IL-10 in improving abnormal pregnancy outcomes caused by T. gondii infection was still to be further investigated. Sufficient quantity of the Treg cells is necessary to the maintaining of normal pregnancy [33,34]. While apoptosis of Treg cells at the maternal-fetal interface was increased with T. gondii infection [17,35]. One study showed that T. gondii infection could cause Treg cells apoptosis by intrinsic apoptosis pathway [35]. In this study, we observed cleaved Caspase-3 and Caspase-8 expressions were increased significantly with T. gondii infection, suggesting that T. gondii infection could trigger the decidual Treg cells apoptosis via extrinsic apoptosis pathway as well, leading to adverse pregnancy outcomes. Some results showed that IL-10 have regulation functions to apoptosis of Treg cells via down-regulating cleaved Caspase-3 and Caspase-8 expressions [26]. However, whether the IL-10 could regulate the apoptosis of decidual Treg cells induced by T. gondii infection through extrinsic pathway is still unknown. In this study, the levels of cleaved Caspase-3 and Caspase-8 were lower in rIL-10treated group while higher in IL-10/ group than in infected group. These results revealed that IL-10 could obviously decrease the apoptosis of decidual Treg cells induced by T. gondii infection through extrinsic apoptosis pathway, and thus improve the abnormal pregnancy outcomes with T. gondii infection. All these results revealed that IL-10 may play an immunoprotective role in improving abnormal pregnancy outcomes induced by T. gondii infection through reducing the decidual Treg cells apoptosis. It may provide a new insight into the mechanism of IL-10 in improving the adverse pregnancy outcomes caused by T. gondii infection. Conflict of interest All authors approved submission of this manuscript to Microbial Pathogenesis and have no conflicts of interest. Acknowledgments This study was supported in part by funds from the National Natural Science Foundation of China 81171591, 81273243 and 81201307, Natural Science Foundation of Shandong Province ZR2013HQ023, ZR2014HP010 and Taishan Scholar Foundation. References [1] L.C. Gavrilescu, E.Y. Denkers, IFN-gamma overproduction and high level apoptosis are associated with high but not low virulence Toxoplasma gondii
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