Roles Of Oxidative Stress And Endoplasmic Reticulum Stress In Placental Trophoblast Apoptosis Induced By Toxoplasma Infection

Background Toxoplasma gondii can infect all warm-blooded animals, includingapproximately30%of the human population worldwide. Infection is usuallyasymptomatic, but pregnant women infected with T.gondii may result in abortion,stillbirth, severe mental retardation and retinal or neurologic damage later in life.Although birth defects caused by T. gondii could be attributed to structural damage,endocrine disorders and increased cell apoptosis of placental tissue, but the exactmechanisms and key events underlying congenital toxoplasmosis remain unclear. T.gondii has a fascinating dual involvement in host cell apoptosis. Some previous studiesdemonstrated that T. gondii-infected cells became relatively resistant to some apoptoticstimuli. On the other hand, it has been observed that T. gondii infection can induceapoptosis, as with CD4+splenocytes during acute infection of mice with T. gondii. Highlevels of apoptosis and an increased mortality rate have been proven to be associatedwith T. gondii infection with high-virulence strains. In contrast, in chronic Toxoplasmaencephalitis, only a few apoptotic cells were observed. Therefore, the initiation and thedegree of cell apoptosis may play crucial roles in the pathogenesis and outcomes oftoxoplasmosis, but the exact mechanisms and key events underlying congenitaltoxoplasmosis remain unclear.The endoplasmic reticulum(ER) is the primary intracellular organelle for proper protein synthesis, folding and assembly. The accumulation of unfolded or misfoldedproteins in the lumen of the ER, which induces an adaptive program called the unfoldedprotein response (UPR), leads to ER stress (ERS). Increasing evidence shows that ERSplays a key role in the regulation of apoptosis caused by a variety of toxic insults,including reactive oxygen species (ROS), chemicals and heavy metals. Oxidative stressis described as an imbalance between ROS generation and antioxidant capacity, andsuch stress triggers apoptosis through a variety of signaling pathways, such as ERSresponse and the activation of the ASK1/JNK pathway. These pathological processescan be simultaneously observed in some diseases, such as diabetes, cadmium poisoningand neurodegenerative diseases. Additionally, there is growing evidence that oxidativestress plays an important role in infection-induced apoptosis.Generally, pregnancy is a state of mild oxidative stress arising from increasedplacental mitochondrial activity and ROS production for maternal and fetal metabolism.In the first trimester, establishment of blood flow into the intervillous space results in aburst of oxidative stress. The placenta is subjected to hypoxia and thenhypoxia/reoxygenation; the inability to mount an effective antioxidant defensecontributes to early pregnancy loss. In late gestation, increased oxidative stress isobserved in pregnancies complicated by diabetes, intrauterine growth restriction andpreeclampsia in association with escalated trophoblast apoptosis. Previous studiesrevealed that T. gondii infection can lead to oxidative stress and immune suppression inblood donors. Th2immune bias can be seen in the normal maternal-fetal interface,which is unfavorable to the elimination of pathogens,and enhances susceptibility totoxoplasmosis. Thus, T. gondii infection or the reactivation of latent infection duringpregnancy will increase oxidative stress in the placenta, contribute to cell apoptosis andplacenta damage, and, finally, lead to more serious outcomes than would be seen duringnormal physiological state. The relationship between oxidative stress and cell apoptosisin gestation-related disease has been reported. Previous studies demonstrated that LPS could induce oxidative stress in several tissues, leading to preterm labor in mice. Somestudies discovered that trophoblasts could be productively infected by a virulent strainof T. gondii, and uninfected, but not infected, cells undergo apoptosis, which unveiled T.gondii-induced apoptosis was not due to a direct action of the parasite at thematernofoetal interface.Objective To explore the effect T.gondii infection on the apoptosis level oftrophoblasts in vitro and in vivo; to clarify the exact mechanisms and key eventsunderlying congenital toxoplasmosis and T.gondii-triggered apoptosis; to observe thenegative effect of antioxidants on T.gondii-triggered apoptosis, protective effect onpathological damage to placenta tissues, and potential usage as therapeutic regimen forthe treatment of T. gondii-related diseases.Methods (1) In the present study, we used ICR pregnant mice to set up the mousemodels on pregnant infection of RH tachyzoites. Pregnant mice were randomly dividedinto T. gondii infection, N-acetylcysteine (NAC) pretreatment plus T. gondii infectionand control groups. On GD9and GD10, mice in the NAC pretreatment group wereinfused with NAC (100mg/kg) respectively. On GD8, each mouse in the T.gondiiinfection group and NAC pretreatment group was injected intraperitoneally with200tachyzoites. Mice were killed and placentas were carefully dissected out; their weightswere recorded, and processed into cell suspension, or fixed in Bouins fluid forimmunohistochemical analysis, and snap-frozen in liquid nitrogen for further variousanalyses. We investigated the gene expression profile of placenta tissues from GD12mice in T. gondii infection group with a96-Well RT²Profiler^TM PCR Array containing84key genes related to oxidative stress. MDA, GSH and8-OHdG in placentahomogenates were detected as the manufactures' instructions. Tachyzoites burden inblood samples and placenta tissues, and oxidative stress-associated molecules were detected by real-time PCR.(2) For experimental infection, primary trophoblasts werecultured and identified by immunostaining for cytokeratin-7and vimentin.Experimental infection was performed with a transwell system. Parasites and placentalcells were added to the upper well (1×10⁶:1×10⁶). The placental trophoblasts (1×10⁶) inthe lower wells remained uninfected and were co-cultured with infected cells from theupper well on a12-well plate at37°C for the indicated time intervals. Cell suspensionsfrom placental tissues or primary cultured trophoblasts were harvested, and theapoptosis levels were analyzed by FCM or TUNEL. The mRNA levels of oxidativestress an ERS markers were detected by real-time RT-PCR.(3)Placental tissues weredissected and homogenized in ice-cold lysis buffer. Primary cultured trophoblasts weredirectly treated with100μl lysis buffer. Total cell lysates were subjected to SDS-PAGEand immunoblotting using several Abs, such as GRP78), anti-phospho-JNK,antiphospho-p38, anti-p38MAPK, anti-caspase-12and anti-phospho-ASK1. To observethe negative effect of NAC on trophoblast apoptosis and the activation of pro-apoptosispathways, the placenta tissues and primary cultured trophoblast from NAC pretreatmentwere also analyzed by real-time RT-PCR, FCM and Western blotting.Results (1) The pregnant mice model of T.gondii infection was successfully set up.When injected intraperitoneally with200tachyzoites each mouse on GD8, the miceappeared typical symptoms of T.gondii infection after GD14, but the majority survivedafter GD16.(2) Our studies discovered that twenty-seven genes were at least2-fold upregulated, and9genes were at least2-fold down regulated in T.gondii infection group,compared to the uninfected group, based on the PCR-Array data ofoxidative-response-related gene expression profile in placental tissues. NADPH oxidase1(Nox1) and glutathione peroxidase6(Gpx6) were most significantly increased, about25times as the result of T.gondii gondii infection. The above mentioned genes werealmost recovery to the levels of control groups when subjected mice were pretreated on G9and G10by100mg/kg NAC injected intraperitoneally. By quantitative PCR analysis,T.gondii was not detectable in the placentas of GD10and GD12. A dramatical increaseof parasite load, however, was found in those of GD14. Additionally, our data alsoshowed that the parasiteamia occurred and the GSH level of placentas decreased beforeGD12in T.gondii infection group.The levels of GSH decreased, from9.29±2.26μM/gprot in control group to4.18±1.54μM/gprot in infection group (p<0.01).Severe oxidative stress also resulted in peroxidation of lipids and DNA, presenting withincreased levels of MDA,8-OHdG. The levels of MDA and8-OHdG increased from0.41±0.16μM/gprot and1.77±0.65μg/gprot in control group to0.52±0.23μM/gprotand3.88±0.89μg/gprot in infection group respectively (p<0.05). Usage of NAC couldeffectively alleviate the peroxidation of lipids and DNA, especially in the early stage.The levels of GSH, MDA and8-OHdG in pretreatment of NAC group were9.42±2.40μM/gprot,0.45±0.21μM/gprot and3.42±1.63μg/gprot respectively (p<0.01,0.05and0.05respectively, compared to those of infection group).(3) Primary trophoblast cellsappeared irregular polygon or round, most cells (90.5±3.5%) showed positive stainingfor cytokeratin-7, although about5.1%of cultured cells were macrophage, by analysisof FCM. Additionally, positively reactivity to vimentin had not checked out, whichindicated that the cell cultures did not contain large numbers of contaminating fibroblastor endothelial cells. T.gondii infection also contributed to the significantly increasedapoptosis level of placenta trophoblast. The levels of total apoptosis, early apoptosis andlate apoptosis in control group were11.04±0.91%,9.77±0.99%and1.27±0.43%respectively, but in infection group, apoptosis rate significantly increased(60.71±16.96%,38.57±14.66%and22.14±3.20%respectively). However, pretreatmentof NAC could significantly decrease the total apoptosis level (40.28±8.96%, p<0.05),especially late apoptosis level (4.71±2.81%, p<0.05), when compared with these ofinfection group. The above results were further confirmed by in situ TUNEL detection.(4)By real time RT-PCR and western blot analysis, we found that ER-stress markers, such as GRP78, CHOP and caspase-12, and JNK/ASK1pathway were up-regulated oractivated by acute T.gondii infection; pretreatment of NAC could inhibit the expressionof these genes and activation of these signaling cascades. However, the up-regulation oractivation of p38was not detected in this process.Conclusions (1) A pregnant T.gondii infection mice model was establishedsuccessfully by injected intraperitoneally with200tachyzoites each mouse on GD8.(2)RH T.gondii infection during pregnancy can contribute to a burst of oxidative stress,and finally to escalated trophoblasts apoptosis.(3)Placental trophoblast apoptosis can befound and initiated by ROS-mediated ER stress via activation of caspase12, CHOP andJNK pathway in acute T.gondii infection.(4) Antioxidant has protective effect againstpathological damage of placenta tissues and trophoblast apoptosis.