Gure 2C) and trophoblast cells (Figure 2D) than in uninfected cells. But their was no difference in the levels of HLA-G with IL-10 treated uninfected cells compared to uninfected cells. When infected cells were treated with IL-10, a decreased in levels of HLA-G protein compared to infected, untreated cells was observed at 16, 24, and 36 hr. We observed higher levels of HLA-G in treated, infected cells than in untreated, infected cells at 48 hr and 60 hr.T. gondii nduced apoptosis inhibited by IL-10 treatmentT. gondii induced apoptosis in both trophoblast and BeWo cells as shown by annexin-V-PE/PI staining. Apoptosis of infected cell was obvious at 16 hr after infection and increased with the infection time. The number of apoptotic infected cells decreased following IL-10 treatment compared to infected cells while their was no difference between uninfected cells and IL-10 treated uninfected cells (Figure 3). We examined BeWo cells and trophoblast cells cultures at 24 hr after challenge for proximity of obvious vacuoles to CP21 site distorted (indented) nuclei stained with Hoechst 33258. At 24 hr post infection, most nuclei directly adjacent to parasitophorous vacuoles were not distorted while farHLA-G expression increases upon infection and decreases with IL-10 treatmentReal-time PCR analysis showed that HLA-G mRNA levels were higher in infected cells than uninfected BeWo cells (Figure 2A) and human primary trophoblast cells (Figure 2B). Levels of HLA-G protein were higher in infected BeWo cellsFigure 1. Fluorescence analysis of infection of BeWo cells and trophoblasts with YFP-T. gondii. Infected and uninfected (A) BeWo cells and (B) trophoblasts were observed under fluorescence microscope. The infection with T. gondii expressing yellow fluorescent protein in BeWo and trophoblasts were observed by fluorescence microscopy at (C, D) 16 hr, (E, F) 24 hr, (G, H) 36 hr, and (I, J) 48 hr. At 16 hr, coupled or ternate tachyzoites were observed inside the cells. Tachyzoites MedChemExpress A 196 arranged in a chrysanthemum shape were observed at 24 hr. Broken cells and scattered tachyzoites were observed at 48 hr. doi:10.1371/journal.pone.0056455.gIL-10 Protects T. gondii-Infected TrophoblastsFigure 2. HLA-G expression in infected cells decreased by IL-10 treatment. HLA-G mRNA expression was analyzed in infected and uninfected BeWo cells (A) and primary human trophoblast cells (B) with and without IL-10 treatment. (C) HLA-G protein expression was analyzed in infected and uninfected BeWo cells with and without IL-10 treatment. (D) HLA-G protein expression was analyzed in infected and uninfected trophoblasts with and without IL-10 treatment. * p,0.05; ** p,0.01. doi:10.1371/journal.pone.0056455.gaway from the infected cells were distorted 15900046 badly. In addition, the abnormal nucleus in IL-10 treated infected cells decreased compared to infected cells, while IL-10 had no role in decreasing the uninfected cells without T. gondii infection (Figure 4), suggesting that IL-10 treatment of cells in infected groups is associated, at least initially, with resistance to T. gondii timulated apoptosis in the cultures. To analyze the key molecules involved in apoptosis, we evaluated levels of caspase-3, caspase-8, and c-FLIP mRNAs expression in BeWo cells (Figure 5) and primary human trophoblast cells (Figure 6). In infected cells, c-FLIPL and c-FLIPS mRNA levels were decreased and caspase-3 and caspase-8 levels were increased relative to uninfected cells. In infected cells treatedwith IL-10,.Gure 2C) and trophoblast cells (Figure 2D) than in uninfected cells. But their was no difference in the levels of HLA-G with IL-10 treated uninfected cells compared to uninfected cells. When infected cells were treated with IL-10, a decreased in levels of HLA-G protein compared to infected, untreated cells was observed at 16, 24, and 36 hr. We observed higher levels of HLA-G in treated, infected cells than in untreated, infected cells at 48 hr and 60 hr.T. gondii nduced apoptosis inhibited by IL-10 treatmentT. gondii induced apoptosis in both trophoblast and BeWo cells as shown by annexin-V-PE/PI staining. Apoptosis of infected cell was obvious at 16 hr after infection and increased with the infection time. The number of apoptotic infected cells decreased following IL-10 treatment compared to infected cells while their was no difference between uninfected cells and IL-10 treated uninfected cells (Figure 3). We examined BeWo cells and trophoblast cells cultures at 24 hr after challenge for proximity of obvious vacuoles to distorted (indented) nuclei stained with Hoechst 33258. At 24 hr post infection, most nuclei directly adjacent to parasitophorous vacuoles were not distorted while farHLA-G expression increases upon infection and decreases with IL-10 treatmentReal-time PCR analysis showed that HLA-G mRNA levels were higher in infected cells than uninfected BeWo cells (Figure 2A) and human primary trophoblast cells (Figure 2B). Levels of HLA-G protein were higher in infected BeWo cellsFigure 1. Fluorescence analysis of infection of BeWo cells and trophoblasts with YFP-T. gondii. Infected and uninfected (A) BeWo cells and (B) trophoblasts were observed under fluorescence microscope. The infection with T. gondii expressing yellow fluorescent protein in BeWo and trophoblasts were observed by fluorescence microscopy at (C, D) 16 hr, (E, F) 24 hr, (G, H) 36 hr, and (I, J) 48 hr. At 16 hr, coupled or ternate tachyzoites were observed inside the cells. Tachyzoites arranged in a chrysanthemum shape were observed at 24 hr. Broken cells and scattered tachyzoites were observed at 48 hr. doi:10.1371/journal.pone.0056455.gIL-10 Protects T. gondii-Infected TrophoblastsFigure 2. HLA-G expression in infected cells decreased by IL-10 treatment. HLA-G mRNA expression was analyzed in infected and uninfected BeWo cells (A) and primary human trophoblast cells (B) with and without IL-10 treatment. (C) HLA-G protein expression was analyzed in infected and uninfected BeWo cells with and without IL-10 treatment. (D) HLA-G protein expression was analyzed in infected and uninfected trophoblasts with and without IL-10 treatment. * p,0.05; ** p,0.01. doi:10.1371/journal.pone.0056455.gaway from the infected cells were distorted 15900046 badly. In addition, the abnormal nucleus in IL-10 treated infected cells decreased compared to infected cells, while IL-10 had no role in decreasing the uninfected cells without T. gondii infection (Figure 4), suggesting that IL-10 treatment of cells in infected groups is associated, at least initially, with resistance to T. gondii timulated apoptosis in the cultures. To analyze the key molecules involved in apoptosis, we evaluated levels of caspase-3, caspase-8, and c-FLIP mRNAs expression in BeWo cells (Figure 5) and primary human trophoblast cells (Figure 6). In infected cells, c-FLIPL and c-FLIPS mRNA levels were decreased and caspase-3 and caspase-8 levels were increased relative to uninfected cells. In infected cells treatedwith IL-10,.