Toxoplasma gondii prevalent in China induce weaker apoptosis of neural stem cells C17.2 via endoplasmic reticulum stress (ERS) signaling pathways

Abstract

Background: Toxoplasma gondii, an obligate intracellular pathogen, has a strong affinity for the nervous system. TgCtwh3, a representative Chinese 1 Toxoplasma strain prevalent in China, has the polymorphic features of the effectors ROP16I/III with type I and GRA15II with type II Toxoplasma strains. The interaction of this atypical strain with host cells remains extremely elusive.

Methods: Using a transwell system, neural stem cells C17.2 were co-cultured with the tachyzoites of TgCtwh3 or standard type I RH strain. The apoptosis levels of C17.2 cells and the expression levels of related proteins in the endoplasmic reticulum stress (ERS)-mediated pathway were detected by flow cytometry and Western blotting.

Results: The apoptosis level of C17.2 cells co-cultured with TgCtwh3 had a significant increase compared to the negative control group; however, the apoptosis level in the TgCtwh3 group was significantly lower than that in the RH co-culture group. Western blotting analyses reveal that, after the C17.2 cells were co-cultured with TgCtwh3 and RH tachyzoites, the expression levels of caspase-12, CHOP and p-JNK in the cells increased significantly when compared to the control groups. After the pretreatment of Z-ATAD-FMK, an inhibitor of caspase-12, the apoptosis level of the C17.2 cells co-cultured with TgCtwh3 or RH tachyzoites had an apparent decline, and correspondingly, the expression levels of those related proteins were notably decreased.

Conclusions: Our findings suggest that TgCtwh3 may induce the apoptosis of the C17.2 cells by up-regulation of caspase-12, CHOP, and p-JNK, which are associated with ERS signaling pathways. This work contributes to better understanding the possible mechanism of brain pathology induced by T. gondii Chinese 1 isolates prevalent in China, and also reveals the potential value of ERS inhibitors to treat such related diseases in the future.

Figure 1

Detection of GFP-RH tachyzoites in co-culture system. (A) Fluorescent image of the upper chamber (×400); (B) fluorescent image of lower chamber (×400); (C) bright field image of the C17.2 cells in the lower chamber (×200).

Figure 2

Apoptosis detected by flow cytometry. (A) The C17.2 cells were co-cultured with 5 × 10⁵ TgCtwh3 tachyzoites, heat-inactivated tachyzoites or Apopida (apoptosis inducer A) for 6 h, 12 h, and 24 h; (B) The C17.2 cells were co-cultured with various doses of TgCtwh3 tachyzoites (2 × 10⁴, 1 × 10⁵ and 5 × 10⁵) for 24 h. The cells were then collected, stained with Annexin V/PI, and analyzed by FCM. The plots are from a representative measurement and the graphics represent the mean and S.D. on three different assays (n = 3). ^* P < 0.05; ^** P < 0.01 vs. Negative controls.

Figure 3

Apoptosis detected by Western blotting and nucleus staining. (A) The C17.2 cells were collected after they were co-cultured with 5 × 10⁵ RH tachyzoites for 24 h, and the activity of caspase-3 was detected by Western blotting; (B) The nuclei of the apoptotic cells were stained by propidium iodide. The arrows represent a shrinking nucleus or apoptotic body.

Figure 4

Effect of inhibitors on the apoptosis levels of C17.2 cells. After the C17.2 cells were pretreated with or without Z-ATAD-FMK (ZAF) for 6 h, they were co-cultured with 5 × 10⁵ TgCtwh3 or RH tachyzoites for 24 h. The cells were then collected, stained with Annexin V/PI, and analyzed by FCM. TgCtwh3 or RH + ZAF stands for the C17.2 cells pretreated with Z-ATAD-FMK, and then co-cultured with either TgCtwh3 or RH tachyzoites. The data are from three independent experiments. ^* P < 0.05 vs. TgCtwh3 co-cultured group; ^** P < 0.05 vs. RH co-cultured group; ^# P < 0.05 vs. control.

Figure 5

Expression levels of caspase-12, JNK, and CHOP in C17.2 cells. After the C17.2 cells were pretreated with or without Z-ATAD-FMK (ZAF), they were co-cultured with 5 × 10⁵ TgCtwh3 or RH tachyzoites for 24 h. The presented figures are from a representative study and the graphics represent the mean and SD on different assays (n = 3). The C17.2 cells without the co-culture of tachyzoites or only pretreated with ZAF were used as controls. TgCtwh3 or RH + ZAF stands for the C17.2 cells pretreated with Z-ATAD-FMK, and then co-cultured with either TgCtwh3 or RH tachyzoites. The experiments were repeated three times. ^# P < 0.05 vs. control; *P < 0.05 vs. TgCtwh3 co-cultured group; & P < 0.05 vs. RH co-cultured group.