Yonsei Medical Journal Vol. 47, No. 6, pp. 862 - 869, 2006 Toxoplasma gondii Inhibits Apoptosis in Infected Cells by Caspase Inactivation and NF-κB Activation Ji-Young Kim, Myoung-Hee Ahn, Hye-Sun Jun, Jai-Won Jung, Jae-Sook Ryu, and Duk-Young Min Department of Parasitology, Institute of Biomedical Science, Hanyang University College of Medicine, Seoul, Korea. Our experiments aimed to clarify the mechanism by which host cell apoptosis is inhibited by infection with the intracel- lular protozoan parasite, Toxoplasma gondii (T. gondii). Mouse spleen cells were cultured in 6-well plates with RPMI 1640/ 10% FBS at 37 , in a 5% CO2 atmosphere. Apoptosis of spleen cells was induced by actinomycin-D (AD) treatment for 1 h prior to infection with T. gondii. A variety of assays were used to assess the progression of apoptosis: DNA size analysis on agarose gel electrophoresis, flow cytometry with annexin V/PI staining, and analysis of expression levels of Bcl-2 family and NF-κB mRNA and proteins by RT-PCR, Western blotting, and EMSA. Additionally, transmission electron microscopy (TEM) was performed to observe changes in cell morphology. Fragmentation of DNA was inhibited in spleen cells treated with AD and T. gondii 5 h and 18 h post infection, respec- tively, and flow cytometry studies showed a decreased apoptotic rates in AD and T. gondii treated spleen cells. We observed decreased expression of Bax mRNA and protein, while levels of Bcl-2 mRNA remained constant in spleen cells treated with AD and T. gondii. Caspase 3 and PARP were inactivated in cells treated with AD and T. gondii, and increased levels of cleaved caspase 8 were also observed. Analysis of EMSA and Western blot data suggests that activation of transcription factor NF-κB may be involved in the blockade of apoptosis by T. gondii. TEM analysis showed nuclear fragmentation and chromatin condensation occurring in spleen cells treated with AD; however, such apoptosis- associated morphological changes were not observed in cells treated with both AD and T. gondii tachyzoites. Together, these data show that T. gondii infection inhibits AD induced apoptosis via caspase inactivation and NF-κB activation in Received February 3, 2006 Accepted June 7, 2006 This work was supported by the Anti-Communicable Diseases Programs of the National Institute of Health (NIH 348-6111-215) and Ministry of Health and Welfare, Korea. Reprint address: requests to Dr. Myoung-Hee Ahn, Department of Parasitology, Institute of Biomedical Science, Hanyang University College of Medicine, 17 Haengdang-dong, Seongdong- gu, Seoul 133-791, Korea. Tel: 82-2-2220-0682, Fax: 82-2-2281- 6519, E-mail: mhahn@hanyang.ac.kr Yonsei Med J Vol. 47, No. 6, 2006 mouse spleen cells. Key Words: Toxoplasma, apoptosis, Bcl-2 family, caspase, NF-κB, TEM INTRODUCTION Toxoplasma gondii (T. gondii) is an obligate intra- cellular protozoan parasite that causes congenital infections in newborns and opportunistic infec- tions in immunocompromised patients, such as those with AIDS. T. gondii infects all nucleated cells, eventually causing the host cell to rupture following replication of the tachyzoite. Cell survival or death, as induced by apoptosis, is a physiological response to cell damage caused by external and internal stimuli, and the exact fate of a cell depends on the balance of pro-apoptotic and anti-apoptotic proteins. Apoptosis limits the growth of infectious agents and plays a crucial role in the immune response. There are two path- ways of apoptosis of cells: one triggered by death signals, and another involving the mitochondria. Activation of death receptors triggers the cleavage of caspase 8, whereas release of cytochrome c from mitochondria activates caspase 9; both pathways eventually converge to activate the ex- ecution caspase, caspase 3.1 While the pathogens Acanthamoeba sp. and E. histolytica can induce host cell apoptosis,2-4 the anti- or pro-apoptotic activi- ties associated with T. gondii remain controversial. Induction of apoptosis in Peyer's patch T cells and the ocular tissue of mice infected with T. gondii has been reported.5,6 The Fas/Fas L pathway may play a crucial role in the induction of IFN-γ mediated apoptosis in N. caninum and T. gondii infected cells,7 and infection with T. gondii (RH