Roles of calpain in the apoptosis of Eimeria tenella host cells at the middle and late developmental stages.

This study investigated the role of calpain in Eimeria tenella-induced host cell apoptosis. Chick embryo cecal epithelial cell culture technology, flow cytometry, enzyme-linked immunosorbent assays, and fluorescence quantitative PCR were used to detect the E. tenella host cell apoptotic rate, Bax and Bid expression levels, and calpain activity. The results demonstrated that Bax, Bid, and calpain levels were upregulated and apoptosis was increased following E. tenella infection at 24-120 h. Calpain levels were reduced by pharmacological inhibition of calpain using SJA6017 or by blocking Ca2+ entry into the cell using BAPTA/AM at 24-120 h. The mRNA and protein levels of Bax and Bid, the E. tenella infection rate, and the early apoptotic and late apoptotic (necrosis) rates were decreased by using SJA6017 at 24-120 h. These results indicated that E. tenella-promoted host cell apoptosis is regulated by calpain via Bid and Bax at 24-120 h. Thus, manipulation of calpain levels could be used to manage E. tenella infection in chickens in the middle and late developmental stages.

Dynamic expression of death receptor adapter proteins tradd and fadd in Eimeria tenella-induced host cell apoptosis.

The present study aimed to investigate the dynamic expression patterns of death-receptor adapter proteins TNF-receptor-associated death-domain protein (TRADD) and Fas-associated death-domain protein (FADD) in E. tenella-induced host-cell apoptosis. Culture techniques for primary chick embryo cecum epithelial cells, ELISA, hematoxylin-eosin staining, fluorescence quantitative PCR techniques, and Hoechst-Annexin V-PI apoptosis staining were used to detect the apoptosis rates and dynamic expression patterns of TRADD and FADD in E. tenella host cells at 4, 24, 48, 72, 96, and 120 h. The rates of early apoptosis, late apoptosis, and necrosis of E. tenella-infected group (group T0) were significantly lower (P < 0.05) or highly significantly lower (P < 0.01) than those of control group (group C) at 4 h, but higher (P < 0.05 or P < 0.01) at varying degrees than those of the same group at 24 to 120 h. Compared with group C, both the mRNA and protein expression levels of TRADD in the group T0 cells increased highly significantly (P < 0.01) at 4 and 24 h, and significantly (P < 0.05) at 48, 72, and 120 h. Compared with the mRNA expression in the group C cells, that of TRADD in the group T0 cells increased significantly (P < 0.05) at 96 h. Both the mRNA and protein expression of FADD in the group T0 cells displayed no significant difference (P > 0.05) from those in the group C cells at 4 h. However, FADD expression in the group T0 cells were significantly (P < 0.05) or highly significantly higher (P < 0.01) than those in the group C cells at 24 to 120 h. These observations indicate that in the early developmental stages of E. tenella, the host-cell apoptosis rate decreased, and TRADD expression increased. In the middle and later developmental stages of E. tenella, the host-cell apoptosis rate increased and expression of TRADD and FADD increased. The variation trends of TRADD and FADD expression were significantly positively correlated with the change rule of the host-cell apoptosis rate, respectively. These results indicate that TRADD and FADD may play an important role in E. tenella-induced host-cell apoptosis.

Mitochondrial pathways are involved in Eimeria tenella-induced apoptosis of chick embryo cecal epithelial cells.

Accumulating evidence suggests that Eimeria tenella severely damages the intestinal mucosa in infected poultry, resulting in deadly haemorrhagic typhlocolitis and major economic losses. Damage to host tissue is believed to arise mainly from apoptosis, which is, in general, intimately related to mitochondrial function. However, it is unclear whether mitochondria-dependent apoptotic pathways are specifically involved in parasite-induced apoptosis of chick embryo cecal epithelial cells. Because the mitochondrial permeability transition pore (MPTP) and caspase-9 are important elements in these pathways, we studied the effects of their respective inhibitors (i.e., cyclosporine A [CsA] and Z-LEHD-FMK, respectively) in primary cultures of chicken embryonic cecum epithelial cells using histopathological techniques, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays, flow cytometry (FCM) and ELISA. Results indicated that the inhibitors significantly decreased (p < 0.01) DNA injury, apoptosis and caspase-9 and caspase-3 activity of chick embryo cecal epithelial cells at 24, 48, 72, 96 and 120 h after E. tenella infection. Thus, our data supported that mitochondria-dependent apoptotic pathways were involved in apoptosis of parasitised chick embryo cecal epithelial cells.

Dynamic changes in the main regulatory genes of mitochondrial permeability transition pore in Eimeria tenella host cells.

The purpose of the present study was to investigate the dynamic changes in the main regulatory genes of the mitochondrial permeability transition pore in E. tenella host cells. Primary chick embryo cecum epithelial cell culture techniques, spectrophotometer technology, Hoechst-Annexin V-PI apoptosis staining and ELISA were used to detect the apoptosis rate and dynamic changes of Bcl-2, Bcl-xl, Bax, Bak, Bid, Bad, HK-II, and ATP content in E. tenella host cells at 4, 24, 48, 72, 96, and 120 h. The rates of early apoptosis, late apoptosis, and necrosis of group T0 were significantly lower (P < 0.05) or highly significantly lower (P < 0.01) than those of group C at 4 h, but higher (P < 0.05 or P < 0.01) at varying degrees than those of the same group at 24-120 h. Compared to group C, the amount of Bcl-2, ATP, Bax and Bad in group T0 were visibly lower (P < 0.05 or P < 0.01) at 4 h, whereas Bcl-xl/Bax was highly significantly higher (P < 0.01) at 4 h. In addition, group T0 had less ATP at 24-120 h than group C, whereas the amount of Bcl-2, Bcl-xl, Bax, Bak, Bid, Bad and HK-II in group T0 inversely increased in varying degrees at 24-120 h compared with group C. Moreover, Bcl-2/Bax was lower (P < 0.01) at 24, 48, and 96 h, and Bcl-xl/Bax was lower (P < 0.05) at 48 h in group T0 than in group C, respectively. Taken together, these observations indicate that in the early developmental stages of E. tenella, the host-cell apoptosis rate decreased; although the amount of anti- and pro-apoptotic genes in host cells decreased, the ratios of anti-apoptotic to pro-apoptotic bcl-2 gene-family members increased. In the middle and later developmental stages of E. tenella, the host-cell apoptosis rate increased; the amount of anti- and pro-apoptotic genes increased, while the ratios of anti-apoptotic to pro-apoptotic bcl-2 gene-family members decreased. In addition, ATP decreased at all developmental stages of E. tenella.

The effect of the mitochondrial permeability transition pore on apoptosis in Eimeria tenella host cells.

Although the mitochondrial permeability transition pore (MPTP) is associated with cellular apoptosis and necrosis, its effect in host response to Eimeria infections is not well understood. In an effort to better understand the effect of MPTP on apoptosis in Eimeria tenella host cells, an MPTP inhibitor (cyclosporin A) was used to inhibit MPTP opening in vitro. Cecal epithelial cells from chick embryos, which were either treated or non-treated with cyclosporin A, were used as Eimeria tenella host cells. In addition, primary chick embryo cecum epithelial cell culture techniques and flow cytometry were used to detect the dynamic changes in MPTP opening, mitochondrial transmembrane potential, and cell apoptosis rate of Eimeria tenella host cells. Compared with the control group, cytometric techniques showed that untreated host cells exhibited a significantly higher (P < 0.01) degree of MPTP opening but lower (P < 0.01 or P < 0.05) mitochondrial transmembrane potential. Moreover, untreated group cells had less apoptosis (P < 0.01) at 4 h and more apoptosis (P < 0.05 or P < 0.01) at 24 to 120 h as compared with control group cells. After the application of cyclosporin A, the degree of MPTP opening in the treated group was significantly lower (P < 0.01) at 4 to 120 h compared to the untreated group, whereas the treated group had higher (P < 0.05 or P < 0.01) mitochondrial transmembrane potentials at 24 to 120 h. Flow cytometry assays also showed that there was less (P < 0.05 or P < 0.01) apoptosis after 24 h in the treated group than in the untreated group. Taken together, these observations indicate that MPTP is a key node that plays a predominant role in the mitochondrial apoptosis pathway in the host cell induced by Eimeria tenella.

Calcium homeostasis in mitochondrion-mediated apoptosis of chick embryo cecal epithelial cells induced by Eimeria tenella infection.

In this study, the process of Eimeria tenella-induced apoptosis and the effect of calcium homeostasis were investigated in chick embryo cecal epithelial cells. In particular, we examined cytochrome c release into the cytoplasm, mitochondrial permeability transition pore (MPTP) opening, and changes in [Ca(2+)]c and apoptosis in host cells. Apoptosis, MPTP opening, cytochrome c release, and [Ca(2+)]c in host cells increased following infection. This trend was reversed by blocking the increase in [Ca(2+)]c using BAPTA/AM and EGTA (intra- and extracellular chelators of Ca(2+), respectively) and by applying heparin sodium and ryanodine (blockers of the inositol triphosphate and ryanodine receptors of the endoplasmic reticulum, respectively). These results indicate that [Ca(2+)]c plays a significant role in host cell mitochondrial apoptosis, which is induced via modulation of extracellular Ca(2+) levels and endoplasmic reticulum Ca(2+) channels. Thus, agents that restore Ca(2+) homeostasis may be useful for managing E. tenella infection in chickens.

Relationship between Eimeria tenella development and host cell apoptosis in chickens.

Coccidiosis causes considerable economic losses in the poultry industry. At present, the pathology of coccidiosis is preventable with anticoccidials and vaccination, although at considerable cost to the international poultry industry. The purpose of the present study was to elucidate the relationship between Eimeria tenella development and host cell apoptosis in chickens, which provides a theoretical basis for further study of the injury mechanism of E. tenella and the prevention and treatment of coccidiosis. Cecal epithelial cells from chick embryo were used as host cells in vitro. In addition, flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling, and histopathological assays were used to detect the dynamic changes in E. tenella infection rates, DNA injury rates, and apoptosis rates in groups treated with and without the caspase-9 inhibitor Z-LEHD-FMK. Following E. tenella infection, we demonstrated that untreated cells had less apoptosis at 4 h and, inversely, more apoptosis at 24 to 120 h compared with control cells. Furthermore, after the application of Z-LEHD-FMK, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays, and translation of phosphatidyl serines to the host cell plasma membrane surface, the treated group chick embryo cecal epithelial cells exhibited decreased apoptosis and DNA injuries (P<0.01) at 24 to 120 h. However, light microscopy showed that E. tenella infection rates of treated cells were higher (P<0.01) than untreated cells during the whole experimental period. Together, these observations suggest that E. tenella can protect host cells from apoptosis at early stages of development but can promote apoptosis during the middle to late stages. In addition, the inhibition of host cell apoptosis can be beneficial to the intracellular growth and development of E. tenella.

The effect of mitochondrial ATP-sensitive potassium channels on apoptosis of chick embryo cecal cells by Eimeria tenella.

The objective of this study was to investigate the effect of mitochondrial ATP-sensitive potassium (mitoKATP) channels on apoptosis induced by Eimeria tenella. At 24, 48, 72, 96 and 120 h after Eimeria tenella infection, TUNEL assays and translation of phosphatidyl serines to the host cell plasma membrane surface showed that diazoxide-treated chick embryo cecal cells underwent less apoptosis (P <0.05), while light microscopy showed that infection rates of treated cells were higher (P <0.01) than untreated cells. Caspase 9 and caspase 3 of infected cells were activated less (P <0.01) in diazoxide-treated cells than untreated cells. These results indicate that opening mitoKATP channels can protect chick embryo cecal cells from mitochondria-dependent apoptosis induced by Eimeria tenella by inhibiting activations of caspase 9 and caspase 3.