Influence of Different Death Receptor Signaling Pathways on Apoptosis of Eimeria tenella Host Cells

Abstract

The aim of this study was to investigate the regulatory roles of distinct signaling cascades within the death receptor pathway in host cell apoptosis induced by Eimeria tenella (E. tenella); to this end, primary chicken embryo cecal epithelial cell culture, gene silencing, enzyme-linked immunosorbent assay (ELISA), Hoechst–Annexin V/PI apoptosis staining, hematoxylin–eosin (HE) staining, and quantitative real-time polymerase chain reaction (qRT-PCR) were employed. At 4, 24, 72, and 120 h post-inoculation (hpi) with E. tenella sporozoites, the proportion of apoptosis in six treatment groups [Group C, Group T0 (E. tenella infection group), Group T1 (E. tenella + Fas SiRNA), Group T2 (E. tenella + TRAIL SiRNA), Group T3 (E. tenella + TNFR1 SiRNA), and Group T4 (E. tenella + NC SiRNA)] and the dynamic changes in Fas cell surface death receptor (Fas), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), tumor necrosis factor receptor 1 (TNFR1), TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD), and death domain-associated protein (Daxx) expression and caspase-8 activity in the cells were determined. The results demonstrated that, from 4 to 120 hpi, the Fas and TNFR1 mRNA expression levels in Group T0’s host cells were significantly higher than those in Group C (p < 0.05 or p < 0.01). At 72 and 120 hpi, TRAIL mRNA expression in Group T0’s host cells was significantly or highly significantly elevated compared to that in Group C (p < 0.05 or p < 0.01). From 24 to 120 hpi, the expression levels of the FADD and Daxx genes, caspase-8 activity, and apoptotic rates in Group T1’s host cells were significantly lower than those in Group T4 (p < 0.05). At 72 and 120 hpi, the FADD expression, caspase-8 activity, and apoptotic rates in Group T2’s host cells were significantly reduced relative to Group T4 (p < 0.05). Additionally, at 4 hpi, TRADD gene expression in Group T3’s host cells was significantly lower than that in Group T4 (p < 0.05), while the apoptotic rate was significantly higher (p < 0.05). However, from 24 to 120 hpi, the TRADD expression, caspase-8 activity, and apoptotic rates in Group T3’s host cells were significantly lower than those in Group T4 (p < 0.05). The results indicated that, in the early stages of E. tenella development, TNFR1 overexpression promoted TRADD mRNA expression, thereby inhibiting the apoptosis of E. tenella host cells. In the middle and late developmental stages of E. tenella, the Fas-FADD, Fas-Daxx, TRAIL-FADD, and TNFR1-TRADD apoptotic pathways were all activated, collectively facilitating host cell apoptosis. The pro-apoptotic effects of these pathways were ranked in descending order, as follows: Fas signaling pathway > TNFR1 signaling pathway > TRAIL signaling pathway.

1. Introduction

Chicken coccidiosis is a parasitic disease primarily characterized by intestinal lesions, caused by protozoa of the genus Eimeria. It is distributed globally and predominantly affects chickens aged 15–50 days, with a prevalence of up to 70% and a mortality ranging from 20% to 40%. In addition to causing mortality, recovered chickens often fail to fully rehabilitate, leading to considerable impairments in weight gain and egg production—inflicting substantial economic losses on the global poultry industry [1]. Among the etiological agents, Eimeria tenella (E. tenella) is the most virulent species of coccidia. It primarily damages the mucosa of the chicken cecum and adjacent intestinal segments, inducing microvilli shedding, swelling, necrosis, disintegration, and detachment of mucosal epithelial cells. The development of E. tenella involves two rounds of schizogony, both of which are completed within the host’s cecal epithelial cells. An increase in the number of apoptotic epithelial cells in the cecal mucosal layer of E. tenella-infected chickens is a critical event in the pathogenesis of chicken coccidiosis [2].

The death receptor signaling pathway is induced by extracellular signals and is thus also known as the extrinsic apoptosis pathway; it is one of the main apoptotic signal transduction pathways [3]. Among them, the TNF-α/TNFR signaling pathway, the Fas/FasL signaling pathway, and the TRAIL/TRAILR signaling pathway are the three main death receptor apoptosis pathways. After the TNF-α trimer interacts with tumor necrosis factor receptor 1 (TNFR1), it induces the aggregation of the death domain (DD) of TNFR1, which can interact with the DD binding region of the adaptor protein TNF receptor-associated death domain (TRADD), recruiting TRADD to the activated receptor molecule. TRADD, as an adaptor platform, further aggregates the Fas-associated death domain (FADD) signaling molecule. FADD links with the corresponding DED region of procaspase-8 through its own DED region, activating caspase-8 and further activating caspase-3, cleaving PARP, initiating apoptosis [4]. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein that can bind to the related receptors TRAIL-R1 and TRAIL-R2, recruiting FADD intracellularly to form a DISC, activating procaspase-8 into caspase-8, and thereby initiating apoptosis. Moreover, Fas is a type I transmembrane protein located on the cell surface. Fas contains a DD, which can interact with signal adaptors such as FADD and death domain-associated protein (Daxx), promoting apoptosis. Studies have shown that the death receptor signaling pathway is one of the main apoptosis pathways in E. tenella host cells. Fas, TRAIL, and TNFR1, as the key death receptors in the three death receptor apoptosis signaling pathways, have been shown to play crucial roles in the process of pathogen-induced host cell apoptosis [3]. Currently, the effects of the three death receptor signaling pathways on host cell apoptosis caused by E. tenella have not been systematically reported. Therefore, this project was carried out to lay a theoretical foundation for exploring the process and regulatory mechanism of host cell apoptosis induced by the death receptor pathway of E. tenella, which is of great scientific significance for further clarifying the mechanisms of damage and immune evasion of coccidiosis in chickens.

2. Materials and Methods

2.1. Experimental Animals

A total of 150 specific-pathogen-free (SPF) chicken embryos at 15 days of incubation were purchased from Beijing Merial Vital Laboratory Animal Technology Co., Ltd. (Beijing, China). Additionally, 1-day-old SPF chicks (n = 20) were hatched in-house from SPF eggs.

2.2. Parasite Strain

The virulent E. tenella strain (Henan strain) was provided and maintained by the Laboratory of Animal Parasitology, Henan Institute of Science and Technology.

2.3. Isolation and Culture of Chicken Embryo Cecal Epithelial Cells

Fifteen-day-old SPF chicken embryos were disinfected aseptically, and ceca were aseptically excised. The tissues were washed three times with phosphate-buffered saline (PBS) containing double antibiotics (penicillin and streptomycin), minced into tissue fragments of approximately 1 mm³, and processed according to previously described protocols [5].

2.4. Preparation of Sporozoites of E. tenella

The 20 SPF chicks reared in isolators were inoculated orally with 5 × 10³ sporulated oocysts of E. tenella per bird at 20 days of age. Subsequent experimental procedures followed established methods as previously reported [6].

2.5. Transfection Procedure

The cultured cells were washed twice, and then 1.5 mL of cell culture medium without penicillin–streptomycin or serum and 0.5 mL of transfection complex (siRNA and lipofectamine 2000) were added. The mixture was incubated in a 8% CO₂ incubator for 6 h. After discarding the transfection solution, 2 mL of freshly prepared complete culture medium was added, and the samples were further incubated for 42 h. Fas siRNA, TRAIL siRNA, TNFR1 siRNA, and NC siRNA were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China), the sequences of which are shown in

Table 1. siRNA sequences.

Name		Sequence
Fas siRNA	Sense	CCACACACACUGCACAUAATT
	Antisense	UUAUGUGCAGUGUGUGUGGTT
TRAIL siRNA	Sense	GCCACUCCUUCCUCUACAATT
	Antisense	UUGUAGAGGAAGGAGUGGCTT
TNFR1 siRNA	Sense	GCUCGUGUGCUAAUGGAAUTT
	Antisense	AUUCCAUUAGCACACGAGCTT
NC siRNA	Sense	UUCUCCGAACGUGUCACGUTT
	Antisense	ACGUGACACGUUCGGAGAATT

.

2.6. Experimental Groups and Treatments

When primary chicken embryo cecal epithelial cells cultured in 6-well plates reached over 85% confluency, they were randomly assigned to six groups: blank control group (Group C, without E. tenella infection), infected non-treated group (Group T0), Fas siRNA-treated group (Group T1), TRAIL siRNA-treated group (Group T2), TNFR1 siRNA-treated group (Group T3), and negative control siRNA-treated group (Group T4). Except for Group C, all other groups were inoculated with 4 × 10⁵ sporozoites per well. Cells were continuously cultured for 120 h in an incubator maintained at 41 °C and 8% CO₂, with medium replaced every 48 h. For the siRNA treatment groups (T1–T4), transfection was performed at four time points: 44 h and 24 h before sporozoite inoculation, and 24 h and 72 h after inoculation. Each siRNA (Fas siRNA, TRAIL siRNA, TNFR1 siRNA, or NC siRNA) was transfected using Lipofectamine 2000 at a ratio of 0.5 μL of siRNA to 0.5 μL of Lipofectamine 2000. The mRNA silencing efficiencies of Fas siRNA, TRAIL siRNA, and TNFR1 siRNA were 85.60%, 83.50%, and 86.20%, respectively. At 4 h, 24 h, 72 h, and 120 h post-inoculation, cells from each group were collected to determine rates of E. tenella infection and host cell apoptosis. Additionally, dynamic changes in the mRNA expression levels of Fas, TRAIL, TNFR1, TRADD, FADD, and Daxx, as well as caspase-8 activity in host cells, were analyzed. All measurements were performed in five biological replicates.

2.7. Dynamic Detection of E. tenella Development

Cell slides from each group at different time points were taken, stained with H.E, and the E. tenella infection rate was counted. The specific procedure was the same as was previously reported [5].

2.8. Apoptosis Assay

The experiment adopted the triple apoptosis staining method with Hoechst 33342 (beyotime, Shanghai, China; cat: C1025), AnnexinV-FITC, and PI (BD Biosciences, San Diego, CA, USA; cat: 556547, lot: 6033840). The subsequent experimental procedures were carried out in accordance with previous reports [7]. Normal cells were characterized by low blue/low green (Hoechst 33342+/Annexin V+), early apoptotic cells by low blue/high green (Hoechst 33342+/Annexin V++), and late apoptotic and necrotic cells by high green/high red (Hoechst 33342++/PI++).

2.9. Detection of Caspase-8 Activity

The dynamic changes in caspase-8 activity in E. tenella-infected host cells were determined using a chicken caspase-8 assay kit (Beyotime, Lot No.: C1152) according to the manufacturer’s instructions. An enzyme-linked immunosorbent assay (ELISA) was employed as the detection method.

2.10. Quantitative Real-Time PCR

Total RNA was extracted using the TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed with a kit (Takara, Japan), followed by quantitative real-time PCR (qPCR) using SYBR Green PCR Master Mix (Roche, Germany), according to the manufacturer’s instructions. The primer sequences for target genes are listed in

Table 2. Real-time PCR primer sequence.

Name		Sequence	Length	Reference Sequence
Fas	F	5′-ACCGTCTATCGGAACCTACCA-3′	126	AF296875.1
	R	5′-AGGTTTCGTAGGCTCCTCCC-3′	126	AF296875.1
TRAIL	F	5′-GCGTCCCCGCACATAGATTA-3′	206	NM_204379.2
	R	5′-AACAAACCCGAGTCCTCGTC-3′	206	NM_204379.2
TNFR1	F	5′-ATACTGTGTGTGGCTGTCGG-3′	205	XM_015294558.1
	R	5′-AAGCACTCTTCTCCAACGCA-3′	205	XM_015294558.1
TRADD	F	5′-ACAACAGAACGCTCACACCA-3′	152	XM_004944140.2
	R	5′-TCCCTCTCGGTCGTATTCGT-3′	152	XM_004944140.2
FADD	F	5′-ACTGGAAAATGCTGATGCGA-3′	113	XM_421073.5
	R	5′-CCACTCTCGAAGCGACTGAAA-3′	113	XM_421073.5
Daxx	F	5′-TCGAGGACTTCCACCCCAAA-3′	116	XM_015274711.1
	R	5′-CCGTGGGGTCTTTTCATTGG-3′	116	XM_015274711.1
β-actin	F	5′-CACCACAGCCGAGAGAGAAAT-3′	135	L08165.1
	R	5′-TGACCATCAGGGAGTTCATAGC-3′	135	L08165.1

, and the gene sequences were determined by Beijing Liuhe Huada Gene Technology Co., Ltd. (Beijing, China).

2.11. Statistical Analysis

The inter-group data in the experimental results were analyzed using independent sample t-test and ANOVA variance difference significance analysis with SPSS19.0 data statistics software. The data distribution conforms to the normal distribution. The data in each group of experimental results were expressed as mean ± standard error.

3. Results

3.1. Changes in the Expression of Fas, TRAIL, and TNFR1 Genes in E. tenella Host Cells

From 4 to 120 h post-sporozoite infection, the Fas and TNFR1 mRNA expression levels in Group T0’s host cells were significantly or highly significantly greater than those in Group C (p < 0.05 or p < 0.01). At 72 h and 120 h post-infection, the TRAIL mRNA expression in Group T0 was also significantly elevated compared to Group C (p < 0.05). See Figure 1 for details.

3.2. Relationship Between Fas, TRAIL, TNFR1 and E. tenella Development

From 4 to 120 h post-infection with E. tenella sporozoites, no significant differences in E. tenella infection rates were observed between Groups T4 and T0 (p > 0.05). At 24 h and 120 h post-infection, the infection rates in Group T1 were significantly higher than those in Group T4 (p < 0.05). At 72 h post-infection, the infection rate in Group T2 was significantly elevated compared to that in Group T4 (p < 0.05). From 72 to 120 h post-infection, the infection rates in Group T3 remained significantly higher than those in Group T4 (p < 0.05). See Figure 2 and Figure 3 for details.

3.3. Relationship Between Fas, TRAIL, and TNFR1 and Apoptosis of E. tenella Host Cells

At 4 h post-sporozoite infection, the early apoptosis rate in Group T0’s host cells was significantly lower than that in Group C (p < 0.05). From 24 to 120 h post-infection, the early apoptosis rate in Group T0 was highly significantly higher than that in Group C (p < 0.01). From 4 to 120 h post-infection, no significant differences in the early apoptosis rates were observed between Groups T4 and T0 (p > 0.05). From 24 to 120 h post-infection, the early apoptosis rate in Group T1 was significantly lower than that in Group T4 (p < 0.05). At 72 h and 120 h post-infection, Group T2 exhibited a significantly lower early apoptosis rate compared to Group T4 (p < 0.05). At 4 h post-infection, the early apoptosis rate in Group T3 was significantly higher than that in Group T4 (p < 0.05), whereas from 72 to 120 h post-infection, it was significantly lower than in Group T4 (p < 0.05). See Figure 4 and Figure 5 for details.

At 4 h post-infection, the late apoptosis and necrosis rates in Group T0 were significantly lower than those in Group C (p < 0.05). From 24 to 120 h post-infection, these rates in Group T0 were highly significantly elevated compared to those in Group C (p < 0.01). From 4 to 120 h post-infection, no significant differences in late apoptosis and necrosis rates were observed between Groups T4 and T0 (p > 0.05). From 24 to 120 h post-infection, Group T1 showed significantly lower late apoptosis and necrosis rates than Group T4 (p < 0.05). At 72 h and 120 h post-infection, both Groups T2 and T3 exhibited significantly lower rates than Group T4 (p < 0.05). See Figure 4 and Figure 5 for details.

In addition, via a comparison with Group T4, the data providing reductions in apoptosis rates for each treatment group were obtained from 24 to 120 h post-infection. At 24 h and 72 h, the early apoptosis reduction rate in Group T3 was significantly lower than that in Group T1 (p < 0.05). Compared to Group T3, Group T2 also exhibited a significantly lower early apoptosis reduction rate (p < 0.05). At 120 h, although the early apoptosis reduction rate in Group T3 was lower than that in Group T1, the difference was not statistically significant (p > 0.05). Similarly, Group T2 showed a reduced rate compared to Group T3, but without statistical significance (p > 0.05). See

Table 3. Comparison of the proportions of early apoptosis rate reduction in E. tenella host cells via different signaling pathways of the death receptor pathway (%).

The Reduction Ratio of Early Apoptosis Rate (%)	T1	T2	T3
24 h	15.63 ± 1.19	7.81 ± 0.85 +	10.93 ± 0.98 *
72 h	15.11 ± 1.16	9.30 ± 0.95 +	11.63 ± 0.86 *
120 h	13.25 ± 0.96	10.84 ± 0.92	12.05 ± 1.01

Note: Compared with the T1 group, “*” indicates significant difference (p < 0.05); compared with the T3 group, “⁺” indicates significant difference (p < 0.05).

for details.

At 24 h, 72 h, and 120 h, the late apoptosis and necrosis reduction rates in Group T3 were significantly lower than those in Group T1 (p < 0.05). At 24 h and 72 h, Group T2 showed lower late apoptosis and necrosis reduction rates than Group T3, but the differences were not significant (p > 0.05). However, at 120 h, the reduction rate in Group T2 was significantly lower than that in Group T3 (p < 0.05). See

Table 4. Comparison of the ratios of reduction in late apoptosis and necrosis rates of E. tenella host cells by different signaling pathways of death receptor pathways (%).

The Reduction Ratio of Late Apoptosis and Necrosis Rate (%)	T1	T2	T3
24 h	17.50 ± 2.16	5.00 ± 0.97	6.25 ± 0.92 *
72 h	13.25 ± 1.56	8.43 ± 1.15	9.64 ± 1.26 *
120 h	21.74 ± 2.82	10.87 ± 1.22 +	15.22 ± 1.26 *

Note: Compared with the T1 group, “*” indicates significant difference (p < 0.05); compared with the T3 group, “⁺” indicates significant difference (p < 0.05).

for details.

These results indicated that, during the mid-to-late stages of E. tenella development, the Fas signaling pathway exerted a stronger pro-apoptotic effect on the host cells than the TNFR1 pathway, while the TNFR1 pathway had a greater influence than the TRAIL pathway.

3.4. Effects of Fas and TRAIL on FADD Protein Expression in E. tenella Host Cells

From 24 to 120 h post-sporozoite infection, the FADD protein expression in Group T0’s host cells was highly significantly higher than that in Group C (p < 0.01). From 4 to 120 h post-infection, no significant difference in FADD protein expression was observed between Groups T4 and T0 (p > 0.05). From 24 to 120 h post-infection, the FADD protein levels in Group T1 were significantly lower than those in Group T4 (p < 0.05). At 72 h and 120 h post-infection, Group T2 also exhibited significantly reduced FADD protein expression compared to Group T4 (p < 0.05). See Figure 6 for details.

3.5. Effect of Fas on Daxx Expression in E. tenella Host Cells

At 72 h and 120 h post-sporozoite infection, Daxx mRNA expression in the host cells of Group T0 was significantly higher than that in Group C (p < 0.05). From 4 to 120 h post-infection, no significant differences in Daxx mRNA expression were observed between Groups T4 and T0 (p > 0.05). At 72 h post-infection, the Daxx mRNA levels in Group T1 were significantly lower than those in Group T4 (p < 0.05). See Figure 7 for details.

3.6. Effect of TNFR1 on TRADD mRNA Expression in E. tenella Host Cells

From 4 to 120 h post-sporozoite infection, the TRADD mRNA expression in Group T0’s host cells was significantly or highly significantly greater than that in Group C (p < 0.05 or p < 0.01). From 4 to 120 h post-infection, no significant differences in TRADD mRNA expression were observed between Groups T4 and T0 (p > 0.05). Similarly, from 4 to 120 h post-infection, the TRADD mRNA levels in Group T3 were significantly lower than those in Group T4 (p < 0.05). See Figure 8 for details.

3.7. Effects of Fas, TRAIL, and TNFR1 on Caspase-8 Activity in E. tenella Host Cells

At 4 h post-infection with E. tenella sporozoites, no significant difference in caspase-8 activity was observed between Groups T0 and C (p > 0.05). From 24 to 120 h post-infection, the caspase-8 activity in Group T0 was significantly or highly significantly greater than that in Group C (p < 0.05 or p < 0.01). From 4 to 120 h post-infection, there were no significant differences in caspase-8 activity between Groups T4 and T0 (p > 0.05). Compared to Group T4, the caspase-8 activity in Group T1 was significantly reduced at 24 h and 120 h post-infection (p < 0.05); Groups T2 and T3 also exhibited significantly reduced caspase-8 activity at 72 h and 120 h post-infection (p < 0.05). See Figure 9 for details.

4. Discussion

4.1. Changes in Fas, TRAIL, and TNFR1 Expression in E. tenella Host Cells

The results of this study indicated that E. tenella infection promoted the expression of Fas in host cells. Previous studies have reported that Toxoplasma gondii infection upregulated the expression of Fas/CD95 and increased FasL secretion in THP-1 cells [8]. Similarly, following Schistosoma infection, elevated expression of Fas/FasL has been observed in skin-draining lymph node (sdLN) cells in mice, accompanied by a reduced number of surviving CD4⁺ T cells [9]. The findings of the present study are consistent with those of previous reports on the regulatory effects of other pathogens on intracellular Fas expression [8,9].

The experimental results also indicated that, in the mid-to-late developmental stages, E. tenella significantly promoted TRAIL expression in host cells. It has been reported that Cryptosporidium parvum infection induces high TRAIL and caspase-8 expression in HCT-8 cells, and TRAIL-mediated apoptosis of HCT-8 cells occurs in a concentration-dependent manner [10]. Reovirus infection could trigger TRAIL secretion in human tissue cells and upregulate the expression of death receptors DR5 and DR4 [11]. In dendritic cells infected with attenuated measles virus (MV), moderate IFN-α secretion was observed, accompanied by enhanced TRAIL expression [12]. The present findings demonstrated that E. tenella in its mid-to-late development stage enhanced TRAIL expression in host cells, which is consistent with previous reports on the regulatory effects of other pathogens on TRAIL expression [10,11,12].

In addition, the results showed that E. tenella infection promoted TNFR1 expression in host cells. TNFR1 is expressed in nearly all cell types [13], and it has been found that, following E. tenella infection in chickens, increased TNF-α expression is detected in cecal epithelial tissues [14]. TNF-α exerts its biological effects primarily through TNFR1, and stimulation with TNF-α leads to upregulation of TNFR1 expression in host cells [15]. Evidence indicates that at 36 h post-infection with infectious bursal disease virus (IBDV) in renal tubular epithelial cells, the expression levels of TNFR1, TRADD, and FADD are significantly elevated [16]. In peripheral blood mononuclear cells with low-level bovine leukemia virus (BLV) infection, TNFR1 mRNA expression was found to significantly increase [17]. Conversely, some studies have reported that exposing human neutrophils to TNF-α leads to significant downregulation of the pro-apoptotic genes TNFR1 and TNFR2 [18]. Therefore, TNFR1 expression levels might vary depending on cell type and context. The current study demonstrated that E. tenella infection enhanced TNFR1 expression in host cells, a finding consistent with the effects of other pathogens on TNFR1 regulation [16,17].

4.2. Relationship Between Fas, TRAIL, TNFR1 and E. tenella Development

The results of this study show that, during E. tenella infection, all cell groups exhibited relatively high parasite infection rates, indicating successful model establishment. The parasite infection rate in the host cells treated with NC siRNA did not differ significantly from that in the sporozoite-inoculated control group without drug treatment, demonstrating that NC siRNA has no significant effect on parasite infectivity.

In this study, at 24 h and 120 h post-E. tenella infection, the parasite infection rate in host cells treated with Fas siRNA significantly increased, indicating that, during the mid-to-late developmental stages of E. tenella, Fas siRNA enhanced parasite infectivity in the host cells. This also suggested that low expression levels of Fas promote parasite replication. Additionally, at 72 h post-infection, the parasite infection rate in the host cells treated with TRAIL siRNA was significantly elevated, indicating that, during the mid-developmental stage of E. tenella, reduced TRAIL expression is associated with increased parasite burden. Previous studies have shown that TRAIL overexpression in human intestinal epithelial cells during late-stage Cryptosporidium infection markedly reduces the parasite load [10]. TRAIL not only induces apoptosis but also inhibits hepatitis B virus (HBV) replication [19]. In the present study, TRAIL siRNA-mediated downregulation of TRAIL expression led to increased parasite infection rates, which was consistent with TRAIL’s reported suppressive effects on the development of other pathogens [10,19].

Furthermore, at 72 h and 120 h post-infection, the parasite infection rates in the host cells treated with TNFR1 siRNA significantly increased, indicating that high levels of TNFR1 expression inhibit E. tenella development during its mid-to-late developmental stages. It has been reported that in TNFR1−/− hosts infected with Plasmodium chabaudi, gametocyte numbers increase [20]. The current findings (where TNFR1 siRNA knockdown resulted in enhanced parasite infectivity) were consistent with the known regulatory role of TNFR1 in pathogen development [20].

4.3. Relationship Between Fas, TRAIL, TNFR1, and Apoptosis

The results showed that, during the early developmental stage of E. tenella, the rates of early apoptosis, late apoptosis, and necrosis in the host cells were significantly reduced. In contrast, during the mid-to-late developmental stages of E. tenella, the rates of early apoptosis, late apoptosis, and necrosis in host cells significantly increased.

The present study also demonstrated that, compared to the NC siRNA control group, when E. tenella was in its early developmental stage, there were no significant differences in the rates of early apoptosis, late apoptosis, and necrosis between the host cells treated with Fas siRNA and those in the control group. However, during the parasite’s mid-to-late developmental stages, the host cells treated with Fas siRNA exhibited significantly reduced levels of early and late apoptosis as well as necrosis. These findings indicated that Fas overexpression had no significant effect on host cell apoptosis during the early phase of E. tenella development, whereas it markedly promoted apoptosis during the mid-to-late stages. Upon trimerization, Fas recruits cytoplasmic FADD [a protein containing a homologous death domain (DD)] through DD–DD interaction. The death effector domain (DED) of FADD then binds to the DED of pro-caspase-8, forming the death-inducing signaling complex (DISC). This leads to proteolytic processing and activation of caspase-8 (FLICE), which subsequently cleaves other procaspases into their active subunits, thereby activating the caspase cascade and promoting apoptotic cell death. Previous studies have shown that silencing Kindlin-2 significantly upregulates the expression of both Fas and Fas ligand (FasL), thereby activating the Fas/FasL signaling pathway, ultimately inducing apoptosis [21]. In Hong Kong oysters (Crassostrea hongkongensis), Vibrio alginolyticus or V. hemolyticus stimulation induced a significant increase in the relative expression of ChFasL in hemocytes, and ChFasL knockdown using dsRNA resulted in a marked reduction in hemocyte apoptosis [22]. Furthermore, Fas siRNA has been shown to effectively suppress Fas gene expression and inhibit cardiomyocyte apoptosis [23]. The current findings are consistent with previous reports on the role of Fas in regulating apoptosis in host cells infected with other pathogens [22].

The experimental results also showed that, during E. tenella’s mid-to-late developmental stages, the host cells treated with TRAIL siRNA exhibited significantly or highly significantly reduced rates of early apoptosis, late apoptosis, and necrosis, indicating that TRAIL overexpression promoted host cell apoptosis at these stages. TRAIL initiates apoptosis by binding to its death receptors DR4 or DR5, leading to DISC formation and subsequent activation of caspase-8. Notably, TRAIL-mediated apoptosis could bypass the p53-dependent pathway, selectively inducing death in tumor cells while sparing most normal cells [24]. Clarke et al. demonstrated that neutralizing antibodies against TRAIL blocked reovirus-induced TRAIL secretion and prevented reovirus-mediated apoptosis [11]. Moreover, astrocyte-secreted TRAIL bound to death receptor 5 (DR5) on motor neurons, resulting in caspase-8 activation and neuronal death [25]. Human cytomegalovirus (HCMV)-encoded glycoprotein gpUL4 bound to TRAIL with low molar affinity, acting as a decoy receptor by preventing TRAIL from interacting with its functional receptors, thereby inhibiting soluble TRAIL-induced NK cell apoptosis [26]. The present results aligned with prior evidence demonstrating the pro-apoptotic function of TRAIL in host cells during infections with other pathogens [11,26].

Additionally, this study showed that, in the early developmental stage of E. tenella, host cells treated with TNFR1 siRNA displayed significantly increased rates of early and late apoptosis and necrosis. However, during the mid-to-late stages, the TNFR1 siRNA treatment led to significantly or highly significantly reduced apoptosis and necrosis rates. Silencing of the TNFR1 or RIPK1 genes was found to attenuate p38 activation and prevent PARP cleavage, thereby inhibiting apoptosis [27]. siRNA-mediated knockdown of TNFR1 significantly reduced the expression of TNFR1, caspase-8, TRADD, and the overall apoptosis rate [28]. Transfection with TNFR1 siRNA also markedly decreased the HBV-enhanced transcriptional activity of SREBP1 and PPAR, confirming that HBV induces hepatocyte apoptosis through the TNFR1-mediated apoptotic pathway [29]. These results suggested that, during early E. tenella development, TNFR1 overexpression activated anti-apoptotic signaling pathways and suppressed host cell apoptosis, whereas during its mid-to-late stages of development, TNFR1 overexpression promoted apoptosis—findings consistent with those of previous reports [28,29].

Furthermore, in terms of the extent of apoptosis induced in the host cells, the Fas signaling pathway induced a higher level of apoptosis than the TNFR1 pathway, while the TNFR1 pathway exerted a greater pro-apoptotic effect than the TRAIL pathway. This indicated that different death receptor-mediated apoptotic pathways contribute distinctly to E. tenella-regulated host cell death, with the Fas and TNFR1 pathways being the predominant contributors.

4.4. Relationship Between Fas, TRAIL, and FADD Gene Expression

The results showed that, during the early developmental stage of E. tenella, there was no significant effect on FADD mRNA expression in the host cells. However, during the mid-to-late stages, E. tenella promoted the expression of FADD mRNA. In E. tenella’s mid-to-late developmental phases, the host cells treated with Fas siRNA exhibited significantly reduced FADD mRNA levels, indicating that Fas siRNA effectively suppressed FADD mRNA expression in E. tenella-infected host cells. This also suggested that upregulated Fas enhanced FADD mRNA expression. As a key adaptor protein in the Fas-mediated apoptotic pathway, abnormal FADD expression directly impairs Fas/FasL-induced apoptosis. Cells lacking FADD expression are resistant to Fas-triggered cell death [30,31]. Following RNAi-mediated silencing of Fas, cell proliferation remains unchanged, while the expression levels of the FADD, FLIP, and TRAF proteins markedly decrease, with no significant change in NF-κB expression [32]. Moreover, transfection of Fas-siRNA into mouse brain microvascular endothelial cells significantly inhibits both Fas and downstream FADD expression [28]. Thus, the present findings are consistent with previous reports [28,32].

Following TRAIL siRNA transfection, FADD mRNA expression in host cells was significantly downregulated during the mid-to-late stages of E. tenella development, indicating that the suppression of TRAIL reduced FADD expression. FADD functions as a critical signal transduction protein in the TRAIL-mediated apoptotic pathway. TRAILR2 signals through FADD, and FADD-deficient cells failed to undergo apoptosis induced by DR3 or DR4 activation [33]. It has been reported that upon silencing of c-FLIP or RIP, increasing the TRAIL concentration progressively enhances its pro-apoptotic effect [34]. The engagement of TRAIL or Fas death receptors could trigger the assembly of a cytoplasmic signaling complex—caspase-8/FADD/RIPK1—which promotes the activation of NF-κB [35]. The current study demonstrated that, during the mid-to-late stages of E. tenella infection, elevated TRAIL expression leads to increased FADD expression, which aligns with prior evidence on TRAIL’s regulatory role in modulating FADD expression [35].

4.5. Relationship Between Fas and Daxx mRNA Expression

The results indicated that, during the early developmental stage of E. tenella, there was no significant impact on Daxx mRNA expression. However, during its mid-to-late developmental stages, E. tenella promoted Daxx mRNA expression. Furthermore, transfection with Fas siRNA led to a significant reduction in Daxx mRNA levels during the mid-to-late stages, suggesting that Fas siRNA effectively suppresses Daxx expression. Daxx is recognized as a pro-apoptotic protein downstream of Fas that activates the JNK pathway independently of FADD [36]. Daxx overexpression markedly enhances Fas-mediated apoptosis, which depends on pathway activation [37]. Additionally, in host cells infected with respiratory enteric viruses and exhibiting high Fas expression, Daxx expression is upregulated, highlighting its important role in Fas-induced apoptotic processes [38]. These findings are consistent with previous observations regarding the regulation of Daxx expression by Fas in host cells infected with other pathogens [38].

4.6. Relationship Between TNFR1 and TRADD Protein Expression

This study showed that TRADD protein expression in E. tenella-infected host cells was significantly or highly significantly greater than in Group C, consistent with previous findings [4]. Moreover, transfection with TNFR1 siRNA resulted in significantly reduced TRADD mRNA expression, indicating that upregulated TNFR1 promotes TRADD expression. TNF-α exerts its biological effects primarily through TNFR1, and upon TNF-α stimulation, both TNFR1 and TRADD expression are upregulated in host cells [15]. Previous studies have shown that TNFR1-siRNA significantly suppresses TNFR1, caspase-8, and TRADD expression, along with markedly decreasing the apoptosis rate [28]. During early E. tenella infection, TRADD exerts anti-apoptotic effects, whereas in later stages, it promotes apoptosis [4]. The present results confirm that TNFR1 siRNA significantly inhibits TRADD expression in E. tenella-infected host cells, supporting the previously reported roles of TNFR1 in regulating TRADD expression [15,28].

4.7. Relationship Among Fas, TRAIL, TNFR1 and Caspase-8 Activity

The experimental data showed that, during the early stages of E. tenella development, caspase-8 activity in host cells did not change significantly. However, during the mid-to-late stages, caspase-8 activity was markedly increased. Additionally, following the silencing of the Fas, TRAIL, or TNFR1 genes, caspase-8 activity remained unchanged in the early phase but was significantly reduced to varying degrees during the mid-to-late stages of E. tenella development. These results indicate that, during the parasite’s mid-to-late developmental stages, Fas, TRAIL, and TNFR1 all contribute to enhanced caspase-8 activity. In the extrinsic apoptotic pathway, caspase-8 serves as a hallmark executioner protease, with FADD acting as its upstream activator; FADD thus promotes apoptosis by activating caspase-8 [39]. Oxidative stress has been shown to upregulate intracellular expression of FasL, Fas, FADD, caspase-8, and caspase-3 [40]. In studies on FADD-induced SMC apoptosis, overexpression of FADD under FasL and cycloheximide treatment has been found to correlate with increased caspase-8 activity [41]. Prior research has established that both Fas and TRAIL could regulate FADD expression in host cells [28,35]. Additionally, TRADD requires interaction with FADD to exert its pro-apoptotic function [42], and silencing TRADD results in significantly reduced FADD expression and apoptosis rate in E. tenella-infected host cells [4]. Furthermore, elevated TNFR1 expression enhances TRADD levels. Therefore, in E. tenella-infected host cells, Fas, TRAIL, and TNFR1 might each regulate FADD expression through their respective signaling pathways, thereby modulating caspase-8 activity.

5. Conclusions

In conclusion, when E. tenella was in its early developmental stages, Fas and TNFR1 expression in host cells increased. Upregulated TNFR1 promoted TRADD expression and exerted an anti-apoptotic effect. During the mid-to-late stages of E. tenella development, Fas, TRAIL, and TNFR1 expression was elevated. Overexpressed Fas, TRAIL, and TNFR1 activate distinct apoptotic pathways (Fas-FADD, Fas-Daxx, TRAIL-FADD, and TNFR1-TRADD), leading to increased caspase-8 activity and collectively promoting host cell apoptosis. In terms of the magnitude of these pro-apoptotic effects, each pathway contributes in the following order: Fas signaling pathway > TNFR1 signaling pathway > TRAIL signaling pathway. This lays a theoretical foundation for investigating the regulatory mechanisms by which E. tenella induces host cell apoptosis via the death receptor pathway.