You are viewing the site in preview mode

Skip to main content
Download PDF

Immunomodulatory effects of Eimeria maxima surface antigen (EmSAG) as an IFN-γ inhibitory molecule on peripheral blood mononuclear cells (PBMCs) and T cell subsets in chickens

Veterinary Research volume 56, Article number: 103 (2025) Cite this article

Abstract

Eimeria maxima (E. maxima) infection inhibits the expression of IFN-γ, a cytokine that is essential for the Th1 immune response and plays a key role in combating this parasite. In our preliminary investigations, we identified the E. maxima surface antigen (EmSAG) as an inhibitory molecule of IFN-γ. EmSAG was screened and characterised from an E. maxima sporozoite cDNA expression library. The present study aimed to evaluate the immunomodulatory effects of EmSAG on chicken peripheral blood mononuclear cells (PBMCs) and various T cell subsets. We analysed cell proliferation, nitric oxide (NO) release, and cytokine transcription. The results revealed that EmSAG boosts PBMC proliferation and promotes differentiation of CD4+/CD8+ T cells. Additionally, stimulation with EmSAG significantly inhibited NO release and IFN-γ transcription while enhancing the transcription of IL-4, IL-10, and TGF-β1 in chicken PBMCs. The sorting purity of T cell subsets was as follows: CD8+ (96.90%), CD4+ (86.25%), CD4+CD25− (89.14%), and CD4+CD25+ regulatory T cells (Tregs; 92.16%). These purified subsets were co-incubated with EmSAG to analyse the transcription of hallmark cytokines associated with Th1, Th2, and Treg responses. EmSAG significantly inhibited the transcription of IFN-γ and IL-2 in both CD4+ and CD8+ T cells, while promoting the expression of IL-10, TGF-β1, and CTLA-4 in Tregs. Moreover, depletion of CD25+ cells reversed the EmSAG-induced suppression of IL-2 transcription and reduced its stimulating effects on IL-4 and IL-10 transcription in CD4+CD25− T cells. These findings highlight the role of EmSAG as an inhibitor of IFN-γ, facilitating immune evasion by attenuating the Th1 immune response and modulating Treg cell function. This study provides critical insights into the immune evasion mechanisms utilised by chicken coccidia.

Introduction

Chicken coccidiosis is a parasitic enteric disease caused by single or mixed infections of various Eimeria species. It continues to impose significant economic burdens on the global poultry industry, leading to serious clinical symptoms such as mortality, bloody feces, weight loss, and reduced egg production [1]. A particular concern is the disease’s ability to make chickens more susceptible to secondary infections by opportunistic pathogens such as Clostridium perfringens [2, 3].

The rise of widespread drug resistance in Eimeria strains, along with increasing regulatory restrictions on anticoccidial drug residues in poultry products, has significantly hindered current control strategies [4, 5]. Although traditional live attenuated vaccines offer some protection, their practical application is fraught with challenges, such as the risk of parasite spread, high production costs, and manufacturing limitations [1, 4].

Therefore, gaining a deeper understanding of the competition between chicken coccidia and the host immune system, as well as the possible mechanisms of immune evasion, is essential for advancing the study of parasite pathogenicity and developing novel vaccines.

In the long-term evolution process of parasites, they have developed sophisticated strategies to evade the host's immune system. This is achieved by secreting immunomodulatory factors and manipulating immune responses, which enables them to survive, reproduce, and induce diseases. Parasite-derived enzymes, or surface glycoproteins, exert various immunosuppressive effects through antioxidant activity, inflammatory suppression, immune cell apoptosis induction, and inhibition of lymphocyte activation.

Protozoan infections, for example, can inhibit the expression of Th1 cytokines in the host, and various cytokine inhibitory molecules have been identified [6,7,8,9]. For instance, the Trypanosoma cruzi (T. cruzi) membrane glycoprotein AGC10 (glycosylphosphatidylinositol-anchored) impairs macrophage functions and suppresses the production of IFN-γ/IL-2 in PBMCs [10]. Similarly, the lipophosphoglycan (LPG) from Leishmania shawi (L. shawi) modulates the cytokine profiles of peritoneal macrophage by reducing IL-12 and elevating IL-10 [11].

These coordinated mechanisms collectively create an immunosuppressive microenvironment that supports parasitic survival and chronic infection. In the context of chicken coccidia infections, Cornelissen et al. reported that, at 4 days post-infection (dpi) with E. maxima, acervulina and tenella, there was a suppression of IFN-γ mRNA across different intestinal segments (jejunum/duodenum/cecum), along with increased expression of IL-4/IL-10 [12]. Additionally, Walston's study demonstrated a significant reduction in IFN-γ mRNA levels in the spleen by 11 dpi, which decreased to half the level observed in uninfected controls [13].

These studies suggest that other molecules may regulate or inhibit IFN-γ and related cytokines during chicken coccidia infections, although the molecular mediators involved remain largely uncharacterised. Notably, Chen et al. identified E. maxima surface antigen (EmSAG) as an IFN-γ inhibitory molecule. They demonstrated that EmSAG could inhibit IL-12p40 secretion in chicken HD11 macrophages and bone marrow-derived dendritic cells (BMDCs). Mechanistic studies showed that EmSAG activates phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) within the mitogen-activated protein kinase (MAPK) cascade, establishing a regulatory axis for IL-12 suppression [14].

It is suggested that molecules secreted by Eimeria parasites may suppress the host's IFN-γ response by inhibiting its expression, ultimately enhancing the survival of the parasites. The limited identification of Eimeria’s IFN-γ inhibitory molecules highlights the urgent need to characterise such molecules. Doing so could reveal new targets for disrupting Eimeria’s immune evasion and inform the development of next-generation control strategies.

Pathogens evade host immunity by suppressing essential immune cell functions, which include activation, proliferation, differentiation, and regulatory processes. CD4+CD25+ regulatory T cells (Tregs) play a crucial role as mediators through contact-dependent mechanisms and the production of immunosuppressive cytokines.

Parasite-activated Tregs orchestrate immune suppression by secreting regulatory cytokines (IL-10, TGF-β, and IL-35). They also release cytotoxic mediators (granzyme B and perforin-1), and upregulate cytotoxic T lymphocyte-associated protein 4 (CTLA-4) [15,16,17]. These effectors can inhibit Th1/Th2 polarisation, CTLs cytotoxicity, and NO production, all of which contribute to parasite survival and prevent excessive immunopathological reactions.

Additionally, avian coccidia trigger a significant increase in the proportion of Tregs in chickens, suggesting that Eimeria induces the activation of regulatory T cell subsets [18]. An oral challenge with mixed Eimeria spp. (E. tenella, E. acervulina, and E. maxima) reduces the population of CD4+ T cells in the cecal mucosa while increasing the proportion of Tregs and their secreted specific cytokines (IL-10 and TGF-β) [13, 19]. These findings indicate that chicken Tregs and the immunosuppressive molecules they produce may play a role in the pathogenesis of chicken coccidiosis.

A potential mechanism for immune evasion by Eimeria spp. involves their induction of Treg activation in the host, leading to high expression of immune inhibitory cytokines (IL-10 and TGF-β) and a subsequent suppression of IFN-γ-mediated Th1 responses. This process promotes parasite survival and infection [20].

Building on previous evidence that EmSAG, an E. maxima-derived IFN-γ inhibitor, suppresses IL-12 in innate immune cells [14], this study explores its role in T cell-mediated adaptive immunity. We first assessed the functionality of PBMCs by measuring cell proliferation, CD4+/CD8+ T cells proportion, NO release, and cytokines transcription. We then analysed the specificity of cytokine responses induced by rEmSAG stimulation in chicken CD4+, CD8+, and CD4+CD25+ T cells. Finally, we conducted a CD25+ cell depletion assay to examine the dynamic changes in Th1/Th2/Treg cytokines, thereby elucidating the role of Tregs in modulating CD4+ T cell functionality. This study not only provides critical insights into the immune evasion strategies of Eimeria spp., but also establishes a theoretical foundation for improving the effectiveness of coccidiosis vaccines in chickens.

Materials and methods

Animals, strain and plasmid

In this study, newborn Hy-Line white chicks used for experiments and were raised in a thoroughly cleaned feeding room. The floors, walls, and cages were sanitized using hot water at temperatures exceeding 90 °C. All feed troughs and utensils in the room were soaked in hot water above 90 °C for more than 10 min to inactivate any potential remaining coccidia oocysts. Additionally, the room and all utensils were routinely disinfected with a 0.1% benzalkonium bromide solution to eliminate any potential contamination from other pathogens. The chicks had unrestricted access to drinking water and feed, which did not contain anticoccidial drugs.

Sporulated oocysts of the E. maxima Jiangsu strain were stored in a 2.5% (w/v) potassium dichromate solution at 4 ℃. Eimeria-free chicks were orally infected every three months for rejuvenation and propagation purposes. The pET-32a-EmSAG plasmid, which encodes the EmSAG antigen (RefSeq ID: XP_013337465.1), was constructed and maintained in our laboratory.

Purification of rEmSAG

E. coli BL21 (DE3) containing the pET-32a-EmSAG plasmid was cultured in LB medium supplemented with ampicillin (100 µg/mL) at 37 ℃ with shaking at 180 r/min until the OD600 value reached approximately 0.6. The recombinant protein EmSAG (rEmSAG) was expressed by adding Isopropyl β-D-thiogalactoside (IPTG; 100 mM; Yfxbio Biotech. Co., Ltd., Nanjing, China) and shaking the culture for 5 h under the same conditions. The bacterial cells were collected and then disrupted ultrasonically.

Subsequently, rEmSAG was purified using a His-tag purification column (Cytiva, Marlborough, MA, USA), and purity verification was performed using 12% (w/v) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). To eliminate endotoxin from the purified protein, a ToxinEraser™ Endotoxin Removal Kit (GenScript, Nanjing, China) was utilised. Finally, the protein concentration was determined using a Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA).

Immunoblot analysis of rEmSAG

Sera were harvested from E. maxima-experimentally infected chickens as well as coccidia-free chickens using a previously established method [21]. The recognition of rEmSAG was evaluated through immunoblot analysis, utilising serum from E. maxima-infected chickens and a mouse-derived anti-His tag monoclonal antibody (Proteintech, Wuhan, China).

In brief, rEmSAG was separated using 12% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Merck, Darmstadt, Hessen, Germany) using a Trans-Blot® Turbo™ Transfer System (Bio-Rad, Hercules, CA, USA). After the transfer, the PVDF membrane was blocked with 5% (w/v) skimmed milk (Solarbio, Beijing, China) for 2 h at room temperature. The membrane was then incubated overnight at 4 °C on a rotary shaker (80 r/min) with either E. maxima-infected chicken serum (1:100 dilution) or mouse-derived anti-His tag monoclonal antibody (1:8000 dilution). Coccidia-free chicken serum served as a negative control.

Following the antibody incubation, the membrane was rinsed six times with TBST (5 min per rinse) and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-chicken IgY (H + L) (Abbkine, Atlanta, GA, USA) (1:5000 dilution) or anti-mouse IgG (H + L) (Affinity Biosciences, Cincinnati, OH, USA) (1:10 000 dilution) at 37 °C for 1 h. After the secondary antibody incubation, the membrane received additional TBST rinses and was reacted with Chemistar™ High-sig ECL Western Blotting Substrate (Tannon, Shanghai, China). The protein bands were visualised using a Tannon 5200 series automatic chemiluminescence imaging system (Tannon, Shanghai, China).

Separation and culture of chicken PBMCs

Chicken PBMCs were isolated using a previously described density gradient centrifugation protocol [22]. The freshly isolated PBMCs were washed with sterile phosphate-buffered saline (PBS) and then resuspended in RPMI 1640 complete culture medium (Gibco, Waltham, MA, USA). This medium contained 10% (v/v) fetal bovine serum (FBS; Excell Bio, Shanghai, China) and 1% (v/v) penicillin–streptomycin solution (Gibco, Waltham, MA, USA). The cell concentration was counted and adjusted to 1 × 106 cells per 1 mL in the complete culture medium.

Effect of rEmSAG on the proliferation of chicken PBMCs

To assess the proliferation effect of rEmSAG on chicken PBMCs, we used the CellTrace™ Cell Proliferation Kit (Invitrogen, Waltham, MA, USA) following the manufacturer’s instructions. First, a suspension of PBMCs was prepared based on experimental requirements and labelled with 1 μM CellTrace™ Far Red dye (Invitrogen, Waltham, MA, USA) working solution. The cell suspension was incubated with the dye at 37 °C with 5% CO2 for 20 min. Staining was then terminated by washing the cells with PBS (5 times suspension volume), followed by centrifugation and a brief incubation with complete medium at 4 °C for 5 min.

Next, the PBMCs were resuspended in freshly pre-warmed culture medium (37 °C) and seeded into 12-well cell culture plates. The cells were incubated for 48 h at 37 °C with 5% CO2 in the presence of the following treatments: rEmSAG at final concentrations of 10, 20, 40, and 80 μg/mL, PBS (negative control), pET-32a tag protein, and lipopolysaccharides (LPS; 2 μg/mL; positive control; Sigma-Aldrich, Saint Louis, MO, USA).

After incubation, the cells were transferred to 1.5 mL tubes, centrifuged at 300 × g for 10 min at 4 °C, rinsed twice with 1 mL PBS, and resuspended in 400 μL PBS. Additionally, two control groups were established: one group was stained but not cultured to determine the fluorescence intensity of the parent generation, while the other was cultured but not stained to determine the auto-fluorescence intensity of the progeny generation. Finally, the fluorescence intensity of all cells was analysed by flow cytometry (Beckman Coulter, Brea, CA, USA) to assess the cell proliferation index.

Impacts of rEmSAG on the proportion of CD4+/CD8+ T lymphocytes in chicken PBMCs

PBMCs suspension was seeded into a 12-well cell culture plate and treated with varying final concentrations of rEmSAG (10, 20, 40, and 80 μg/mL) in a cell incubator for 24 h at 37 °C. Control groups included PBS, His-tagged pET-32a protein, and 2 μg/mL LPS.

After treatment, the cells were centrifuged at 300 × g for 10 min at 4 °C and resuspended in 100 μL PBS. Surface staining was performed by adding 1 μL of mouse-derived anti-chicken CD3, CD4 and CD8 antibodies (Southernbiotech, Birmingham, AL, USA) conjugated with fluorescein isothiocyanate (FITC), allophycocyanin (APC), and P-phycoerythrin (PE). The cells were incubated with the antibodies in the dark at 4 °C for 30 min.

Following the incubation, the cells were rinsed with 900 μL of PBS and then resuspended in 400 μL PBS. Multiple control groups need to be established for the subsequent flow cytometry serial gating strategy, including a blank control without any fluorescent staining, single-staining controls with different fluorescent antibodies, and fluorescence minus one (FMO) control. Finally, to determine the proportion of CD4+/CD8+ T lymphocytes, all cells were analysed using flow cytometry.

Effects of rEmSAG on total nitric oxide release and cytokines transcription in chicken PBMCs

Fresh chicken PBMCs were resuspended in DMEM complete medium (Gibco, Waltham, MA, USA) containing 1% penicillin–streptomycin solution and 10% FBS, adjusting the cell density to 1 × 106 cells per 1 mL. The chicken PBMCs were then stimulated with rEmSAG at concentrations of 10, 20, 40, and 80 μg/mL (n = 3 independent replicates per group) in a 12-well cell culture plate. Control groups included PBS, pET-32a tag protein, and 2 ug/mL LPS.

After incubation, the cell culture was centrifuged at 300 × g for 10 min at 4 °C to separate the supernatant and pellet for the determination of NO release and cytokine transcription levels. The concentrations of NO2− and NO3−, which are stable metabolites of NO, in the supernatant were measured using a total nitric oxide (NO) assay kit (Beyotime, Nanjing, China) following the nitrite reductase method. The absorbance value at OD540 wavelength was recorded using a microplate reader (ThermoFisher Scientific, Waltham, MA, USA), and the total NO release levels for the different treatment groups were calculated by preparing a standard curve with sodium nitrite solution standards at concentrations of 0, 1, 2, 5, 10, 20, 40, 60, and 80 μM.

The cell pellet was transferred to 1.5 mL RNase-free tubes, and total RNA was extracted and converted into cDNA using the Total RNA Extraction Reagent (Vazyme, Nanjing, China) and HiScript III RT SuperMix (Vazyme, Nanjing, China), respectively. In brief, 1 μg of total RNA was reverse transcribed into 20 μL of cDNA, which was then diluted 5 times with RNase-free water (Transgen, Beijing, China).

Following our established protocol [22], a Quantitative PCR (qPCR) reaction system was prepared with a total volume of 10 μL using cytokine-specific primers detailed and validated in Table 1. The reaction mixture contained 5 μL of 2 × PerfectStartTM Green qPCR Super Mix (+ Dye II) (Transgen, Beijing, China), 0.2 μL of forward and reverse primers at a concentration of 10 μM each, 1 μL of diluted cDNA, and 3.6 μL of RNase-free water.

Table 1 Primer sequences used for quantitative real-time PCR
Full size table

The qPCR reaction program was set up for a two-step qPCR assay. The initial step involved pre-denaturation at 94 °C for 30 s, followed by 40 cycle reactions of 94 ℃ for 5 s, and 60 ℃ for 30 s. The final melting curve consisted of 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. The experimental data were analysed using the 2−ΔΔCt method [23] to assess the effect of rEmSAG on cytokine transcription in chicken PBMCs.

Immunomagnetic bead sorting of chicken T cell subsets

CD8+ T cells, CD4+CD25− T cells, and CD4+CD25+ Tregs were sorted from freshly isolated chicken PBMCs following our established protocol [22]. The PBMCs were first incubated with mouse-derived anti-chicken CD8α or CD4 antibodies labelled with PE, along with anti-PE antibodies labelled with MicroBeads (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany) for 30 min at 4 °C. The labelled cells were then transferred to a magnetic bead sorting system (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany) for the sorting of chicken CD8+ or CD4+ T cells.

In a similar manner, chicken PBMCs were incubated with FITC-conjugated human anti-chicken CD25 antibodies (Bio-Rad, Hercules, CA, USA) and anti-FITC MultiSort MicroBeads antibodies (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany) for 30 min at 4 °C. This step allowed for the separation of CD25+ cells and CD25− PBMCs through a magnetic bead sorting system.

Subsequently, the sorted CD25+ cells or CD25− PBMCs were further incubated with PE-labelled mouse anti-chicken CD4 antibodies and anti-PE MicroBeads to isolate CD4+CD25+ or CD4+CD25− T cells. Finally, the purity of the various T cell subsets obtained through immunomagnetic bead sorting was confirmed using flow cytometry.

Effects of rEmSAG on cytokines transcription in chicken T cell subsets

Freshly sorted chicken T cell subsets, including CD8+, CD4+, and CD4+CD25+ T cells were stimulated with 20 μg/mL rEmSAG at 37 °C for 6 h in 12-well cell culture plates. The control groups included PBS, pET-32a tagged protein, and 2 µg/mL LPS. After the incubation period, the cells were transferred to 1.5 mL tubes by centrifugation at 300 × g at 4 °C for 10 min. Total RNA was extracted from the cell pellet and reverse transcribed into cDNA. Subsequently, a qPCR assay was performed using the cDNA and cytokine-specific primers, detailed and validated in Table 1. Finally, the effects of rEmSAG on the transcription of cytokines associated with Th1, Th2, and Treg responses in different T cell subsets of chickens were assessed using the 2−ΔΔCt method.

Effects of rEmSAG on cytokines transcription of chicken CD4+CD25− T cells following CD25+ cell depletion

CD4+CD25− T cells were isolated by depleting CD25+ cells from chicken CD4+ T cells. The isolated cells were incubated with 20 μg/mL of rEmSAG at 37 °C with 5% CO2 for 6 h. Control groups included PBS, His-tagged pET-32a protein, and 2 µg/mL LPS in a 12-well cell culture plate. After incubation, total RNA was extracted from the cells and reverse transcribed into cDNA for qPCR experiments. CD4+ T cells were used as a reference to assess the potential regulatory effects of CD25+ cells by measuring the transcription levels of Th1, Th2, and Treg cytokines.

Statistical analysis of data

The experimental data was analysed using SPSS 23.0 system software (IBM, Armonk, NY, USA). To assess the differences between treatment groups, we applied Duncan’s test within a one-way ANOVA framework. The results are presented as the mean ± standard deviation (SD). Different letters indicate a significant difference (p < 0.05), while the same letters denote no significant difference (p > 0.05). Furthermore, we compared the transcription of cytokines in CD4+ T cells before and after the depletion of CD25+ cells using an independent sample t-test. The levels of significance were defined as follows: *p < 0.05, **p < 0.01, and “ns” for nonsignificant results.

Results

Purification and immunoblot analysis of rEmSAG

The rEmSAG protein was expressed in E. coli BL21 (DE3) and purified using a His-tag purification column. The purification was verified through SDS-PAGE and immunoblot assays. As shown in Figure 1, the results indicated that compared with before purification (Figure 1, lane 1), the molecular weight of purified rEmSAG is 43 kDa, consistent with theoretical predictions, confirming successful purification (Figure 1, lane 2).

Figure 1
figure 1

Purification and immunoblot analysis of rEmSAG. lane M: standard molecular marker for protein; lane 1: rEmSAG before purification; lane 2: purified rEmSAG; lane 3: His-tag in purified rEmSAG was identified by mouse-derived anti-His tag antibody; lane 4: purified rEmSAG was identified by serum from E. maxima-infected chicken; lane 5: purified rEmSAG was identified by serum from coccidia-free chicken.

Full size image

Furthermore, the immunoblot analysis demonstrated that rEmSAG is recognised by a mouse-derived anti-His tag antibody and serum from E. maxima-infected chickens (Figure 1, lane 3 and lane 4), but not by serum from coccidia-free chickens (Figure 1, lane 5). This indicates that rEmSAG possesses satisfactory antigenicity and can elicit an immune response in the host against EmSAG.

Regulatory effects of rEmSAG on the immune function of chicken PBMCs

Effect of rEmSAG on the proliferation of chicken PBMCs

Parent generation cells were labeled with a fluorescent dye, and subsequent generations were tracked using flow cytometry through the dye dilution method. The percentage of progeny cells generated by rEmSAG-stimulated chicken PBMCs is presented in Additional file 1. After stimulation with rEmSAG at concentrations of 10, 20, and 40 μg/mL, the percentages of neogenic PBMCs were found to be 54.65 ± 0.8491 (%), 56.86 ± 1.564 (%), and 56.58 ± 0.9765 (%), respectively. These values were all significantly higher than those observed in the pET-32a tag protein (49.31 ± 0.6232) (%) and PBS (50.30 ± 0.3988) (%) control groups (p < 0.05). In contrast, the percentage of neogenic PBMCs stimulated with rEmSAG at a concentration of 80 μg/mL was 47.87 ± 0.7139 (%), which was not significantly different from the pET-32a tag protein control group (p > 0.05).

The flow cytometry histogram data are illustrated in Figures 2A–F. Figures 2A and B display the auto-fluorescence intensity of progeny generation cells and the fluorescence intensity of parent generation cells, respectively. The proliferation percentages of chicken PBMCs after 48 h stimulation are shown in Figures 2C-E (PBS, pET-32a-tagged protein, and LPS) and Figures 2F1–4 (10–80 μg/mL rEmSAG). To quantify the impact of rEmSAG on the proliferative capability of PBMCs, the percentages were calculated and converted into a cell proliferation index, as shown in Figure 2G. Notably, rEmSAG at concentrations of 10, 20, and 40 μg/mL significantly promoted the proliferation of chicken PBMCs (p < 0.05). However, the 80 μg/mL rEmSAG concentration did not show a significant difference compared to the PBS and His-tagged pET-32a protein groups (p > 0.05).

Figure 2
figure 2

Detection of cell proliferation of chicken PBMCs by flow cytometry. A: The auto-fluorescence intensity of the progeny generation cells in chicken PBMCs. B: The fluorescence intensity of the parent generation cells in chicken PBMCs. C: The percentage of neogenic chicken PBMCs following stimulation with PBS. D: The percentage of neogenic chicken PBMCs following stimulation with pET-32a tag protein. E: The percentage of neogenic chicken PBMCs following stimulation with LPS. F1-4: The percentage of neogenic chicken PBMCs following stimulation with 10, 20, 40, and 80 μg/mL rEmSAG, respectively. G: The cell proliferation index was used to indicate the effect of rEmSAG on the proliferation in chicken PBMCs.

Full size image

Effects of rEmSAG on T lymphocyte subsets proportion in chicken PBMCs

After incubating rEmSAG-stimulated chicken PBMCs with fluorescent antibodies, we analysed the cell surface markers (CD3, CD4, and CD8) in each treatment group using flow cytometry. The gating strategy used for detecting the proportions of CD4+/CD8+ T lymphocytes is illustrated in Figure 3. The contour plots in Figure 4A and C display the proportions of CD3+CD4+ and CD3+CD8+ T lymphocytes, respectively. As shown in Figure 4B, all concentrations of rEmSAG induced a significant increase in the proportion of CD4+ T lymphocytes in chicken PBMCs compared to both the PBS and His-tagged pET-32a protein stimulation groups (p < 0.05). Similarly, the proportion of CD8+ T lymphocytes was also significantly elevated in response to rEmSAG stimulation (p < 0.05), as depicted in Figure 4D.

Figure 3
figure 3

The flow cytometry serial gating strategy for detection of the proportion of CD4+ and CD8+ T lymphocytes. Chicken PBMCs stimulated by different treatment groups were collected and detected by flow cytometry. Cell debris were excluded by gating according to FSC/SSC, and then singlet cells were obtained by gating according to FSC-A/FSC-H. The CD3+ T cells were gated by the addition of the isotype control and FITC-labelled specific fluorescent antibodies, and the proportion of CD4+ and CD8+ T lymphocytes was measured by extracellular labelling of CD4 or CD8 with specific fluorescent antibodies. Results shown are from a representative experiment. FSC: forward scatter, SSC: side scatter, FITC: fluorescein isothiocyanate, APC: allophycocyanin, PE: P-phycoerythrin.

Full size image
Figure 4
figure 4

Investigation of the proportion of CD4+/CD8+ T lymphocytes in chicken PBMCs. A: CD4+ T lymphocytes were detected by contour plot of flow cytometry using extracellular surface molecular antibodies (anti-CD3 and anti-CD4). B: Proportion of CD4+ T cell subset stimulated by different concentrations of rEmSAG. C: CD8+ T lymphocytes were detected by contour plot of flow cytometry using extracellular surface molecular antibodies (anti-CD3 and anti-CD8). D: Proportion of CD8+ T cell subset stimulated by different concentrations of rEmSAG.

Full size image

Effects of rEmSAG on nitric oxide release and cytokines transcription in chicken PBMCs

The effect of rEmSAG on NO release from chicken PBMCs was evaluated using the Griess assay (nitrite quantification). As demonstrated in Figure 5, rEmSAG significantly inhibited NO release from chicken PBMCs at various concentrations (p < 0.05). Additionally, the impact of rEmSAG on cytokine transcription in chicken PBMCs was assessed using qPCR. The results shown in Figure 5 indicate that rEmSAG significantly inhibited the transcription of IFN-γ while promoting the transcription of IL-2, IL-4, IL-10, and TGF-β1 in chicken PBMCs (p < 0.05).

Figure 5
figure 5

Detection of total nitric oxide release and cytokines transcription in chicken PBMCs. The nitrite reduction method was used to determine the concentration of total nitric oxide in chicken PBMCs following incubation with 10, 20, 40, and 80 μg/mL rEmSAG. qPCR was used to detect the transcription of cytokines including IFN-γ, IL-2, IL-4, IL-10, and TGF-β1 after chicken PBMCs were stimulated with 10, 20, 40, and 80 μg/mL rEmSAG.

Full size image

Regulatory effects of rEmSAG on the immune function of chicken T cell subsets

Assessment of the purity of chicken T cell subsets by flow cytometry

Different T cell subsets were sorted using immunomagnetic bead sorting with indirect labeling, and flow cytometry was employed to assess the purity of the positive cells. The purity levels achieved were as follows: CD8+ for 96.90%, CD4+ for 86.25%, CD4+CD25− for 89.14%, and CD4+CD25+ Tregs for 92.16% (Figure 6). These purified populations were considered suitable for subsequent co-incubation experiments with rEmSAG.

Figure 6
figure 6

Detection of cell sorting purity of T cell subsets in chicken by flow cytometry. A: Magnetic bead sorting of chicken CD8+ T cells. B: Magnetic bead sorting of chicken CD4+ T cells. C: Magnetic bead sorting of chicken CD4+CD25− T cells. D: Magnetic bead sorting of chicken CD4+CD25+ T cells. 1: Lymphocytes in chicken peripheral blood. 2: Singlet cells. 3: Blank cells incubated without fluorescent antibody. 4: CD8+, CD4+, CD4+CD25−, and CD25+ cells before cell sorting. 5: CD8+, CD4+, CD4+CD25−, and CD25+ cells after cell sorting. 6: CD4+CD25+ T cells before cell sorting. 7: CD4+CD25+ T cells after cell sorting.

Full size image

Effects of rEmSAG on cytokines transcription in chicken T cell subsets

In this study, we investigated chicken T cell subsets, including CD8+, CD4+, and CD4+CD25+ Tregs, by stimulating them with rEmSAG for qPCR assays. The results demonstrated that stimulation with rEmSAG significantly inhibited the transcription of IFN-γ and IL-2 while promoting the transcription of IL-4 and IL-10 in CD4+ T cells (p < 0.05; Figure 7A).

Figure 7
figure 7

Effects of rEmSAG on cytokines transcription of T cell subsets in chicken. A: Relative fold changes in IFN-γ, IL-2, IL-4, IL-10, and TGF-β1 transcription were defined in chicken CD4+ T cells following stimulation with 20 μg/mL rEmSAG. B: Relative fold changes in IFN-γ, IL-2, and TNF-α transcription were defined in chicken CD8+ T cells following stimulation with 20 μg/mL rEmSAG. C: Relative fold changes in IL-10, TGF-β1, and CTLA-4 transcription were defined in chicken CD4+CD25+ T cells following stimulation with 20 μg/mL rEmSAG.

Full size image

Similarly, in CD8+ T cells, rEmSAG significantly reduced the transcription levels of IFN-γ and IL-2 (p < 0.05; Figure 7B). However, there was no statistically significant effect observed on the transcription level of TNF-α (p > 0.05; Figure 7B). Additionally, rEmSAG significantly enhanced the expression of hallmark cytokines (IL-10 and TGF-β1) and the surface marker (CTLA-4) in chicken Tregs (p < 0.05; Figure 7C).

Effects of rEmSAG on cytokines transcription of chicken CD4+CD25− T cells following CD25+ cell depletion

To investigate the regulatory role of CD25⁺ T cells, we stimulated both CD4⁺CD25⁻ T cells (which were depleted of the CD25⁺ population) and conventional CD4⁺ T cells with 20 μg/mL rEmSAG for a comparative analysis of the transcriptional analysis of Th1, Th2, and Treg-related cytokines. The results showed that rEmSAG significantly increased the transcription level of IL-2 in chicken CD4+CD25− T cells compared to chicken CD4+ T cells (p < 0.05; Figure 8B). Conversely, rEmSAG significantly decreased the transcription levels of IL-4 (p < 0.05; Figure 8C) and IL-10 (p < 0.05; Figure 8D). However, there were no significant differences in the transcription levels of IFN-γ (p > 0.05; Figure 8A) and TGF-β1 (p > 0.05; Figure 8E). These findings suggest that the depletion of CD25+ T cells can significantly reverse the inhibitory effect of rEmSAG on IL-2 transcription in chicken CD4+CD25− T cells, while also reducing its promotion of IL-4 and IL-10.

Figure 8
figure 8

Effects of rEmSAG on cytokines transcription of chicken CD4+CD25− T cells following CD25+ cell depletion. qPCR was used to detect the transcription of cytokines including IFN-γ, IL-2, IL-4, IL-10, and TGF-β1 in chicken CD4+CD25− T cells stimulated with 20 μg/mL rEmSAG following the depletion of CD25+ cells from CD4+ T cells.

Full size image

Discussion

Chicken coccidiosis is a major disease that poses a significant threat to the global poultry industry. The prevention and control of this disease is increasingly challenging due to rising drug resistance and concerns over drug residues [4, 5]. Additionally, similar to other parasitic protozoa, Eimeria spp. employ various strategies to evade the host immune system. One effective strategy is the secretion of immunomodulatory molecules that inhibit the production of pro-inflammatory cytokines while regulating the activation, proliferation, and differentiation of T and B lymphocytes.

Our prior research identified four molecules that inhibit IFN-γ, one of which is EmSAG (unpublished data). Given the crucial role of the IFN-γ-mediated Th1 immune response in anticoccidial immunity [1], the suppression of IFN-γ by pathogen-derived factors may be a key mechanism for Eimeria spp. to evade the immune response. Moreover, IL-10 produced by Tregs is recognised as another contributor to Eimeria’s immune evasion [20].

This study investigates the dual immunoregulatory function of EmSAG in chicken PBMCs and T cell subsets, specifically its role in suppressing IFN-γ production and inducing IL-10 and TGF-β1. Notably, the depletion of CD25⁺ cells partially reduced the EmSAG-mediated inhibition of IFN-γ, suggesting that CD25⁺ cells are involved in this regulatory network. Mechanistically, EmSAG appears to facilitate immune evasion by coordinating the suppression of the Th1 response and the activation of Tregs. Our research provides important insights into the immune evasion mechanisms used by Eimeria.

The proliferation, differentiation, and functional collaboration of immune cells are crucial for host defense against Eimeria infections. In chickens, PBMCs (mainly lymphocytes and monocytes) and CD4+/CD8+ T cells can be activated to respond to various Eimeria infections [1, 24, 25]. To investigate the immunomodulatory effects of EmSAG, we assessed its impact on PBMC proliferation and the differentiation of CD4+/CD8+ T cells using flow cytometry. Our results demonstrate that rEmSAG, at concentrations ranging from 10–40 μg/mL (with the peak effect observed at 20 μg/mL), effectively stimulated PBMC proliferation. Additionally, rEmSAG enhanced the surface expression of CD4 and CD8 markers and promoted the differentiation and maturation of both T cell subsets.

These findings regarding the proliferation of chicken immune cells induced by EmSAG align with established mechanisms of anticoccidial immunity. For instance, CD4+ intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) proliferate in the duodenum and cecum during infections caused by E. acervulina and E. tenella [26,27,28]. Moreover, CD8+ T cells assist in the transport and control of sporozoite spread, migrating to the cecal tonsils in infected chickens [27, 29, 30]. Overall, these results suggest that EmSAG may activate the immune response, establish immune memory, or enhance immune protection, ultimately helping chickens resist coccidiosis.

Studies conducted both in vitro and in animal models have shown that nitric oxide, which is produced in response to cytokine stimulation, plays a vital role in mediating immune protection against various protozoa infections [31,32,33]. In avian coccidiosis models, Allen observed increased plasma levels of NO2− and NO3− at 5 days after E. tenella infection [34]. Furthermore, chicken spleen macrophages stimulated by E. tenella sporozoites exhibited heightened NO synthesis, particularly at parasitic sites [35].

In experiments with E. tenella-infected primary chicken kidney cells (PCKs), the addition of exogenous NO from sodium nitroferricyanide (III) dihydrate (SNP) led to increased excretion of free sporozoites within just 30 min, suggesting that NO stimulates rapid sporozoite release [36]. On the other hand, impaired NO synthesis can increase the host’s susceptibility to parasitic protozoa [37,38,39,40,41].

In this study, we quantified NO metabolites (NO2− and NO3−) in the supernatant of chicken PBMCs stimulated with E. maxima recombinant SAG protein (rEmSAG) using the Griess assay. The results showed that rEmSAG significantly inhibited NO secretion. This finding is supported by research conducted by Chow et al. on seven E. tenella SAG family members, which revealed that macrophages produced low levels of NO when stimulated by these SAG family members, including rEtSAG2 and rEtSAG3 [42]. This aligns with the NO-inhibitory phenotype we observed in our study.

Th1 immune response plays a crucial role in resisting infections caused by parasitic protozoa. However, these parasites have developed mechanisms to weaken the Th1 immune response by inhibiting the secretion of IFN-γ or IL-12, which allows them to evade the immune system and sustain persistent infections [7,8,9,10,11]. In particular, the early activation of IFN-γ is essential for combating E. maxima infections [43]. Our earlier research showed that Eimeria spp. can disrupt the Th1 immune response by suppressing IFN-γ levels. When chickens were injected with a recombinant plasmid and protein encoding the SAG gene of E. maxima, serum IFN-γ levels decreased, while IL-4, IL-10, and TGF levels increased at 6 dpi (unpublished data). Mechanistically, in vitro experiments demonstrated that rEmSAG inhibits IL-12p40 expression in HD11 cells and BMDCs by regulating phosphorylated ERK1/2, which subsequently reduces IFN-γ secretion [14]. This study builds on those findings by systematically assessing the immunomodulatory effects of EmSAG in chicken PBMCs and T cell subsets. The qPCR assay showed significant inhibition of IFN-γ transcription in chicken PBMCs, as well as in CD4+ and CD8+ T cells, following EmSAG stimulation, highlighting its effectiveness as an IFN-γ inhibitory molecule.

Notably, as the concentration of rEmSAG increased, its inhibitory effect on IFN-γ was gradually diminished. At a concentration of 80 μg/mL, the transcription level of IFN-γ in chicken PBMCs showed no significant reduction. This lack of effect is likely due to the functional concentration exceeding the optimal range (10–40 μg/mL). We maintain that the inhibitory effect of rEmSAG on IFN-γ production by chicken PBMCs requires maintaining an appropriate concentration. If this concentration surpasses a specific threshold, the protein's impact on IFN-γ secretion may change direction.

The functional concentration range of EmSAG is supported by complementary cell proliferation assays. Data presented in Figure 2G shows that rEmSAG at concentrations of 10, 20, and 40 μg/mL significantly promoted chicken PBMC proliferation, whereas 80 μg/mL did not exhibit any proliferative effect tended to be inhibitory. This could explain why the results deviate from the initial working hypothesis. However, further studies are needed to explore the more complex mechanisms that may be involved.

Another important factor to consider is the potential deviations in the relative quantification method of qPCR, which occur due to variations in the selection of standard housekeeping genes. For example, some anti-parasitic drugs inhibit the housekeeping gene β-tubulin. This could lead to errors in detecting the relative fold changes of gene expression in drug-resistant strains if β-tubulin is used as a housekeeping gene.

In the present study, we used the qPCR relative quantification method to measure the relative fold changes of cytokine mRNA levels in PBMCs and T cell subsets of healthy chickens. It is important to note that the chickens used in this experiment were not administered any anticoccidial drugs. Therefore, we are inclined to move away from the initial working hypothesis due to the high concentration (80 μg/mL) of rEmSAG.

Research on the immune evasion strategies of parasitic protozoan emphasises the role of host CD4+CD25+FoxP3+ regulatory T cells (Tregs; in chickens, defined as CD4+CD25+ T cells due to the lack of FoxP3 gene). Tregs regulate the proliferation and activation of immune cells through various mechanisms that include the release of regulatory cytokines (IL-10, TGF-β, IL-35), as well as the action of granzyme B, perforin-1, and specific surface markers (CTLA-4, PD-1, LAG-3) [44]. These molecules are notably upregulated in Tregs during infections with parasitic protozoa and act as key players in immunosuppression [16, 45,46,47,48,49].

In the case of avian coccidiosis, infection with Eimeria triggers the expansion of Tregs, which is associated with increased expression of IL-10, TGF-β, and CTLA-4. For instance, Selvaraj et al. demonstrated that coccidia infection induces Treg differentiation [18]; Han et al. found an accumulation of intestinal Tregs during E. tenella infection, with heightened IL-10 production contributing to immunosuppression [50]. Additionally, Yu et al. reported that Tregs-secreted IL-10, TGF-β, and CTLA-4 in the cecum peaked at 6 days post-E. tenella infection, while splenic IL-10 peaked at 6 dpi and TGF-β/CTLA-4 peaked at 10 dpi [19].

These findings closely align with our experimental results. Specifically, rEmSAG stimulation led to a significant increase in the expression of IL-10 and TGF-β1 in PBMCs, IL-10 in CD4+ T cells, and IL-10, TGF-β1, and CTLA-4 in Tregs. This suggests that the SAG of E. maxima may play a role in the disease process of chicken coccidiosis. One possible hypothesis is that coccidia have developed mechanisms to stimulate Tregs to produce IL-10, thereby inhibiting the IFN-γ-mediated Th1 immune response and enhancing parasite survival. However, current evidence does not directly support the notion that EmSAG-induced inhibition of IFN-γ is mediated through IL-10 induction.

We temporarily hypothesised that rEmSAG may promote Treg activation, leading to the secretion of IL-10 or TGF-β, which in turn inhibits the expression of IFN-γ and IL-2. To validate this mechanism, we will conduct CD25+ cell depletion studies to explore the correlation between the inhibition of IFN-γ/IL-2 and the activation of Tregs.

Given the role of Tregs in promoting disease progression, researchers have started to explore strategies to disrupt Tregs-mediated immunosuppression through approaches such as cellular depletion, cytokine neutralisation, and the inhibition of surface molecules. One strategy involves depleting host Tregs to reduce susceptibility to infections such as Plasmodium, Leishmania, and Trypanosoma [51,52,53].

Therapeutic neutralisation of cytokines produced by Tregs, including IL-10 [54, 55], TGF-β [16], IL-35 [16] and checkpoint inhibitors-CTLA-4 [56], PD-1 [57], LAG-3 [57, 58] via monoclonal antibodies (mAbs), has the potential to reduce host damage and enhance parasite clearance. In avian coccidia models, for instance, the intraperitoneal injection of anti-CD25 mAbs leads to an approximate 80% reduction in intestinal Tregs, which subsequently elevates mRNA levels of IFN-γ and IL-2, and promotes the proliferation of CD4+CD25− T cells [59, 60].

Similarly, the use of anti-IL-10/IL-10R antibodies has been shown to increase IFN-γ levels, reduce intestinal lesions, and decrease immunosuppression induced by IL-10 [61,62,63,64]. In this study, depleting CD25+ cells significantly reversed the inhibition of IL-2 and lessened the promotion of IL-4/IL-10 caused by rEmSAG, while higher levels of IFN-γ were observed. These results suggest that rEmSAG predominantly inhibits IFN-γ expression directly, with some involvement in the induction of Tregs-secreted IL-10, which also inhibits IFN-γ. This conclusion is further supported by the observation that the transcription level of IFN-γ did not show a significant difference after CD25+ cell depletion.

However, the mechanism of rEmSAG-mediated IL-2 inhibition operates via dual pathways: a Tregs-dependent inhibition via IL-10 production and a direct inhibition of IL-2 expression in CD4+CD25− T cells or CD8+ T cells. The reversal of IL-2 inhibition following CD25+ cell depletion could confirm the existence of this dual regulatory pathway.

In conclusion, this study clarified the effects of the E. maxima IFN-γ inhibitory molecule, EmSAG, on the immune functions of chicken PBMCs and T cell subsets, with a focus on adaptive immunity. Stimulation with rEmSAG suppressed NO release and IFN-γ levels while upregulating IL-4, IL-10, and TGF-β1 in PBMCs. Additionally, it inhibited IFN-γ in CD4+/CD8+ T cells and promoted IL-10 level in CD4+/CD4+CD25+ subsets. Depleting CD25+ cells reversed the suppression of IL-2 and diminished the rEmSAG-induced promotion of IL-4/IL-10, while also enhancing PBMC proliferation and the differentiation of CD4+/CD8+ T cells. These findings provide critical insights into the immune evasion mechanisms utilised by chicken coccidia through the Th1 immune response and the function of Treg cells, paving the way for potential therapeutic strategies against E. maxima infections.

Availability of data and materials

All data generated or analysed in this research are included in this paper and its additional information files.

References

  1. Lee Y, Lu M, Lillehoj HS (2022) Coccidiosis: recent progress in host immunity and alternatives to antibiotic strategies. Vaccines 10:215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Pham HHS, Matsubayashi M, Tsuji N, Hatabu T (2021) Relationship between Eimeria tenella associated-early clinical signs and molecular changes in the intestinal barrier function. Vet Immunol Immunopathol 240:110321

    Article  CAS  PubMed  Google Scholar 

  3. Tomal F, Sadrin G, Gaboriaud P, Guitton E, Sedano L, Lallier N, Rossignol C, Larcher T, Rouille E, Ledevin M, Guabiraba R, Silvestre A, Lacroix-Lamandé S, Schouler C, Laurent F, Bussière FI (2023) The caecal microbiota promotes the acute inflammatory response and the loss of the intestinal barrier integrity during severe Eimeria tenella infection. Front Cell Infect Microbiol 13:1250080

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zaheer T, Abbas RZ, Imran M, Abbas A, Butt A, Aslam S, Ahmad J (2022) Vaccines against chicken coccidiosis with particular reference to previous decade: progress, challenges, and opportunities. Parasitol Res 121:2749–2763

    Article  PubMed  PubMed Central  Google Scholar 

  5. Sharma MK, Kim WK (2024) Coccidiosis in egg-laying hens and potential nutritional strategies to modulate performance, gut health, and immune response. Animals 14:1015

    Article  PubMed  PubMed Central  Google Scholar 

  6. Saeij JP, Coller S, Boyle JP, Jerome ME, White MW, Boothroyd JC (2007) Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue. Nature 445:324–327

    Article  CAS  PubMed  Google Scholar 

  7. Butcher BA, Fox BA, Rommereim LM, Kim SG, Maurer KJ, Yarovinsky F, Herbert DR, Bzik DJ, Denkers EY (2011) Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1-dependent growth control. PLoS Pathog 7:e1002236

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hunter CA, Sibley LD (2012) Modulation of innate immunity by Toxoplasma gondii virulence effectors. Nat Rev Microbiol 10:766–778

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lima TS, Lodoen MB (2019) Mechanisms of human innate immune evasion by Toxoplasma gondii. Front Cell Infect Microbiol 9:103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kierszenbaum F, de Diego JL, Fresno M, Sztein MB (1999) Inhibitory effects of the Trypanosoma cruzi membrane glycoprotein AGC10 on the expression of IL-2 receptor chains and secretion of cytokines by subpopulations of activated human T lymphocytes. Eur J Immunol 29:1684–1691

    Article  CAS  PubMed  Google Scholar 

  11. Passero LF, Assis RR, da Silva TN, Nogueira PM, Macedo DH, Pessoa NL, Campos MA, Laurenti MD, Soares RP (2015) Differential modulation of macrophage response elicited by glycoinositolphospholipids and lipophosphoglycan from Leishmania (Viannia) shawi. Parasitol Int 64:32–35

    Article  CAS  PubMed  Google Scholar 

  12. Cornelissen JB, Swinkels WJ, Boersma WA, Rebel JM (2009) Host response to simultaneous infections with Eimeria acervulina, maxima and tenella: a cumulation of single responses. Vet Parasitol 162:58–66

    Article  CAS  PubMed  Google Scholar 

  13. Walston M, Shanmugasundaram R, Selvaraj R (2016) Effect of infection with mixed Eimeria species on T cells and T regulatory cell properties. J Appl Poult Res 25:407–413

    Article  CAS  Google Scholar 

  14. Chen C, Chen Y, Lu M, Xu L, Yan R, Li X, Song X (2024) IFN-γ inhibitory molecules derived from Eimeria maxima inhibit IL-12 secretion by modulating MAPK pathways in chicken macrophages. Poult Sci 103:103359

    Article  CAS  PubMed  Google Scholar 

  15. Hall AO, Beiting DP, Tato C, John B, Oldenhove G, Lombana CG, Pritchard GH, Silver JS, Bouladoux N, Stumhofer JS, Harris TH, Grainger J, Wojno ED, Wagage S, Roos DS, Scott P, Turka LA, Cherry S, Reiner SL, Cua D, Belkaid Y, Elloso MM, Hunter CA (2012) The cytokines interleukin 27 and interferon-γ promote distinct Treg cell populations required to limit infection-induced pathology. Immunity 37:511–523

    Article  PubMed  PubMed Central  Google Scholar 

  16. Asad M, Sabur A, Shadab M, Das S, Kamran M, Didwania N, Ali N (2019) EB1-3 chain of IL-35 along with TGF-β synergistically regulate anti-leishmanial immunity. Front Immunol 10:616

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Gomes PS, Bhardwaj J, Rivera-Correa J, Freire-De-Lima CG, Morrot A (2016) Immune escape strategies of malaria parasites. Front Microbiol 7:1617

    Article  PubMed  PubMed Central  Google Scholar 

  18. Selvaraj RK (2013) Avian CD4(+)CD25(+) regulatory T cells: properties and therapeutic applications. Dev Comp Immunol 41:397–402

    Article  CAS  PubMed  Google Scholar 

  19. Yu H, Zou W, Mi C, Wang Q, Dai G, Zhang T, Zhang G, Xie K, Wang J, Shi H (2021) Research note: expression of T cell-related cytokines in chicken cecal and spleen tissues following Eimeria tenella infection in vivo. Poult Sci 100:101161

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kim WH, Chaudhari AA, Lillehoj HS (2019) Involvement of T cell immunity in avian coccidiosis. Front Immunol 10:2732

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Yu Z, Xu L, He K, Lu M, Yan R, Song X, Li X (2022) Actin depolymerizing factor-based nanomaterials: a novel strategy to enhance E. mitis-specific immunity. Front Immunol 13:1080630

    Article  PubMed  PubMed Central  Google Scholar 

  22. Pu X, Pan Y, Xiang Q, Lu M, Xu L, Yan R, Li X, Song X (2023) Inhibitory effect of Eimeria maxima IFN-γ inhibitory molecules on the immune function of T cell subsets in chickens. Poult Sci 102:103098

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  24. del Cacho E, Gallego M, Lee SH, Lillehoj HS, Quilez J, Lillehoj EP, Sánchez-Acedo C (2012) Induction of protective immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina infections using dendritic cell-derived exosomes. Infect Immun 80:1909–1916

    Article  PubMed  PubMed Central  Google Scholar 

  25. Kasem SM, Mira NM, Helal IB, Mahfouz ME (2024) Prophylactic and therapeutic efficacy of ultrasonicated rosmarinus officinalis ethanolic extract and its chitosan-loaded nanoparticles against Eimeria tenella infected broiler chickens. Acta Parasitol 69:951–999

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lillehoj HS (1994) Analysis of Eimeria acervulina-induced changes in the intestinal T lymphocyte subpopulations in two chicken strains showing different levels of susceptibility to coccidiosis. Res Vet Sci 56:1–7

    Article  CAS  PubMed  Google Scholar 

  27. Vervelde L, Vermeulen AN, Jeurissen SH (1996) In situ characterization of leucocyte subpopulations after infection with Eimeria tenella in chickens. Parasite Immunol 18:247–256

    Article  CAS  PubMed  Google Scholar 

  28. Choi KD, Lillehoj HS, Zalenga DS (1999) Changes in local IFN-gamma and TGF-beta4 mRNA expression and intraepithelial lymphocytes following Eimeria acervulina infection. Vet Immunol Immunopathol 71:263–275

    Article  CAS  PubMed  Google Scholar 

  29. Trout JM, Lillehoj HS (1996) T lymphocyte roles during Eimeria acervulina and Eimeria tenella infections. Vet Immunol Immunopathol 53:163–172

    Article  CAS  PubMed  Google Scholar 

  30. Sandholt AKS, Wattrang E, Lilja T, Ahola H, Lundén A, Troell K, Svärd SG, Söderlund R (2021) Dual RNA-seq transcriptome analysis of caecal tissue during primary Eimeria tenella infection in chickens. BMC Genomics 22:660

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Witola WH, Kim CY, Zhang X (2017) Inherent oxidative stress in the lewis rat is associated with resistance to toxoplasmosis. Infect Immun 85:e00289-17

    Article  PubMed  PubMed Central  Google Scholar 

  32. Ye C, Zhang L, Tang L, Duan Y, Liu J, Zhou H (2023) Host genetic backgrounds: the key to determining parasite-host adaptation. Front Cell Infect Microbiol 13:1228206

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhao X-Y, Lempke SL, Urbán Arroyo JC, Brown IG, Yin B, Magaj MM, Holness NK, Smiley J, Redemann S, Ewald SE (2024) iNOS is necessary for GBP-mediated T. gondii clearance in murine macrophages via vacuole nitration and intravacuolar network collapse. Nat Commun 15:2698

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Allen PC (1997) Nitric oxide production during Eimeria tenella infections in chickens. Poult Sci 76:810–813

    Article  CAS  PubMed  Google Scholar 

  35. Lillehoj HS, Li G (2004) Nitric oxide production by macrophages stimulated with Coccidia sporozoites, lipopolysaccharide, or interferon-gamma, and its dynamic changes in SC and TK strains of chickens infected with Eimeria tenella. Avian Dis 48:244–253

    Article  PubMed  Google Scholar 

  36. Yan X, Han W, Liu X, Suo X (2021) Exogenous nitric oxide stimulates early egress of Eimeria tenella sporozoites from primary chicken kidney cells in vitro. Parasite 28:11

    Article  PubMed  PubMed Central  Google Scholar 

  37. Omar M, Abdelal HO (2022) Nitric oxide in parasitic infections: a friend or foe? J Parasit Dis 46:1147–1163

    Article  PubMed  PubMed Central  Google Scholar 

  38. da Rocha R, LaRocque-de-Freitas IF, Arcanjo AF, Logullo J, Nunes MP, Freire-de-Lima CG, Decote-Ricardo D (2019) B-1 cells may drive macrophages susceptibility to Trypanosoma cruzi infection. Front Microbiol 10:1598

    Article  PubMed  PubMed Central  Google Scholar 

  39. Roman-Campos D, Sales-Junior P, Santos-Miranda A, Joviano-Santos JV, Ropert C, Cruz JS (2020) Deletion of inducible nitric oxide synthase delays the onset of cardiomyocyte electrical remodeling in experimental Chagas disease. Biochim Biophys Acta Mol Basis Dis 1866:165949

    Article  CAS  PubMed  Google Scholar 

  40. Omar M, Abaza BE, Mousa E, Ibrahim SM, Rashed HE, Farag TI (2021) Effect of spiramycin versus aminoguanidine and their combined use in experimental toxoplasmosis. J Parasit Dis 45:1014–1025

    Article  PubMed  PubMed Central  Google Scholar 

  41. da Silva BA, Temple MCR, Varela ELP, Gomes ARQ, Silveira EL, de Carvalho EP, Dolabela MF, Percario S (2021) Inhibition of nitric oxide synthesis promotes increased mortality despite the reduction of parasitemia in Plasmodium berghei-infected mice. Res Soc Dev 10:e27810111805

    Article  Google Scholar 

  42. Chow YP, Wan KL, Blake DP, Tomley F, Nathan S (2011) Immunogenic Eimeria tenella glycosylphosphatidylinositol-anchored surface antigens (SAGs) induce inflammatory responses in avian macrophages. PLoS One 6:e25233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Bremner A, Kim S, Morris KM, Nolan MJ, Borowska D, Wu Z, Tomley F, Blake DP, Hawken R, Kaiser P, Vervelde L (2021) Kinetics of the cellular and transcriptomic response to Eimeria maxima in relatively resistant and susceptible chicken lines. Front Immunol 12:653085

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Barros L, Ferreira C, Veldhoen M (2022) The fellowship of regulatory and tissue-resident memory cells. Mucosal Immunol 15:64–73

    Article  CAS  PubMed  Google Scholar 

  45. McManus CM, Maizels RM (2023) Regulatory T cells in parasite infections: susceptibility, specificity and specialisation. Trends Parasitol 39:547–562

    Article  CAS  PubMed  Google Scholar 

  46. Edwards CL, Ng SS, de Labastida RF, Corvino D, Engel JA, Montes de Oca M, Bukali L, Frame TC, Bunn PT, Chauhan SB, Singh SS, Wang Y, Na J, Amante FH, Loughland JR, Soon MS, Waddell N, Mukhopadhay P, Koufariotis LT, Johnston RL, Lee JS, Kuns R, Zhang P, Boyle MJ, Hill GR, McCarthy JS, Kumar R, Engwerda CR (2023) IL-10-producing Th1 cells possess a distinct molecular signature in malaria. J Clin Invest 133:e153733

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Rostami MN, Khamesipour A (2021) Potential biomarkers of immune protection in human leishmaniasis. Med Microbiol Immunol 210:81–100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Saha A, Roy S, Ukil A (2022) Cytokines and signaling networks regulating disease outcomes in leishmaniasis. Infect Immun 90:e0024822

    Article  PubMed  Google Scholar 

  49. Lucca LE, Dominguez-Villar M (2020) Modulation of regulatory T cell function and stability by co-inhibitory receptors. Nat Rev Immunol 20:680–693

    Article  CAS  PubMed  Google Scholar 

  50. Han M, Li J, Wu Y, Liao J (2024) Correlation of caecal microbiome endotoxins genes and intestinal immune cells in Eimeria tenella infection based on bioinformatics. Front Cell Infect Microbiol 14:1382160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Hisaeda H, Maekawa Y, Iwakawa D, Okada H, Himeno K, Kishihara K, Tsukumo S, Yasutomo K (2004) Escape of malaria parasites from host immunity requires CD4+ CD25+ regulatory T cells. Nat Med 10:29–30

    Article  CAS  PubMed  Google Scholar 

  52. Bhattacharya P, Ghosh S, Ejazi SA, Rahaman M, Pandey K, Ravi Das VN, Das P, Goswami RP, Saha B, Ali N (2016) Induction of IL-10 and TGFβ from CD4+CD25+FoxP3+ T cells correlates with parasite load in Indian Kala-azar patients infected with Leishmania donovani. PLoS Negl Trop Dis 10:e0004422

    Article  PubMed  PubMed Central  Google Scholar 

  53. Araujo Furlan CL, Boccardo S, Rodriguez C, Mary VS, Gimenez CMS, Robson SC, Gruppi A, Montes CL, Acosta Rodríguez EV (2024) CD39 expression by regulatory T cells participates in CD8+ T cell suppression during experimental Trypanosoma cruzi infection. PLoS Pathog 20:e1012191

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Couper KN, Blount DG, Wilson MS, Hafalla JC, Belkaid Y, Kamanaka M, Flavell RA, de Souza JB, Riley EM (2008) IL-10 from CD4CD25Foxp3CD127 adaptive regulatory T cells modulates parasite clearance and pathology during malaria infection. PLoS Pathog 4:e1000004

    Article  PubMed  PubMed Central  Google Scholar 

  55. de Oliveira Cardoso JM, de Brito RCF, Costa AFP, Siqueira Mathias FA, Soares Reis LE, Vieira JFP, de Oliveira Aguiar Soares RD, Reis AB, Roatt BM (2021) IL-10 receptor blockade controls the in vitro infectivity of Leishmania infantum and promotes a Th1 activation in PBMC of dogs with visceral leishmaniasis. Mol Immunol 137:20–27

    Article  PubMed  Google Scholar 

  56. Kurup SP, Obeng-Adjei N, Anthony SM, Traore B, Doumbo OK, Butler NS, Crompton PD, Harty JT (2017) Regulatory T cells impede acute and long-term immunity to blood-stage malaria through CTLA-4. Nat Med 23:1220–1225

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Butler NS, Moebius J, Pewe LL, Traore B, Doumbo OK, Tygrett LT, Waldschmidt TJ, Crompton PD, Harty JT (2011) Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection. Nat Immunol 13:188–195

    Article  PubMed  PubMed Central  Google Scholar 

  58. Edwards CL, Engel JA, de Labastida RF, Ng SS, Corvino D, Montes de Oca M, Frame TC, Chauhan SB, Singh SS, Kumar A, Wang Y, Na J, Mukhopadhyay P, Lee JS, Nylen S, Sundar S, Kumar R, Engwerda CR (2023) A molecular signature for IL-10-producing Th1 cells in protozoan parasitic diseases. JCI Insight 8:e169362

    Article  PubMed  PubMed Central  Google Scholar 

  59. Shanmugasundaram R, Selvaraj RK (2012) Effects of in vivo injection of anti-chicken CD25 monoclonal antibody on regulatory T cell depletion and CD4+CD25- T cell properties in chickens. Dev Comp Immunol 36:578–583

    Article  CAS  PubMed  Google Scholar 

  60. Shanmugasundaram R, Selvaraj RK (2013) In ovo injection of anti-chicken CD25 monoclonal antibodies depletes CD4+CD25+ T cells in chickens. Poult Sci 92:138–142

    Article  CAS  PubMed  Google Scholar 

  61. Sand JM, Arendt MK, Repasy A, Deniz G, Cook ME (2016) Oral antibody to interleukin-10 reduces growth rate depression due to Eimeria spp. infection in broiler chickens. Poult Sci 95:439–446

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Lessard PA, Parker M, Bougri O, Shen B, Samoylov V, Broomhead J, Li X, Raab RM (2020) Improved performance of Eimeria-infected chickens fed corn expressing a single-domain antibody against interleukin-10. Nat Food 1:119–126

    Article  CAS  PubMed  Google Scholar 

  63. Ren Z, Yan J, Whelan R, Liao X, Bütz DE, Arendt MK, Cook ME, Yang X, Crenshaw TD (2022) Dietary supplementation of sulfur amino acids improves intestinal immunity to Eimeria in broilers treated with anti-interleukin-10 antibody. Anim Nutr 10:382–389

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Arendt MK, Knoll LJ, Cook ME (2019) Oral antibody to interleukin-10 receptor 2, but not interleukin-10 receptor 1, as an effective Eimeria species immunotherapy in broiler chickens. Poult Sci 98:3471–3480

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors sincerely express their gratitude to Yangdong Pan for his guidance and assistance in the implementation of flow cytometry.

Funding

This research was funded by the National Key Research and Development Program of China (Grant No. 2023YFD1801200), the National Natural Science Foundation of China (Grant No. 32273034, 31972705).

Author information

Authors and Affiliations

  1. Ministry of Education (MOE) Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China

    Xianglin Pu, Yiyuan Zhang, Mingmin Lu, Lixin Xu, Ruofeng Yan, Xiangrui Li & Xiaokai Song

  2. Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Science, Nanjing, 210014, China

    Xinmei Huang

Authors
  1. Xianglin Pu
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Yiyuan Zhang
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Xinmei Huang
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Mingmin Lu
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Lixin Xu
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Ruofeng Yan
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Xiangrui Li
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Xiaokai Song
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

XS conceptualised and designed the experiments and revised the manuscript. XP performed the experiments, analysed the statistical data and drafted the manuscript. YZ and XH assisted the experiments and participated in statistical data analysis. ML, LX, RY and XL supervised the experiments implementation and provided guidance and support. All authors read and approved of the final manuscript.

Corresponding author

Correspondence to Xiaokai Song.

Ethics declarations

Ethics approval and consent to participate

All animal studies were reviewed and approved by the Experimental Animal Welfare and Ethics Committee of Laboratory Animal Center, Nanjing Agricultural University, China, and conducted in accordance with the guidelines of the committee (Permission: NJAU.No20220926177).

Competing interests

The authors declare that they have no competing interests.

Additional information

Handling editor: Kate Sutton

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pu, X., Zhang, Y., Huang, X. et al. Immunomodulatory effects of Eimeria maxima surface antigen (EmSAG) as an IFN-γ inhibitory molecule on peripheral blood mononuclear cells (PBMCs) and T cell subsets in chickens. Vet Res 56, 103 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13567-025-01535-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13567-025-01535-7

Keywords

Veterinary Research

ISSN: 1297-9716