In vitro effects of 5 recombinant antigens of Eimeria maxima on maturation, differentiation, and immunogenic functions of dendritic cells derived from chicken spleen

Abstract

Eimeria maxima possesses integral families of immunogenic constituents that promote differentiation of immune cells during host-parasite interactions. Dendritic cells (DCs) have an irreplaceable role in the modulation of the host immunity. However, the selection of superlative antigen with immune stimulatory efficacies on host DCs is lacking. In this study, 5 recombinant proteins of E. maxima (Em), including Em14-3-3, rhomboid family domain containing proteins (ROM) EmROM1 and EmROM2, microneme protein 2 (EmMIC2), and Em8 were identified to stimulate chicken splenic derived DCs in vitro. The cultured populations were incubated with recombinant proteins, and typical morphologies of stimulated DCs were obtained. DC-associated markers major histocompatibility complex class II, CD86, CD11c, and CD1.1, showed upregulatory expressions by flow cytometry assay. Immunofluorescence assay revealed that recombinant proteins could bind with the surface of chicken splenic derived DCs. Moreover, quantitative real-time PCR results showed that distinct gene expressions of Toll-like receptors and Wnt signaling pathway were upregulated after the coincubation of recombinant proteins with DCs. The ELISA results indicated that the DCs produced a significant higher level of interleukin (IL)-12 and interferon-γ secretions after incubation with recombinant proteins. While transforming growth factor-β was significantly increased with rEmROM1, rEmROM2, and rEmMIC2 as compared to control groups, and IL-10 did not show significant alteration. Taken together, these results concluded that among 5 potential recombinant antigens, rEm14-3-3 could promote immunogenic functions of chicken splenic derived DCs more efficiently, which might represent an effective molecule for inducing the host Th1-mediated immune response against Eimeria infection.

Keywords: Eimeria maxima; chicken spleen; dendritic cell; immunogenic function; recombinant antigens.

Figure 1

Morphological characteristics of chicken splenic derived dendritic cells (DCs) were observed at Day 1, red arrows indicated that seeded cells were round and smooth in shape. At Day 4, red arrows showed that cell surface projection/veils were seen in complete culture medium (RPMI + chicken serum + recombinant chicken GMCSF + IL-4). After stimulation of rEm antigens or LPS stimulation at day 7, red arrows shown that cultured populations were matured, irregularly shaped cell aggregates with dendrites that became semi-suspended or suspended in the medium and exhibited distinctive morphology of DCs.

Figure 2

The binding ability of recombinant proteins (rEm14-3-3, rEmROM1, rEmROM2, rEmMIC2, and rEm8) with chicken splenic derived dendritic cells by immunofluoresence assay. The cells were incubated with rat sera antirecombinant antigens IgG, anti-pET-32a tag protein, and negative rat sera IgG (control). (a1, b1, c1, d1, e1) Staining of target proteins (rEm14-3-3, rEmROM1, rEmROM2, rEmMIC2, and rEm8) was visualized by cy3-conjugated secondary antibody (red color). (a2, b2, c2, d2, e2) Nuclei of corresponding cells were visualized by 4′,6-diamidino-2-phenylindole (DAPI; blue color). (a3, b3, c3, d3, e3) Merged, red, and blue colors overlapped. No fluorescence was observed in control groups. The results were analyzed by a confocal laser scanning microscope with scale bars 20 μm.

Figure 3

Recombinant antigens of E. maxima (rEm14-3-3, rEmROM1, rEmROM2, rEmMIC2, and rEm8) increased the expression of cell surface markers MHC-II, CD1.1, CD11c, and CD86 in chicken splenic-derived dendritic cells (ChSP-DCs) as determined by flow cytometry. Bar graph representing percentages of A, MHC-II; B, CD86; C, CD11c; D, CD1.1. The data are presented as mean ± SEM and represented of triplicates experiments (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001) and ns represents nonsignificant (P > 0.05).

Figure 4

To characterize the cells population in more detail, double-color flow cytometric analysis was performed using MHC-II as a reference molecule. Bar graph representing proportions of cells: A, CD11c/MHC-II; B, CD86/MHC-II; C, CD1.1/MHC-II. The data are presented as mean ± SEM and represented of triplicates experiments (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001) and ns represents nonsignificant (P > 0.05).

Figure 5

Gene expressions of Wnt and TLRs signaling pathways by QRT-PCR. Total RNA was isolated from chicken splenic derived DCs. A, mRNA expression of key genes β-catenin, APC, DVL, CK2; B, mRNA expression of key genes TLR1, 3, 4, 7, 15, 21, and MYD88. Significant values are obtained as presented (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001). Data are presented as mean ± SEM from 3 independent experiments, with methodical 3 technical triplicates per group, and ns represents nonsignificant (P > 0.05).

Figure 6

Relative analysis of multiple cytokines produced by chicken splenic derived dendritic cells. After coincubation with recombinant proteins (rEm14-3-3, rEmROM1, rEmROM2, rEmMIC2, and rEm8), control groups (PBS, pET-32a), and positive control group (GM-CSF + IL-4+LPS), cytokines secretions in the supernatant of cell culture were measured by ELISA kits. A, Cytokine production level of IL-12; B, cytokine production level of IFN-γ; C, cytokine production level of TGF-β; D, cytokine production level of IL-10. The data were representative of 3 independent experiments, and the values presented here were the means ± SEM (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001); ns represents nonsignificant (P > 0.05).

Supplementary Figure 1

Optimization of different concentrations of anti-rEm-Ags rat sera was evaluated by indirect ELISA. Optimum concentration of A: anti-rat-rEm14-3-3 sera; B: anti-rat-rEmROM1 sera; C: anti-rat-rEmROM2 sera; D: anti-rat-rEmMIC2 sera; E: anti-rat-rEm8 sera. The P/N (OD₄₅₀ value of positive serum/OD₄₅₀ value of negative serum) value of each antigen was shown on each bar (right Y axis). P/N value (>2.1) was determined to be positive results and optimum sera concentration (the highest P/N value) of each antigen was pointed by red arrows.

Supplementary Figure 2

(A to E) Purified recombinant E. maxima (rEm) antigens were resolved on SDS-PAGE and transferred to membranes probed by rat antibodies generated against rEm antigens (rEm14-3-3, rEmROM1, rEmROM2, rEmMIC2, and rEm8). Immuno-blot results revealed that the rEm antigens possess antigenic nature and could be recognized by the polyclonal antibodies generated against rEm antigens. The serum obtained from unimmunized/normal rats was used as negative control.

Supplementary Figure 3

Dot plot analysis of cultured cells treated with PBS, pET-32a tag protein, recombinant antigens of E. maxima (rEm14-3-3, rEmROM1, rEmROM2, rEmMIC2, and rEm8) and GM-CSF + IL-4+LPS were analyzed by flow cytometry at day 7. Cells were gated based on side/forward scatters and dot plot percentages of MHC-II, CD86, CD11c, and CD1.1 were shown. The data shown are representative of 3 independent experiments.

Supplementary Figure 4

Flow cytometric analysis shows the percentage of double-positive cell surface markers of chicken splenic-derived dendritic cells (ChSP-DCs) stimulated with recombinant antigens of E. maxima (rEm14-3-3, rEmROM1, rEmROM2, rEmMIC2, and rEm8), GM-CSF + IL-4+LPS, pET-32a protein, and PBS. Dot plot percentage of CD11c/MHC-II, CD86/MHC-II, and CD1.1/MHC-II. The data shown are representative of 3 independent experiments.