A 21.6 kDa tegumental protein of Clonorchis sinensis induces a Th1/Th2 mixed immune response in mice - PubMed (original) (raw)
A 21.6 kDa tegumental protein of Clonorchis sinensis induces a Th1/Th2 mixed immune response in mice
EunJoo Chung et al. Immun Inflamm Dis. 2018 Dec.
Abstract
Introduction: Clonorchis sinensis is a major parasite affecting the Korea population. Despite the high infection rate and pathogenicity, very few studies have been conducted to investigate the immune responses against the proteins of C. sinensis.
Methods: In this study, in vitro immune response induced by a recombinant 21.6 kDa tegumental protein derived from C. sinensis (rCsTegu21.6) was confirmed in murine dendritic cells and T cells. For the in vivo analysis, each mouse was immunized three times. Total serum IgG and T cell cytokine production were determined by ELISA, while T cell proliferation was detected by a WST (Water-Soluble Tetrazolium salt)-1 assay.
Results: In vitro tests indicated that rCsTegu21.6 treatment increased the expression of surface molecules, such as CD40 (77%), CD80 (52%) and CD86 (46%), on murine dendritic cells and the secretion of cytokines (TNF-α, IL-6, IL-1β, IL-10, and IL-12p70). Moreover, co-culturing dendritic cells activated by rCsTegu21.6 with allogenic T cells induced T cell proliferation over time. rCsTegu21.6 also stimulated specific antibody production and cytokine secretion [IL-2, IL-4, and interferon (IFN)-γ)] from T cells following immunization in vivo. Notably, rCsTegu21.6 predominantly induced IgG1 production and secretion of the Th2 cytokine IL-4, regardless of the type of adjuvant used.
Conclusion: These results serve as a foundation for the development of tegumental protein-based vaccines against C. sinensis.
Keywords: Clonorchis sinensis; T lymphocytes; dendritic cells.
© 2018 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.
Figures
Figure 1
Expression and identification of recombinant CsTegu21.6 protein. (A) SDS–PAGE analysis of rCsTegu21.6 purified using a Ni‐IDA column (1, flow‐through; 2–3, washing; 4–9, elution). (B) Western blot image is showing the purified rCsTegu21.6 protein using anti‐His antibodies (1, flow‐through; 2–3, washing; 4–9, elution). (C) Immunolocalization of rCsTegu21.6 in the tegument of adult C. sinensis worms present in infected rabbit liver tissue sections, using CsTegu21.6‐immunized mouse sera. Sera from naïve mouse (left panel) were used as the negative control. CsTegu21.6 localization is indicated by arrows (right panel). CsTegu21.6 is mainly located in the tegument of adult C. sinensis.
Figure 2
rCsTegu21.6 treatment induced surface marker expression on mBMDCs in a dose‐dependent manner. (A) mBMDCs were isolated from C57BL/6 mice and stimulated with LPS (100 ng/mL) or rCsTegu21.6 (1, 5, or 10 µg/mL) for 18 h. Harvested cells were labeled with FITC‐conjugated anti‐mouse CD11c and PE‐conjugated anti‐mouse CD40, CD80, and CD86 antibodies, individually. Fluorescence intensity was measured by flow cytometry. (B) Statistical results of the surface marker expression on mBMDCs. Data are shown as the mean ± standard error (SEM) and are representative of three independent experiments. *P < 0.05 compared to the PBS‐treated control group.
Figure 3
Cytokine secretion from rCsTegu21.6‐treated mBMDCs increased in a dose‐dependent manner. mBMDCs from C57BL/6 mice were activated with LPS or rCsTegu21.6 (1, 5, or 10 µg/mL) for 18 h. The concentration of secreted cytokines in the supernatant was quantified by ELISA. The results are shown as the mean ± standard error (SEM) and are representative of two independent experiments. *P < 0.05, **P < 0.01 compared to the PBS‐treated control group.
Figure 4
Production of intracellular IL‐12, but not IL‐10, increased with protein dose in rCsTegu21.6‐treated mBMDCs. (A) mBMDCs were cultured for 8 days and then stimulated with 1, 5, or 10 µg/mL rCsTegu21.6 for 18 h. Harvested cells were permeabilized and stained with PE‐conjugated anti‐mouse IFN‐γ (negative control), IL‐10, or IL‐12 antibodies after labeling with FITC‐conjugated anti‐mouse CD11c antibody. Fluorescence intensity was measured by flow cytometry. (B) Statistical results of the surface marker expression on mBMDCs. The results are shown as the mean ± standard error (SEM) and are representative of three independent experiments. **P < 0.01 compared to the PBS‐treated control group.
Figure 5
Proliferation of allogenic T cells, co‐cultured with rCsTegu21.6‐activated mBMDCs, increases over time. However, cytokines were not secreted from T cells by rCsTegu21.6 protein. mBMDCs were stimulated with 5 µg/mL rCsTegu21.6 for 18 h. Harvested mBMDCs (1 × 104 cells/mL) were co‐incubated at a ratio of 1:10 with CFSE‐conjugated T cells derived from BALB/c mice. Co‐cultured cells were harvested, washed with DPBS, and analyzed at the time shown. (A) Flow cytometry analysis indicates that T cell proliferation increases over time in the co‐cultured cells. The _x_‐axis shows the fluorescence intensity, and the _y_‐axis depicts the number of cells. (B) Cytokine secretion from the T cells co‐cultured with CsTegu21.6‐treated mBMDCs was detected by ELISA. Statistical analyses were performed using two‐tailed t tests, and the results are shown as the mean ± standard error (SEM).
Figure 6
rCsTegu21.6 induced specific antibody production in C57BL/6 mice. rCsTegu21.6 (with or without alum adjuvant (Alum) or Freund's adjuvant (FA) was used to immunize C57BL/6 mice (n = 5 per group) three times after two‐week intervals. Total IgG (A) and their isotypes (B) were detected in the immunized serum by ELISA. The results are shown as the mean ± standard error (SEM). *P < 0.05, **P < 0.01, ***P < 0.001 compared to the rCsTegu21.6 only immunization group. IgG1 appears dominant in all groups.
Figure 7
Cytokine secretion, but not the number of T cells, was increased in rCsTegu21.6‐immunized mice. T cells were isolated from rCsTegu21.6‐immunized mice and cultured either in the absence (uninduced) or presence of 10 µg/mL of rCsTegu21.6 (induced) for 70 h. (A) T cell proliferation was measured using a WST‐1 assay. (B–D) Cytokine levels were determined in the supernatants of harvested cells by ELISA. The results are shown as the mean ± standard error (SEM). *P < 0.05, **P < 0.01 compared to the adjuvant only immunization groups. rCsTegu21.6 protein mixed with FA activates IL‐2, IFN‐γ, and IL‐4 secretion, but alum‐mixed rCsTegu21.6 protein induced only an IL‐4 secretion.
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