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. 2011 Nov;5(11):e1399.
doi: 10.1371/journal.pntd.0001399. Epub 2011 Nov 15.

Dynamics of Th17 cells and their role in Schistosoma japonicum infection in C57BL/6 mice

Affiliations

Dynamics of Th17 cells and their role in Schistosoma japonicum infection in C57BL/6 mice

Xiaoyun Wen et al. PLoS Negl Trop Dis. 2011 Nov.

Abstract

Background: The current knowledge of immunological responses to schistosomiasis, a major tropical helminthic disease, is insufficient, and a better understanding of these responses would support vaccine development or therapies to control granuloma-associated immunopathology. CD4(+) T cells play critical roles in both host immune responses against parasitic infection and immunopathology in schistosomiasis. The induction of T helper (Th)1, Th2 and T regulatory (Treg) cells and their roles in schistosome infections are well-illustrated. However, little in vivo data are available on the dynamics of Th17 cells, another important CD4(+) T cell subset, after Schistosoma japonicum infection or whether these cells and their defining IL-17 cytokine mediate host protective responses early in infection.

Methodology: Levels of Th17 and the other three CD4(+) T cell subpopulations and the cytokines related to induction or repression of Th17 cell generation in different stages of S. japonicum infection were observed. Contrary to reported in vitro studies, our results showed that the Th17 cells were induced along with the Th1, Th2, Treg cells and the IFN-γ and IL-4 cytokines in S. japonicum infected mice. The results also suggested that S. japonicum egg antigens but not adult worm antigens preferentially induced Th17 cell generation. Furthermore, decreasing IL-17 with a neutralizing anti-IL-17 monoclonal antibody (mAb) increased schistosome-specific antibody levels and partial protection against S. japonicum infection in mice.

Conclusions: Our study is the first to report the dynamics of Th17 cells during S. japonicum infection and indicate that Th17 cell differentiation results from the integrated impact of inducing and suppressive factors promoted by the parasite. Importantly, our findings suggest that lower IL-17 levels may result in favorable host protective responses. This study significantly contributes to the understanding of immunity to schistosomiasis and may aid in developing interventions to protect hosts from infection or restrain immunopathology.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Kinetics of Th17, Th1, Th2 and Treg cell induction in S. japonicum infection.
For each of three independent experiments, 20 female C57BL/6 mice were infected with 12 cercariae of S. japonicum per mouse. Four mice were randomly chosen and sacrificed at 0 (before infection), 3, 5, 8 or 13 weeks post-infection. Liver sections were stained with H&E for microscopic examination. Single cell suspensions of splenocytes, lymphocytes or liver cells were stimulated with PMA and ionomycin in the presence of Golgistop for 6 h. Cells were surface stained with anti-CD3-APC and anti-CD4-FITC and then intracellularly stained with PE-conjugated antibodies against IL-17A, IFN-γ, IL-4 or isotype IgG2a control antibody for FACS analysis of Th17, Th1 or Th2 cells. Tregs were detected in single cell suspensions of splenocytes, lymphocytes or liver cells using the Mouse Regulatory T Cell Staining Kit. A. Histopathology in the livers (magnification: ×ばつ). Results are representative of three independent experiments. The kinetics of the percentages of Th17, Th1, Th2 and Treg cells in total CD4+ T cells from mouse spleens (B), mesenteric lymph nodes (D) and livers (F). Data are expressed as the mean ± SD of 12 mice from three independent experiments. Flow cytometric analysis of CD4+ T cell subsets in mouse splenocytes (C), mesenteric lymphocytes (E) and hepatocytes (G) from one representative experiment. Cells were gated on the CD3+ population for analysis of Th17, Th1, Th2 cells, or gated on the CD3+CD4+ population for analysis of Treg cells.
Figure 2
Figure 2. Kinetics of cytokines in the supernatant produced by splenocytes from S. japonicum infected mice.
For each of three independent experiments, 20 female C57BL/6 mice were infected with 12 cercariae of S. japonicum per mouse. At 0 (before infection), 3, 5, 8 and 13 weeks post-infection, four mice at each time point were randomly chosen and sacrificed. Single cell suspensions of splenocytes were prepared and then cultured in the presence of PMA and ionomycin. The culture supernatants were collected after 72 h of incubation for detection of IL-17, IFN-γ, IL-4, TGF-β, IL-6, IL-23 and IL-21 by ELISA. Data are expressed as the mean ± SD of 12 mice from three independent experiments.
Figure 3
Figure 3. Proportions of Th17, Th1, Th2 and Treg cells in mice immunized with SEA or SWA.
For each of three independent experiments, C57BL/6 mice (8 per group) were each injected subcutaneously in the back with 100 μl of an IFA emulsified solution containing 50 μg of SEA, 50 μg of SWA or PBS. The immunizations were repeated two times with a 14-day interval. Two weeks after the final vaccination, the mice were sacrificed, and single cell suspensions of splenocytes were prepared. The splenocytes were stimulated with PMA/ionomycin in the presence of Golgistop for 6 h, followed by surface staining with anti-CD3-APC and anti-CD8-FITC and then intracellular staining with PE-conjugated antibodies against IL-17A, IFN-γ, IL-4 or isotype IgG2a control antibody for FACS analysis of Th17, Th1 or Th2 cells. Splenocytes were stained with the Mouse Regulatory T Cell Staining Kit for detection of Treg cells. A. Proportions of Th17, Th1, Th2 and Treg cells in CD4+ T cells. Results are expressed as mean ± SD of 24 mice from 3 independent experiments. *P<0.05; **P<0.01; ***P<0.001, compared to control mice injected with PBS. B. Flow cytometric analysis from one representative experiment. Cells were gated on the CD3+ population for analysis of Th17, Th1 and Th2 cells or on CD3+CD4+ population for analysis of Treg cells.
Figure 4
Figure 4. ELISA detection of cytokines in the supernatant produced by SEA or SWA immunized mice splenocytes.
For each of three independent experiments, C57BL/6 mice (8 per group) were each injected subcutaneously in the back with 100 μl of an IFA emulsified solution containing 50 μg of SEA, 50 μg of SWA or PBS. The immunizations were repeated two times with a 14-day interval. Two weeks after the final vaccination, the mice were sacrificed, and single cell suspensions of splenocytes were prepared. The splenocytes (×ばつ105/well in 200 μl complete media) were cultured in 96 well plates with PMA/ionomycin. Culture supernatants were collected after 72 h, and cytokines were detected by ELISA. Results are expressed as mean ± SD of 24 mice from three independent experiments. *P<0.05; **P<0.01; ***P<0.001, compared to control groups as indicated.
Figure 5
Figure 5. Protection against S. japonicum infection in C57BL/6 mice administered rmIL-17A or anti-IL-17A neutralizing mAb.
rmIL-17A, anti-mouse IL-17A mAb or controls were administered to mice, followed by challenge with 40 S. japonicum cercariae per mouse. A. Six weeks after the challenge, the mice were sacrificed and perfused to collect adult worms. The numbers of eggs extracted from the liver were determined by microscopic examination. Protection was measured by assessing the worm and egg burden. Values are given as mean ± SD of eight mice from two independent experiments. *P<0.05; **P<0.01; ***P<0.001, compared to control mice injected with isotype antibody. B. Paraffin embedded formalin fixed liver sections stained with H&E. Images shown are representative of two independent experiments. C. Sizes of the granulomas were measured by computer-assisted morphometric analysis. Ten sections for each mouse and five microscope fields for each section were measured under a microscope. Only granulomas with a visible central egg, which reflected their true shape and dimension, were analyzed for accuracy. Values are given as mean ± SD of eight mice from two independent experiments. *P<0.05; **P<0.01; ***P<0.001, compared to control mice injected with isotype antibody.
Figure 6
Figure 6. Percentages of granulomatous cells in S. japonicum infected mice administered rmIL-17A or anti-IL-17A neutralizing mAb.
rmIL-17A, anti-mouse IL-17A mAb or controls were administered to mice, followed by challenge with 40 S. japonicum cercariae per mouse. Six weeks after the challenge, the mice were sacrificed, and the livers were processed for H&E staining. Percentages of neutrophils, eosinophils, lymphocytes and macrophages in the granulomas were calculated by microscopic analysis of the same granulomas analyzed for lesion size by randomly counting 200 cells (not including hepatocytes) in each granuloma. Ten sections for each mouse and five microscope fields for each section were counted. Values are given as mean ± SD of eight mice from two independent experiments. *P<0.05; **P<0.01; ***P<0.001, compared to control mice injected with PBS or isotype antibody.
Figure 7
Figure 7. Immune responses in S. japonicum infected mice after administration of rmIL-17A or anti-IL-17A neutralizing mAb.
rmIL-17A, anti-mouse IL-17A mAb or controls were administered to mice, followed by challenge with 40 S. japonicum per mouse. Six weeks after the challenge, the mice were sacrificed, and the splenocytes were prepared. A. Flow cytometric analysis of Th17, Th1, Th2 and Treg cells from one representative experiment. Single cell suspensions of splenocytes were stimulated with PMA/ionomycin in the presence of Golgistop for 6 h, followed by surface staining with anti-CD3-APC and anti-CD4-FITC and intracellularly staining with PE-conjugated antibodies against IL-17A, IFN-γ, IL-4 or isotype IgG2a control antibody for FACS analysis of Th17, Th1 or Th2 cells. Splenocytes were also stained with the Mouse Regulatory T Cell Staining Kit for detection of Treg cells. Cells were gated on the CD3+ population for analysis of Th17, Th1, Th2 cells analysis, or gated on the CD3+CD4+ population for analysis of Treg cells. B. The proportions of Th17, Th1, Th2 and Treg cells in CD4+ T cells. Results are expressed as mean ± SD of eight mice from two independent experiments. *P<0.05; **P<0.01; ***P<0.001, compared to control mice injected with PBS or isotype antibody. C. ELISA detection of cytokines in the supernatant produced by splenocytes from S. japonicum infected mice after administration of rmIL-17A or anti-IL-17A neutralizing mAb. Splenocytes (×ばつ105/well in 200 μl of complete media) were cultured in 96 well plates with PMA/ionomycin. Culture supernatants were collected after 72 h, and cytokines were detected by ELISA. Results are expressed as mean ± SD of eight mice from two independent experiments. *P<0.05; **P<0.01; ***P<0.001, compared to control mice injected with PBS or isotype antibody. SEA (D) and SWA (E) specific IgG, IgG1 and IgG2a antibodies in serum from S. japonicum infected mice after administration of rmIL-17A or anti-IL-17A neutralizing mAb were detected by ELISA. Results are expressed as mean ± SD of eight mice from two independent experiments. *P<0.05; **P<0.01; ***P<0.001, compared to control mice injected with PBS or isotype antibody.

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