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Dynamics of Th17 Cells and Their Role in Schistosoma japonicum Infection in C57BL/6 Mice

Wen, Xiaoyun, He, Lei, Chi, Ying, Zhou, Sha, Hoellwarth, Jason, Zhang, Cui, Zhu, Jifeng, Wu, Calvin, Dhesi, Shawn, Wang, Xuefeng, Liu, Feng, Su, Chuan·2011·PLoS Neglected Tropical Diseases

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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: 400×). 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 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: 400×). 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 (2×105/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 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 (2×105/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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 (2×105/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.
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 (2×105/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.CC BY · PubMed Central

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Authors
Wen, X., He, L., Chi, Y., Zhou, S., Hoellwarth, J., Zhang, C., Zhu, J., Wu, C., Dhesi, S., Wang, X., Liu, F., & Su, C.
Venue
PLoS Neglected Tropical Diseases
Published
2011-11-15
Volume
5
Issue
11
Pages
e1399
ISSN
1935-2735
Cited by
70
References
3
Language
en

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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