Fig 1.
Serum FGL2 levels in E. multilocularis infected mice, effect of IL-17A on FGL2 secretion in spleen cells, and effects of FGL2 on parasite load and proliferation in E. multilocularis infected mice.
(A) Serum levels of FGL2 in infected AE-WT mice at different stages of infection, as compared to non-infected control mice (Control). (B) Different concentrations of recombinant IL-17A (0, 0.5, 1, 2 μg/mL) or anti-IL-17A MAbs (1 μg/ml) were added to primary spleen cells isolated from non-infected WT mice (4 months p.i.). FGL2-levels in culture supernatants were determined by ELISA. (C) Parasite load in AE-WT and AE- fgl2-/- mice assessed by wet weight measurement at 1 month and 4 months post- infection. (D) Representative images of E. multilocularis infection in AE-WT and AE-fgl2-/- mice at 4 months p.i.; arrows point at intraperitoneal metacestode tissue/lesions. (E) Parasite cell proliferation in AE-WT versus AE-fgl2-/- assessed by em14-3-3 qRT-PCR at 4 months p.i, upon comparison to the constitutively expressed house-keeping Em II/3-10 gene. Data represent mean ± SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA for statistical analysis. *P<0.05, ** P<0.01. ‘WT’, wild type mice; ‘fgl2-/-’, fgl2 knock-out mice; ‘AE-WT’, E. multilocularis-infected wild type mice; ‘AE-fgl2-/-’, E. multilocularis-infected fgl2 knock-out mice. ‘Control’, non-infected mice; ‘1m’, 1 month p.i.; ‘4m’, 4 months p.i. AU: arbitrary units. Note: Em 14-3-3 is used as a molecular marker to assess viability and growth activity of the E. multilocularis metacestode tissue, whereas Em II/3-10, so as actin, serve as constitutionally expressed house-keeping gene.
Fig 2.
Treg secretion and function in both AE-WT and AE-fgl2-/- mice after E. multilocularis infection, and in WT mice in response to recombinant FGL2/anti-FGL2-MAb blockade.
(A) Spleen was taken from non-infected WT and AE-WT mice, CD4+ Teffs, CD8+ T cells, CD4+CD25+ Tregs and APCs were isolated by FACS cell sorting, fgl2 mRNA levels were determined by qRT-PCR. (B) Representative images of Foxp3 mean fluorescence intensity (MFI) from AE-WT and AE-fgl2-/- mice, and non-infected mice as controls. (C) Frequency of Foxp3+ T cells within CD4+CD25+T cells in PECs and spleen cells from AE-WT and AE-fgl2-/- mice at 1 month and 4 months post-infection. (D) Foxp3 and IL-10 gene expression in PECs during E. multilocularis infection (measured by qRT-PCR). AU: arbitrary units. Graphs show the mean±SD. Data represent mean±SD of three independent experiments of a total 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA for statistical analysis. (E) 0, 1, and 5 μg/mL of recombinant FGL2 and 1μg/mL of anti-FGL2-MAb were added to primary spleen cells from non-infected WT mice, or spleen cells stimulated with ConA or vesicle fluid (VF). Relative expression levels of Foxp3+/CD4+CD25+ were determined by flow cytometry. (F) CD4+CD25+ Tregs (suppressor cells) and CD4+CD25- Teff cells (responder cells) were isolated from spleen cells of both non-infected and infected AE-WT mice by FACS. The two cell populations were co-cultured at a ratio of 1:1 (suppressor: responder) in the presence of APCs and ConA (2 μg/mL), rFGL2 (1 μg/mL), ConA (2 μg/mL) + rFGL2 (1 μg/mL), or anti-FGL2-MAb (1 μg/mL); cell proliferation was measured using BrdU ELISA. Data represent mean±SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Expression of Foxp3+/CD4+CD25+ was normalized with negative control (cells without rFGL2 and anti-FGL2-MAb (negative control) were considered as base line, e.g. as 1.0). Comparison between groups was performed using a one-way ANOVA with Bonferroni’s multiple comparison post-test for statistical analysis. *P<0.025 for ex vivo assay, and *P<0.006 for in vitro assay. ‘WT’, wild type mice; ‘fgl2-/-’, fgl2 knock-out mice; ‘AE-WT’, E. multilocularis-infected wild type mice; ‘AE-fgl2-/-’, E. multilocularis-infected fgl2 knock-out mice. ‘PEC’, peritoneal exudate cells; ‘Spleen’, spleen cells; VF, vesicle fluid.
Fig 3.
T cell function in both AE-WT and AE-fgl2-/- mice at 1 months and 4 months post-infection.
(A) Frequency of IFN-γ+ T cells within CD4+ T cells in peritoneal and spleen cells from AE-WT and AE-fgl2-/- mice. (B) Representative images of IFN-γ+ T cells within CD4+ T cells in PECs and spleen cells from both AE-WT and AE-fgl2-/- mice at 4 months post-infection. (C) Frequency of IL-4+ T cells within CD4+ T cells in PECs and spleen cells from AE-WT and AE-fgl2-/- mice. (D) Relative level of T cell reactivity markers in spleen cells from AE-WT and AE-fgl2-/- mice, co-cultured with ConA (2 μg/mL), normalized with their corresponding non-infected controls (non-infected control was considered as baseline, e.g. 1.0). (E) Representative images of IL-4+ T cells within CD4+ T cells in PECs and spleen cells from both AE-WT and AE-fgl2-/- mice at 1 month post E. multilocularis infection. The corresponding isotype control antibodies were identically labeled as staining controls. Data represent mean±SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA with Bonferroni’s multiple comparison post-test for statistical analysis. *P<0.025 for ex vivo assay, and *P<0.006 for in vitro assay. ‘WT’, wild type mice; ‘fgl2-/-’, fgl2 knock-out mice; ‘AE-WT’, E. multilocularis-infected wild type mice; ‘AE-fgl2-/-’, E. multilocularis-infected fgl2 knock-out mice. ‘PEC’, peritoneal exudate cells; ‘Spleen’, spleen cells.
Fig 4.
DC maturation in both AE-WT and AE-fgl2-/- mice at 1 month and 4 months post-infection.
(A) Representive images of CD80 mean fluorescence intensity (MFI) in CD11b+ cells from AE-WT and AE-fgl2-/- mice, and non-infected mice as controls (4 months p.i.). (B) CD80 frequency within CD11b+ DCs in PECs and spleen cells of AE-WT and AE-fgl2-/- mice. (C) CD80 frequency within CD11b+ DCs in spleen cells from AE-WT and AE-fgl2-/- mice, co-cultured with ConA (2 μg/mL), normalized with their corresponding non-infected controls (we considered non-infected control as baseline, e.g. 1.0). (D) Representive images of CD86 MFI in CD11b+ cells from AE-WT and AE-fgl2-/- mice, and non-infected mice as controls (4 months p.i.). (E) CD86 frequency within CD11b+ DCs in PECs and spleen cells of AE-WT and AE-fgl2-/- mice. (F) CD86 frequency within CD11b+ DCs in spleen cells from AE-WT and AE-fgl2-/- mice, co-cultured with ConA (2 μg/mL), normalized with their corresponding non-infected controls (we considered non-infected control as baseline, e.g. 1.0). (G) Representive images of CD80 MFI in CD11c+ cells from AE-WT and AE-fgl2-/- mice, and non-infected mice as controls (4 months p.i.). (H) CD80 frequency within CD11c+ DCs in PECs and spleen cells of AE-WT and AE-fgl2-/- mice. (I) CD80 frequency within CD11c+ DCs in spleen cells from AE-WT and AE-fgl2-/- mice, co-cultured with ConA (2 μg/mL), normalized with their corresponding non-infected controls (non-infected control as baseline, e.g. 1.0). The corresponding isotype control antibodies were identically labeled for use as staining controls. Data represent mean±SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA with Bonferroni’s multiple comparison post-test for statistical analysis. *P<0.01 for ex vivo assay, and *P<0.006 for in vitro assay. ‘WT’, wild type mice; ‘fgl2-/-’, fgl2 knock-out mice; ‘AE-WT’, E. multilocularis-infected wild type mice; ‘AE-fgl2-/-’, E. multilocularis-infected fgl2 knock-out mice. ‘PEC’, peritoneal exudate cells; ‘Spleen’, spleen cells.
Fig 5.
T cell reactivity and DC maturation in response to vesicle fluid (VF) in E. multilocularis infected mice at 1 month and 4 months p.i.
(A) Relative expression level of T cell reactivity markers in spleen cells from AE-WT and AE-fgl2-/- mice, co-cultured with VF (10 μg/mL), in the presence of a protein transport inhibitor cocktail (Ebioscience, San Diego, CA, USA), normalized using cells from non-infected controls as baseline (e.g. 1.0). (B) Relative expression level of DC maturation markers in spleen cells from infected AE-WT and AE-fgl2-/- mice, co-cultured with VF (10 μg/mL), normalized using cells from non-infected controls. Data represent mean±SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA with Bonferroni’s multiple comparison post-test for statistical analysis. *P<0.006. ‘WT’, wild type mice; ‘fgl2-/-’, fgl2 knock-out mice; ‘AE-WT’, E. multilocularis-infected wild type mice; ‘AE-fgl2-/-’, E. multilocularis-infected fgl2 knock-out mice.
Fig 6.
Recombinant FGL2 induces down regulation of T cell reactivity and DC maturation in vitro.
Different concentrations of recombinant FGL2 (0, 1, 5 μg/mL), ConA or vesicle fluid (VF), and anti-FGL2-MAb (1 μg/mL) were added to primary spleen cells isolated from non-infected WT mice and cultured in the presence of protein transport inhibitor cocktail. Relative expression level of T cell reactivity (A) and DC maturation markers (B) were determined by flow cytometry. Data represent mean±SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Expression of each maker was normalized with cells without rFGL2 and anti-FGL2-MAb (negative control) as base line (e.g. as 1.0). Comparison between groups was performed using a one-way ANOVA with Bonferroni’s multiple comparison post-test for statistical analysis. *P<0.006.
Fig 7.
A schematic presentation of the hypothetical role of FGL2 in immune regulation during AE: E. multilocularis metabolites induce the release of TNF-α, IFN-γ and IL-17; IFN-γ as demonstrated previously, but also IL-17A as presently demonstrated, contributes to FGL2 secretion by Tregs and other cells; once FGL2 is released, it binds to FcγRIIB receptors, down-regulates the maturation of DCs, decreases co-stimulation of effector T cells, suppresses Th1 and Th17 immune response, accelerates Th2 immune responses, and overall this leads to an immune suppressed status that favors the continuous “tumor-like” progression of the parasitic metacestode.