Cellular and humoral peritoneal immunity to Mesocestoides vogae metacestode infection in mice

Abstract

Background: Here, Mesocestoides (M.) vogae infection in mice is proposed as a suitable experimental model for studying the immunity in the peritoneal cavity of mice.

Methods: To investigate the kinetics of immune parameters in M. vogae-infected mice, we detected, using flow cytometry, the expression of selected lymphoid and myeloid markers within the peritoneal cell population at day 0, 3, 6, 10, 14, 19, 25, 30 and 35 post-infection. Then, using ELISA, we analyzed the cytokine IFN-γ, TGF-β, IL-4 and IL-10 responses and the levels of anti-M. vogae IgG and IgM antibodies in the peritoneal lavage fluid. Cells isolated from the peritoneal cavity were subjected to further molecular analysis. To assess cell activation, peritoneal cells were exposed to LPS, and culture supernatants were collected and assayed for the level of cytokines and production of nitrite. Ly6C+ and Ly6G+ cells were isolated using MACS from the peritoneal cells at day 35 post-infection. Both MACS-isolated subsets were co-cultured with preactivated T cells to measure their suppressive capacity. Next, the role of parasite excretory-secretory antigens in induction of CD11b+ myeloid cells with the suppressive phenotype and the production of IL-10 was examined.

Results: In the peritoneal cavity an initial increase of CD11b+Gr-1+F4/80^highMHC II^high cells, NK, NKT cells and CD8+ cytotoxic T cells was observed in the first week of infection. At day 14 post-infection, an increase in the number of myeloid CD11b+Gr-1+ cells was detected, and most of this cell population expressed low levels of F4/80 and MHC II in later stages of infection, suggesting the impairment of antigen-presenting cell functions, probably through the excretory-secretory molecules. Moreover, we confirmed that peritoneal Gr1+ cells (Ly6C+ and Ly6G+ population) are phenotypically and functionally consistent with myeloid-derived suppressor cells. Metacestode infection elicited high levels of IL-10 and upregulated STAT-3 in peritoneal cells. A higher level of IgM suggests that this isotype may be predominant and is involved in the host protection.

Conclusions: Mesocestoides vogae tetrathyridia induced the recruitment of immunosuppressive cell subsets, which may play a key role in the downregulation of immune response in long-term parasitic diseases, and excretory-secretory antigens seem to be the main regulatory factor.

Keywords: Experimental larval cestodiasis; Host’s immune response; Mesocestoides vogae; Peritoneal cavity.

Fig. 1

Peritoneal lymphocyte cell population in the peritoneal cavities of healthy and Mesocestoides vogae-infected mice at 3, 6, 10, 14, 19, 25, 30 and 35 days post-infection. Peritoneal exudate cells were isolated from the peritoneal cavity, and the expression of lymphocyte surface markers by these cells was analyzed from lymphogate using flow cytometry. a Representative dot plots show the expression of CD3/CD19, CD4/CD8 and CD49b/CD8 on PEC. Proportions of T- and B-lymphocytes (b), T-lymphocyte subpopulations (c), NK and NKT cells (d) in the peritoneal cavities of healthy and infected mice. Data are expressed as the means ± SD. Statistical significance was analyzed using one-way ANOVA with Dunnett's post-test, and significantly different values between control and infected groups are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2

Antibody response of the IgM (a) and IgG (b) subclass specific to Mesocestoides (M.) vogae homogenate (MvH) or excretory/secretory molecules (ES) in the peritoneal exudates of healthy and infected mice at 3, 6, 10, 14, 19, 25, 30 and 35 days post-infection (p.i.). The anti-M. vogae IgM and IgG antibody titers were determined by ELISA methods. Data are presented as the means of OD ± SD. OD values of IgM for uninfected mice (n = 3) were 0.059 ± 0.004 for MvH antigen and 0.067 ± 0.012 for ES. OD values of IgG for uninfected mice (n = 4) were 0.141 ± 0.025 for MvH antigen and 0.079 ± 0.013 for ES. The cut-off values are indicated in the figures. Statistical significance was analyzed using two-way ANOVA followed by Bonferroni's multiple comparison tests, and significantly different values are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001. Detection of immunogenic antigens within MvH and ES preparations immunoreactive to IgG (c MvH; d ES) and IgM antibodies (e MvH; f ES) performed by western blot analysis. Exudates from infected mice obtained on corresponding days p.i. were incubated with nitrocellulose strips with blotted MvH or ES antigens.

Fig. 3

Peritoneal inflammation during Mesocestoides (M.) vogae infection is associated with an increased number of myeloid CD11b+Gr-1+ cells. Myeloid cell population in the peritoneal cavities of healthy and M. vogae-infected mice at 3, 6, 10, 14, 19, 25, 30 and 35 days post-infection. Peritoneal exudate cells (PEC) were isolated from the peritoneal cavity, and the expression of myeloid cell surface markers by these cells was analyzed using flow cytometry (FC). Live cells were gated on CD11b and Gr-1 expression to first identify CD11b+Gr-1+ cells. Then, F4/80 and MHC II expression was identified within the CD11b+Gr-1+ cells. a Representative dot plots obtained by FC analysis show the expression of CD11b/Gr-1 and F4/80/MHC II on PEC. b Proportions of CD11b^highGr-1+ cells in the peritoneal cavities of healthy and infected mice. c Proportions of F4/80+MHC II+ cells within CD11b^highGr-1+ cells in the peritoneal exudate cell population from healthy and infected mice. F4/80 (d) and MHC II (e) mean fluorescence intensity (MFI) within CD11b^highGr-1+ cells in the peritoneal exudate cell population from healthy and infected mice. Data are expressed as the mean ± SD. Statistical significance was analyzed using one-way ANOVA with Dunnett's post-test, and significantly different values between healthy and infected mice are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4

Mesocestoides vogae infection regulates the production of inflammatory cytokines in LPS-induced peritoneal exudate cells (PEC). PEC were isolated from the peritoneal cavity of healthy and infected mice at 3, 6, 10, 14, 19, 25, 30 and 35 days post-infection. Then PEC were cultured in 24-well plates in the presence or absence of LPS (1 µg/ml) at 37 °C 5% CO₂ for 72 h. The concentration of NO (a) in the culture supernatants was determined as nitrite (NO₂⁻) using Griess reagent. Nitrite concentration was determined using 0.1 M NaNO₃ as the standard. Concentrations of cytokines TNF-α, IL-6 and IL-10 (b) in culture supernatants were measured by ELISA and expressed in pg/ml. Data are expressed as the mean ± SD. Statistical significance was analyzed using two-way ANOVA followed by Bonferroni's multiple comparison tests, and significantly different values are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5

Peritoneal exudate cells (PEC) isolated from Mesocestoides vogae-infected mice possess suppressive capacities and can be subdivided into two subsets. Ly6C+ and Ly6G+ cells were isolated using MACS from the peritoneal cell population at day 35 post-infection and cultured with CFSE-labeled T cells stimulated with anti-CD3/ anti-CD28 mAbs for 72 h at different ratios. a Flow cytometry plots of PEC from healthy and infected mice showing the expression of CD11b and Gr-1 before and after MACS sort. b Representative histograms showing suppression of spleen T cells by sorted Ly6C+ or Ly6G+ in vitro (1:4 ratio MDSC to T cells). c The graph represents the proliferation inhibition of CD3/CD28-activated spleen T cells (the ratio of myeloid cell subsets to T cells as indicated in the graph). Results represent means ± SD of triplicates. Statistical significance was analyzed using Student's t test, and significantly different values are indicated as: **p < 0.01, ***p < 0.001. d The sorted cells were subjected to May-Grünwald/Giemsa staining. Representative pictures showing the morphology of sorted subsets are depicted. Original magnification, ×1000, scale bar = 20 μm. e mRNA expression of iNOS and Arg-1 in PEC of healthy mice (control) and sorted Ly6C+ or Ly6G+ cells. Statistical significance was analyzed using two-way ANOVA followed by Bonferroni's multiple comparison tests and significantly different values are indicated as: ***p < 0.001. f Immunofluorescent assay of iNOS and Arg-1 in whole peritoneal cells. Hoechst staining was used to visualize nuclei. Original magnification, ×1000, scale bar = 10 μm.

Fig. 6

Mesocestoides vogae infection is associated with IL-10 production. Concentrations of cytokines IFN-γ (a), IL-4 (b), TGF-β (c) and IL-10 (d) in the peritoneal exudates of healthy and Mesocestoides vogae-infected mice at day 3, 6, 10, 14, 19, 25, 30 and 35 post-infection were measured by ELISA and expressed in pg/ml. Data are expressed as the mean ± SD. Statistical significance was analyzed using one-way ANOVA with Dunnett's post-test and significantly different values between healthy and infected mice are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001. e Relative expression of m-RNA for STAT-1, STAT-3 and STAT-6 in the peritoneal exudate cells of healthy and infected mice was analyzed by real-time PCR. Data are expressed as means ± SD after comparison with uninfected PEC following normalization to β-actin. Statistical significance was analyzed using two-way ANOVA followed by Bonferroni's multiple comparison tests, and significantly different values are indicated as: **p < 0.01, ***p < 0.001.

Fig. 7

Mesocestoides vogae excretory-secretory (ES) products induce the accumulation of CD11b+IL-10+ peritoneal cells. Mice were injected intraperitoneally with 20 µg of ES and PBS as control every day for a total of 6 days. Peritoneal exudate cells (PEC) were isolated and subjected to flow cytometric analysis. a Representative flow cytometry plots showing the expression of IL-10 and CD11b by PEC. Live cells were gated on CD11b expression to first identify CD11b+ cells. Then, IL-10 expression was identified within CD11b+ cells. b Proportions of CD11b+IL-10+ cells in the peritoneal cavities of PBS and ES treated mice. c Concentrations of IL-10 in the peritoneal exudates was measured by ELISA and expressed in pg/ml. d Relative expression of m-RNA for IL-10 in the peritoneal exudate cells was analyzed by real-time PCR. Data are expressed as the mean ± SD. Statistical significance was analyzed using Student's t test, and significantly different values between healthy and ES treated mice are indicated as: *p < 0.05, **p < 0.01.