Trypomastigotes of <i>T. cruzi</i> induce IL-17 production by mouse splenocytes <i>in vitro</i>.

<p>Leukocytes from BALB/c mice (5×10⁶ cells/ml) were cultured with or without trypomastigotes (2.5×10⁷/ml) of <i>T. cruzi</i> for 48 hours and the intracellular expression of IL-17 determined in total splenocytes (<b>A</b>), CD4⁺, CD8⁺, and NK cells (<b>B</b>) (mean ± SEM of cultures in triplicate, 3 animals/group). In <b>A</b>, the empty histograms represent IL-17⁺ cells harvest from cultures in the presence (black line) or absence of parasites (gray line) and the full histogram represents an IgG FITC control. The data are representative of two independent experiments. * <i>P</i><0.05 compared to uninfected mice.</p

IL-17 controls Th1 differentiation in <i>T. cruzi</i>-infected mice.

<p>BALB/c mice were infected, the heart harvested on day 14 p.i., the leukocytes isolated and incubated with monensin for 6 h in culture medium, and the intracellular expression of IL-17 (<b>A</b>) and IFN-γ (<b>B</b>) were determined. Data (mean ± SEM) are representative of two independent experiments using five mice per group. * <i>P</i><0.05 compared to infected mice treated with normal rat IgG.</p

<i>Trypanosoma cruzi</i> infection induces IL-17 expression in mouse splenocytes.

<p>The <i>ex vivo</i> frequencies of IL-17⁺ cells were determined in total splenocytes (<b>A</b>) of BALB/c mice on 0, 7, 14, and 21 days p.i.. In <b>B</b> and <b>C</b>, the percentage (mean ± SEM) of IL-17⁺ lymphocytes and CD4⁺, CD8⁺, NK cells of mice on day 14 p.i. are shown. The data are representative of two independent experiments with 3 mice in each day of infection. * <i>P</i><0.05 compared to uninfected mice.</p

Enhanced cardiac parasitism and mortality in <i>T. cruzi</i> infected mice treated with anti-IL-17.

<p>Parasitemia (<b>A</b>), cardiac parasitism (<b>B</b>), and survival (<b>C</b>) were determined in mice infected with <i>T. cruzi.</i> The quantification of genomic DNA was determined by real time PCR on tissue heart of mice on day 14 p.i. The data (mean ± SEM) are representative of two independent experiments (five mice per group). * <i>P</i><0.05 compared to infected mice treated with normal rat IgG.</p

IL-17 controls IFN-γ, TNF-α, IL-12, and IL-17 production in heart tissue of mice infected with <i>T. cruzi</i>.

<p>IFN-γ, TNF-α, IL-10, IL-12, and IL-17 were quantified in the sera and heart tissue of infected mice (14 days p.i.) that were treated or not with anti-IL-17. For heart cytokine quantification, 50 mg of tissue was homogenized in 1.0 ml of PBS plus protease inhibitors. The data (mean ± SEM) are representative of two independent experiments (five mice per group). * <i>P</i><0.05 compared to infected mice treated with normal rat IgG.</p

Primer sequences and reaction properties.

<p>bp: base pairs of amplicon size.</p

Cross-regulation of IL-12, IFN-γ, and IL-17 during <i>T. cruzi</i> infection.

<p>Splenocytes from BALB/c mice infected (14 days p.i.) or not with <i>T. cruzi</i> were harvested, cultured for 72 hours with or without Con-A (5 µg/ml), <i>T. cruzi</i> antigen (10 ηg/well), anti-IL-17 (10 µg/well) or anti-IFN-γ (10 µg/well). The cytokines IL-17, IL-12, IFN-γ, and TNF-α were determined by ELISA. The data show the mean ± SEM of triplicate cultures with 3 mice per group and are representative of two independent experiments. * <i>P</i><0.05 compared to control culture.</p

Mortality rates of 5 LO^-/-, IL-4^-/-, IL-12p40^-/-, MyD88^-/- and CCR5^-/- STAg-pretreated and infected mice.

<p>5 LO^-/- (A), IL-12p40^-/- (B), MyD88^-/- (C), CCR5^-/- (D) and IL-4^-/- (E) mice were pretreated with STAg and 48h later were infected with 100 <i>T. gondii</i> cysts by oral route. The C57BL/6 that were the WT mice of IL-4^-/-, IL-12p40^-/-, MyD88^-/- and CCR5^-/- and 129/SvEvTac that were the WT of 5 LO^-/- mice were also examined. The mortality rate for 8 mice from each group was determined. 5-LO^-/- (A), or IL-4^-/- (E) STAg-pretreated were significantly more resistant to toxoplasmosis than PBS-pretreated mice (χ²=9.171; <i>p</i> = 0.0018; df = 1; related to 5-LO^-/- mice; and χ²=6.198; <i>p</i> = 0.0128; df = 1; related to IL-4^-/- mice).</p

Mortality rates, tissue parasitism and inflammatory changes of STAg-pretreated C57BL/6 mice orally <i>T. gondii</i> infected.

<p>The mortality rate for 8 mice from each group was determined (A). STAg-pretreated mice were significantly more resistant to toxoplasmosis than PBS-pretreated mice (χ²=10.03; <i>P</i> = .0015; df = 1). Tissue parasitism in the small intestine (B), peripheral organs (C) and brain (D) were detected on day 8 p.i. by immunohistochemistry staining and scored by counting the number of parasitophorous vacuoles per tissue section in the small intestine and brain and per 40 microscopic fields in the other peripheral organs, using a 40 x objective. The small intestine (E,F), lung (G,H) and liver (I,J) of PBS- and STAg-pretreated mice were stained by H&E and analyzed for histological changes. The inflammatory foci in the liver are shown (arrows). Bar scale, 100 µm. The data of inflammatory scores in the organs were obtained by analyzing 40 microscopic fields per section on six sections using a 40 x objective from each mouse (K). Data are representative of at least two independent experiments of 5 mice per group that provided similar results. *<i>p</i> < 0.05 (Significantly different from values obtained from PBS-pretreated mice, Unpaired Student’s <i>t</i>-test). ^&p < 0.03 (Significantly different from values obtained from PBS-pretreated mice, Mann Whitney test); ^#<i>p</i> < 0.05 (Significantly different from values obtained from lung and liver of PBS-pretreated mice, Kruskal-Wallis test and Dunn’s multiple comparisons post-test).</p

<i>Toxoplasma gondii</i> Soluble Tachyzoite Antigen Triggers Protective Mechanisms against Fatal Intestinal Pathology in Oral Infection of C57BL/6 Mice

<div><p><i>Toxoplasma gondii</i> induces a potent IL-12 response early in infection that results in IFN-γ-dependent control of parasite growth. It was previously shown that <i>T. gondii</i> soluble tachyzoite antigen (STAg) injected 48 hr before intraperitoneal infection reduces lipoxin A₄ and 5-lipoxygenase (5-LO)-dependent systemic IL-12 and IFN-γ production as well as hepatic immunopathology. This study investigated the ability of STAg-pretreatment to control the fatal intestinal pathology that develops in C57BL/6 mice orally infected with 100 <i>T. gondii</i> cysts. STAg-pretreatment prolonged the animals’ survival by decreasing tissue parasitism and pathology, mainly in the ilea. Protection was associated with decreases in the systemic IFN-γ levels and IFN-γ and TNF message levels in the ilea and with increased TGF-β production in this tissue, but protection was independent of 5-LO and IL-4. STAg-pretreatment decreased CD4⁺ T cell, NK cell, CD11b⁺ monocyte and CD11b⁺CD11c⁺ dendritic cell numbers in the lamina propria and increased CD8⁺ T cells in the intestinal epithelial compartment. In parallel, decreases were observed in iNOS and IL-17 expression in this organ. These results demonstrate that pretreatment with STAg can induce the recruitment of protective CD8⁺ T cells to the intraepithelial compartment and decrease proinflammatory immune mechanisms that promote intestinal pathology in <i>T. gondii</i> infection.</p> </div