<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

Heme oxygenase-1 activity is involved in the control of Toxoplasma gondii infection in the lung of BALB/c and C57BL/6 and in the small intestine of C57BL/6 mice

Abstract Heme oxygenase-1 (HO-1) is an enzyme that catabolizes free heme, which induces an intense inflammatory response. The expression of HO-1 is induced by different stimuli, triggering an anti-inflammatory response during biological stress. It was previously verified that HO-1 is able to induce indoleamine 2,3-dioxygenase (IDO), an enzyme that is induced by IFN-γ in Toxoplasma gondii infection. To verify the role of HO-1 during in vivo T. gondii infection, BALB/c and C57BL/6 mice were infected with the ME49 strain and treated with zinc protoporphyrin IX (ZnPPIX) or hemin, which inhibit or induce HO-1 activity, respectively. The results show that T. gondii infection induced high levels of HO-1 expression in the lung of BALB/c and C57BL6 mice. The animals treated with ZnPPIX presented higher parasitism in the lungs of both lineages of mice, whereas hemin treatment decreased the parasite replication in this organ and in the small intestine of infected C57BL/6 mice. Furthermore, C57BL/6 mice infected with T. gondii and treated with hemin showed higher levels of IDO expression in the lungs and small intestine than uninfected mice. In conclusion, our data suggest that HO-1 activity is involved in the control of T. gondii in the lungs of both mouse lineages, whereas the hemin, a HO-1 inducer, seems to be involved in the control of parasitism in the small intestine of C57BL/6 mice

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

Mortality rates of nude, MHC II^-/- and MHC I^-/- STAg-pretreated and infected mice.

<p>The mortality rate for 8 mice from each group, nude (A), MHC II^-/- (B) and MHC I^-/- (C) was determined. Nude mice STAg- or PBS-pretreated survived significantly longer than euthymic control <i>T. gondii</i>-infected mice (χ²=10.03; <i>p</i> = 0.0015; df = 2). MHC II^-/- -STAg-pretreated mice were more resistant to infection than PBS-pretreated-MHC II^-/- or -C57BL/6 mice (χ²=4.493; <i>p</i> = 0.034; and χ²=4.120; <i>p</i> = 0.424; df = 2, respectively). MHC I^-/- pretreated with PBS or STAg were more resistant to infection than PBS-pretreated-C57BL/6 mice (χ²=9.069; <i>p</i> = 0.0026; χ²=3.974; <i>p</i> = 0.0462; df = 2, respectively).</p

Cytokine levels in the sera and mRNA expression and cytokine production in the ilea of STAg-pretreated orally <i>T. gondii</i> infected C57BL/6 mice.

<p>The levels of IL-12p70 (A), IFN-γ (B), IL-10 (C), IL-4 (D) and TGF-β (E) in serum samples, and IL-12p70 (J), IFN-γ (K), TNF (L), IL-10 (M), IL-4 (N) and TGF-β (O) in the ilea were measured on day 8 p.i. by ELISA. The values of cytokine production were the mean and SD of 5 mice per data point. The experiments were repeated twice and provided similar results. The cytokine IL-12p40 (F), IFN-γ (G), TNF (H) and IL-10 (I) transcript expressions in the ilea of STAg-pretreated and infected mice was quantified by qPCR on day 8 p.i. Results are demonstrated as mRNA expression of PBS- or STAg-pretreated and <i>T. gondii</i>-infected mice, relative to non-infected mice. The relative levels of gene expression were calculated by reference to the β-actin in each sample, using the threshold cycle (C_t) method; and the data (mean ± SD) represent values from one experiment representative of three independent experiments. * <i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 (ANOVA and Bonferroni multiple comparisons post-test). ^&<i>p</i> < 0.05 (Significantly different from values obtained from PBS-pretreated mice, Unpaired Student’s <i>t</i>-test). NI, non-infected.</p

Phenotypic characterization of leukocytes in the small intestine of STAg-pretreated and infected C57BL/6 mice.

<p>The leukocytes were obtained from the small intestine on day 8 p.i. as described on 'Material and methods' section. The number (mean ± SD) of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺, CD3⁺NK⁺, NK⁺ and CD11b⁺, CD11c⁺, CD11b⁺CD11c⁺ (A) leukocytes were analyzed in the LP. The numbers of CD3⁺, CD3⁺γδ⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ (B) lymphocytes were analyzed in the intraepithelial compartment. The intracellular IFN-γ and IL-17 production, after <i>in </i><i>vitro</i> stimulation with PMA/Ionomicin were detected in cells from the LP (C) and intraepithelial compartment (D) of the small intestine from PBS- or STAg-pretreated C57BL/6 mice on day 8 p.i. The IFN-γ, TGF-β, and RORγt (E) transcript expressions in CD4⁺ or CD8⁺ IELs from a pool obtained of 4 mice per group of naïve, PBS- or STAg-pretreated and infected mice was quantified by qPCR on day 8 p.i. Data are representative of at least two independent experiments of 5 mice per group. *<i>p</i> < 0.05, **<i>p</i> < 0.01 (Statistically different from values observed for the same cell phenotype obtained from PBS-pretreated mice, Unpaired Student’s <i>t</i>-test).</p