<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

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

Anti-IL-4 mAb treatment reduces parasite loads in LTCP393(R)-inoculated BALB/c mice lesions.

<p>The animals were inoculated at right ear dermis with 1×10⁶ stationary phase promastigotes of <i>L. braziliensis</i>, isolates LTCP393(R) (black bars) or LTCP15171(S) (white bars), treated with anti-IL-4 monoclonal antibody (hatched bars) or rat IgG (full bars), and the parasite load estimated at 7 weeks post infection in the ears (A) and draining lymph nodes (B). Results are expressed as mean ± standard error of mean, and are representative of 2 independent experiments. Statistical analysis: ANOVA and Tukey multiple comparision test. *p<0,05.</p

Cytokines and enzymes mRNA expression in the ears from LTCP393(R) and LTCP15171(S)-inoculated mice.

<p>The animals were inoculated with 1×10⁶ stationary phase <i>L. braziliensis</i> promastigotes, isolates LTCP393(R) (black bars) or LTCP15171(S) (white bars). mRNA was extracted from the ears at 3, 5, 7 and 12 weeks post infection, and the specific mRNAs for IFN-γ (A), IL-4 (B), IL-10 (C), TGF-β (D), TNF-α (E), iNOS (F) and Arg I(G) were detected by real time RT PCR. The specific molecules mRNA expression was normalized based in the endogenous expression of the mRNAm for β-actin. Results are listed as the mean of expression in the ears of infected animals in relation to ears of uninfected ones ± standard error of mean, and are representative of 3 independent experiments. *p<0,05.</p

Flow cytometry of infected BALB/c mice ears inflammatory infiltrate.

<p>The animals were inoculated with 1×10⁶ stationary phase <i>L. braziliensis</i> promastigotes, isolates LTCP393(R) (black bars) or LTCP15171(S) (white bars). Analyses were conducted at 3, 5, 7 and 12 weeks post infection, FoxP3, CD103, CTLA-4 and GITR expression on CD4⁺CD25⁺ cells were evaluated at 7 and 12 weeks post infection. Results are expressed as total number of cells in the lesion (A, B, C and D), and as percentage of cells inside CD4⁺CD25⁺ gate (E and F). Results are expressed as mean ± standard error of mean, and are representative of 3 independent experiments. *p<0,05.</p

Anti-IL-4 mAb treatment reduces lesion thickness in LTCP393(R)-inoculated BALB/c mice.

<p>The animals were inoculated with 1×10⁶<i>L. braziliensis</i> stationary phase promastigotes, isolates (A) LTCP393(R) or (B) LTCP15171(S), in the right ear dermis, treated with anti-IL-4 monoclonal antibody (black signs) or rat IgG (white signs), and the course of lesion development was monitored for 7 weeks. Lesion thickness was determined as the difference between the infected ear and the contralateral one, noninfected. Results are expressed as mean ± standard error of mean, and are representative of 2 independent experiments. *p<0,05.</p