Toxoplasma gondii Infection Decreases Intestinal 5-Lipoxygenase Expression, while Exogenous LTB4 Controls Parasite Growth

Abstract

5-Lipoxygenase (5-LO) is an enzyme required for the production of leukotrienes and lipoxins and interferes with parasitic infections. In vitro, Toxoplasma gondii inhibits leukotriene B₄ (LTB₄) production, and mice deficient in 5-LO are highly susceptible to infection. The aim of this study was to investigate the effects of the pharmacological inhibition of the 5-LO pathway and exogenous LTB₄ supplementation during experimental toxoplasmosis. For this purpose, susceptible C57BL/6 mice were orally infected with T. gondii and treated with LTB₄ or MK886 (a selective leukotriene inhibitor through inhibition of 5-LO-activating protein [FLAP]). The parasitism, histology, and immunological parameters were analyzed. The infection decreased 5-LO expression in the small intestine, and treatment with MK886 reinforced this reduction during infection; in addition, MK886-treated infected mice presented higher intestinal parasitism, which was associated with lower local interleukin-6 (IL-6), interferon gamma (IFN-γ), and tumor necrosis factor (TNF) production. In contrast, treatment with LTB₄ controlled parasite replication in the small intestine, liver, and lung and decreased pulmonary pathology. Interestingly, treatment with LTB₄ also preserved the number of Paneth cells and increased α-defensins expression and IgA levels in the small intestine of infected mice. Altogether, these data demonstrated that T. gondii infection is associated with a decrease in 5-LO expression, and on the other hand, treatment with the 5-LO pathway product LTB₄ resulted in better control of parasite growth in the organs, adding to the knowledge about the pathogenesis of T. gondii infection.

Keywords: Paneth cells; antimicrobial peptides; eicosanoids; experimental toxoplasmosis; intestinal immune response.

FIG 1

Toxoplasma gondii impairs 5-lipoxygenase expression in the small intestine. (A) C57BL/6 and BALB/c mice and their congenic strains C57BKs/J and CB10-H2, respectively, were infected with 5 cysts of the ME49 strain of T. gondii. (B and C) On day 7 postinfection, the 5-LO (B) and BLT-1 (C) mRNA expression levels in the ilea were measured by real-time quantitative PCR (qPCR). (D) In the following experiments, C57BL/6 mice were infected with 10 cysts of the ME49 strain of T. gondii and treated daily with MK886 (5 mg/kg/day) or the vehicle (20% ethanolic solution) only. (E and F) On the 7th day after infection, levels of 5-LO mRNA (E) and BLT-1 (F) were measured in the ileum by qPCR. Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Tg, T. gondii infected mice; DI, days of infection. *, P < 0.05 compared to noninfected (NI) mice; &, P < 0.05 compared to vehicle-treated mice (by a Mann-Whitney test [B and C] and one-way analysis of variance [ANOVA] with a Bonferroni posttest [E and F]). Data are presented as means ± standard errors of the means (SEM) and are representative of results from one of two independent experiments.

FIG 2

MK886 or LTB₄ treatment did not increase C57BL/6 mouse survival during Toxoplasma gondii infection. (A) C57BL/6 mice were infected with 10 T. gondii cysts and treated with MK886 (5 mg/kg/day by the oral route), LTB₄ (1 μg/kg/day by the intraperitoneal route), or the vehicle (20% ethanolic solution by the oral route) for 7 days consecutively (n = 5 per group). (B and C) Mice were observed daily for body weight changes (B) and mortality (C) during 60 days postinfection. *, P < 0.05 compared to LTB₄- and vehicle-treated mice (by two-way ANOVA). Data are presented as means ± SEM from one of two independent experiments.

FIG 3

LTB₄ controls Toxoplasma gondii replication in the small intestine and enhances T. gondii-specific fecal IgA levels in C57BL/6 mice. (A) C57BL/6 mice were infected with 10 T. gondii cysts and treated daily with MK886 (5 mg/kg/day by the oral route) or LTB₄ (1 μg/kg/day by the intraperitoneal route). On day 7 postinfection, the small intestines of mice were collected and submitted to histological analysis. (B and C) Small intestine length (B) and parasite quantification (C) by immunohistochemistry of infected mice. (D to F) Representative photomicrographs from small intestine sections stained with hematoxylin and eosin (H&E) (D) and inflammatory scores (E) and goblet cell counts (F) in the small intestine. (G) T. gondii-specific IgA levels in mouse fecal samples were measured by an enzyme-linked immunosorbent assay (ELISA). OD₄₉₂, optical density at 492 nm. (H) Representative photomicrographs from small intestine sections stained with alcian blue. Bars, 200 μm (D) and 100 μm (H). *, P < 0.05 compared to noninfected (NI) mice; &, P < 0.05 for comparison between groups of infected mice (by a Kruskal-Wallis test with Dunn’s posttest [B and C] and one-way ANOVA with a Bonferroni comparison posttest [E to G]). Data are presented as means ± SEM and are representative of results from one of two independent experiments.

FIG 4

LTB₄ treatment attenuates Paneth cell hypoplasia and increases α-defensin 1 expression in the small intestine of T. gondii-infected mice. C57BL/6 mice were infected with 10 T. gondii cysts and treated daily with MK886 (5 mg/kg/day by the oral route) or LTB₄ (1 μg/kg/day by the intraperitoneal route). On day 7 postinfection, the small intestines of mice were collected and analyzed. (A and B) Quantification (A) and representative photomicrographs (B) of Paneth cells obtained from small intestine sections stained with H&E. (C to H) qPCR analysis of α-defensin 3 (C), α-defensin 5 (D), α-defensin 20 (E), α-defensin 21 (F), α-defensin 24 (G), and lysozyme (H) expression in the small intestine of T. gondii-infected mice treated or not with LTB₄. Arrows indicate intestinal crypts with Paneth cells. Bars, 100 μm. log2FC, log₂ fold change, normalized to GAPDH. *, P < 0.05 compared to noninfected (NI) vehicle-treated mice; #, P < 0.05 compared to noninfected LTB₄-treated mice; &, P < 0.05 for comparison between groups of infected mice (by one-way ANOVA with a Bonferroni comparison posttest [A and C to G] and a Kruskal-Wallis test with Dunn’s posttest [H]). Data are presented as means ± SEM and are representative of results from one of two independent experiments.

FIG 5

5-LO inhibition decreases cytokine production during Toxoplasma gondii infection. Cytokine levels were quantified in serum samples (A to F) or small intestine homogenates (G to L) of C57BL/6 mice infected with 10 T. gondii cysts and treated daily with MK886 (5 mg/kg/day by the oral route) or LTB₄ (1 μg/kg/day by the intraperitoneal route). IL-2 (A and G), IL-6 (B and H), IFN-γ (C and I), TNF (D and J), IL-17 (E and K), and IL-10 (F and L) levels were measured using a cytometric bead array (CBA) cytokine kit. *, P < 0.05 compared to noninfected (NI) mice; &, P < 0.05 for comparison between groups of infected mice (by one-way ANOVA). Data are presented as means ± SEM and are representative of results from one of two independent experiments. PBS, phosphate-buffered saline.

FIG 6

LTB₄ controls Toxoplasma gondii replication in the lung and liver of C57BL/6 mice. C57BL/6 mice were infected with 10 T. gondii cysts and treated daily with MK886 (5 mg/kg/day by the oral route) or LTB₄ (1 μg/kg/day by the intraperitoneal route). (A, B, D, and E) On day 7 postinfection, the lung and liver were collected, and tissue parasitism (A and D) and inflammatory scores (B and E) were quantified in tissue sections. (F) ALT levels were measured by an analytic kit in serum samples as an indication of liver damage. (C and G) Photomicrographs showing the histological alterations in the lung (C) and liver (G) of infected and treated mice. Arrowheads indicate inflammatory foci (G). Bars, 50 μm. *, P < 0.05 compared to noninfected (NI) mice; &, P < 0.05 for comparison between groups of infected mice (by a Kruskal-Wallis test with Dunn’s posttest [A, D, and F] or one-way ANOVA [B and E]). Data are presented as means ± SEM and are representative of results from one of two independent experiments.

FIG 7

Schematic representation of the effects of treatment with MK886 or LTB₄ on T. gondii-infected mice. T. gondii infection reduces 5-LO expression in the small intestine of C57BL/6 mice. The inhibition of the 5-LO pathway (MK886 treatment) downregulates IL-6, IFN-γ, and TNF and increases the parasite burden in the small intestine. In contrast, treatment with LTB₄ reduces parasitism in the small intestine, lung, and liver. In the small intestine, treatment with LTB₄ preserved Paneth cell numbers and increased defensin expression and IgA levels. Therefore, treatment with LTB₄ presented a beneficial effect during toxoplasmosis. (Y.C. drew the figure).