Differences in cNOS/iNOS Activity during Resistance to Trypanosoma cruzi Infection in 5-Lipoxygenase Knockout Mice

Abstract

Infection with the protozoan Trypanosoma cruzi causes Chagas disease and consequently leads to severe inflammatory heart condition; however, the mechanisms driving this inflammatory response have not been completely elucidated. Nitric oxide (NO) is a key mediator of parasite killing in T. cruzi-infected mice, and previous studies have suggested that leukotrienes (LTs) essentially regulate the NO activity in the heart. We used infected 5-lipoxygenase-deficient mice (5-LO^-/-) to explore the participation of nitric oxide synthase isoforms, inducible (iNOS) and constitutive (cNOS), in heart injury, cytokine profile, and oxidative stress during the early stage of T. cruzi infection. Our evidence suggests that the cNOS of the host is involved in the resistance of 5-LO^-/- mice during T. cruzi infection. iNOS inhibition generated a remarkable increase in T. cruzi infection in the blood and heart of mice, whereas cNOS inhibition reduced cardiac parasitism (amastigote nests). Furthermore, this inhibition associates with a higher IFN-γ production and lower lipid peroxidation status. These data provide a better understanding about the influence of NO-interfering therapies for the inflammatory response toward T. cruzi infection.

Figure 1

5-LO pathway signaling is essential for mouse resistance and control of parasitism in T. cruzi infection. (a) Parasitemia and (b) survival rate of 129 WT and 5-LO^−/− mice infected with 5 × 10³ trypomastigote forms of T. cruzi Y strain. (c) Representative images from hematoxylin-eosin staining of heart tissue from 129 WT and 5-LO^−/− mice 12 days of postinfection. Black arrows indicate amastigote nests. (d) Cardiac parasitism of three heart sections enumerated per mouse. N = 3‐5 animals in each group and values are the mean ± SEM. ^∗p < 0.001.

Figure 2

Differences in cNOS/iNOs activity considering the resistance to T. cruzi infection. (a–f) Parasitemia of 129 WT and 5-LO^−/− mice infected with 5 × 10³ trypomastigote forms of T. cruzi Y strain. Mice were treated 4 h before infection and were administered i.p. daily for 30 days, employing inhibitors dose as follows: aminoguanidine (AG, iNOS inhibitor, 50 mg/kg/day), Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME, 20 mg/kg/day), or both. Control experimental groups received PBS (0.2 mL/mouse). N = 5 animals in each group and values are the mean ± SEM. ^∗p < 0.001.

Figure 3

Survival rate of T. cruzi-infected mice treated with iNOS/cNOS blockers. (a) 129 WT mice and (b) 5-LO^−/− mice infected with 5 × 10³ trypomastigote forms of T. cruzi Y strain. Mice were treated 4 h before infection and were administered i.p. daily for 30 days, employing inhibitors dose as follows: aminoguanidine (AG, iNOS inhibitor; 50 mg/kg/day), Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME [57], 20 mg/kg/day), or both. Control experimental groups received PBS (0.2 mL/mouse). N = 5 animals in each group and values are the mean ± SEM. ^∗p < 0.001.

Figure 4

Cardiac parasitism. Number of amastigote nests in (a) wild-type (129 WT mice) and (b) 5-LO^−/− mice infected with 5 × 10³ trypomastigote forms of T. cruzi (day 12 of postinfection). (c) Photomicrographs of cardiac sections stained with hematoxylin-eosin. AG: infected mice treated with aminoguanidine (50 mg/kg/day i.p.); LN: infected mice treated with L-NAME (20 mg/kg/day i.p.); AG+LN: infected mice treated with aminoguanidine (50 mg/kg/day i.p. plus L-NAME 20 mg/kg/day). n = 3 animals in each group and values are the mean ± SEM for three independent experiments, ^∗p < 0.001. ns: not significant, p > 0.05 compared with control group (untreated and infected).

Figure 5

Nitric oxide (NO) production. (a) Wild-type (129 WT mice) and (b) 5-LO^−/− mice infected with 5 × 10³ trypomastigote forms of T. cruzi Y strain and treated or untreated with NOS inhibitors. NO was determined by measuring the nitrite levels in plasma employing the cadmium-copper system followed by the Griess reaction. N = 5 animals in each group and values are the mean ± SEM for three independent experiments. ^∗p < 0.001. ^#p < 0.001, ns: not significant, p > 0.05 compared with uninfected group or between groups.

Figure 6

Plasma levels of cytokines after treatment with NOS inhibitors. Three independent samples, each for 129 WT mice (a–c) and 5-LO^−/− mice (d–f), were used. AG: infected mice treated with aminoguanidine (50 mg/kg/day i.p.); LN: infected mice treated with L-NAME (LN; 20 mg/kg/day i.p.); AG+LN: infected mice treated with aminoguanidine (50 mg/kg/day i.p. plus L-NAME 20 mg/kg/day). Mean OD values of Multi-Analyte ELISArray results for indicated cytokines are presented; OD > 1.0 is indicated as 1.0. Data are representative of two independent experiments. N = 5 animals in each group and values are the mean ± SEM. ^∗p < 0.001.

Figure 7

Oxidative stress profile evaluated by tert-butyl hydroperoxide-initiated chemiluminescence on day 12 after T. cruzi infection. (a–d) Mice were infected with 5 × 10³ trypomastigote forms of T. cruzi (Y strain) and were treated or untreated with NOS inhibitors. (a) Uninfected mice and untreated T. cruzi-infected mice were used as controls. (b) AG: infected mice treated with aminoguanidine (50 mg/kg/day i.p.); (c) LN: infected mice treated with L-NAME (LN; 20 mg/kg/day i.p.); (d) AG+LN: infected mice treated with aminoguanidine (50 mg/kg/day i.p. plus L-NAME 20 mg/kg/day). N = 5 animals in each group. Values represent the mean ± SEM and are representative of two independent experiments ^∗p < 0.001.

Figure 8

Plasmatic antioxidant profile. (a) Wild-type (129 WT mice) and (b) 5-LO^−/− mice. Mice were infected with 5 × 10³ trypomastigote forms of T. cruzi (Y strain) and were treated or untreated with NOS inhibitors. AG: infected mice treated with aminoguanidine (50 mg/kg/day i.p.); LN: infected mice treated with L-NAME (LN; 20 mg/kg/day i.p.); AG+LN: infected mice treated with aminoguanidine (50 mg/kg/day i.p. plus L-NAME 20 mg/kg/day). Uninfected mice were used as controls. N = 5 animals in each group. Values represent the mean ± SEM and are representative of two independent experiments ^∗p < 0.001.

Figure 9

Plasma levels of PGE₂. (a) Wild-type (129 WT mice) and 5-LO^−/− mice uninfected and infected and (b) treated or untreated with NOS inhibitors. AG: infected mice treated with aminoguanidine (50 mg/kg/day i.p.); LN: infected mice treated with L-NAME (LN; 20 mg/kg/day i.p.); AG+LN: infected mice treated with aminoguanidine (50 mg/kg/day i.p. plus L-NAME 20 mg/kg/day). Mice were infected with 5 × 10³ trypomastigote forms of T. cruzi (Y strain). N = 3 animals in each group and values are the mean ± SEM. ^∗p < 0.001 compared with the control group.