A Trypsin Inhibitor from Moringa oleifera Flowers Modulates the Immune Response In Vitro of Trypanosoma cruzi-Infected Human Cells

Abstract

Trypanosoma cruzi causes the lethal Chagas disease, which is endemic in Latin America. Flowers of Moringa oleifera (Moringaceae) express a trypsin inhibitor (MoFTI) whose toxicity to T. cruzi trypomastigotes was previously reported. Here, we studied the effects of MoFTI on the viability of human peripheral blood mononuclear cells (PBMCs) as well as on the production of cytokines and nitric oxide (NO) by T. cruzi-infected PBMCs. Incubation with MoFTI (trypsin inhibitory activity: 62 U/mg) led to lysis of trypomastigotes (LC₅₀ of 43.5 µg/mL) but did not affect the viability of PBMCs when tested at concentrations up to 500 µg/mL. A selectivity index > 11.48 was determined. When T. cruzi-infected PBMCs were treated with MoFTI (43.5 or 87.0 µg/mL), the release of the pro-inflammatory cytokine TNF-α and INF-γ, as well as of NO, was stimulated. The release of the anti-inflammatory cytokine IL-10 also increased. In conclusion, the toxicity to T. cruzi and the production of IL-10 by infected PBMCs treated with MoFTI suggest that this molecule may be able to control parasitemia while regulating the inflammation, preventing the progress of Chagas disease. The data reported here stimulate future investigations concerning the in vivo effects of MoFTI on immune response in Chagas disease.

Keywords: Moringa oleifera; cytokines; cytotoxicity; immunomodulatory agent; protease inhibitor; trypanocidal agent.

Figure 1

The Moringa oleifera tree (A) and its flowers (B).

Figure 2

Isolation of MoFTI. (A) Affinity chromatography of M. oleifera flower extract in Trypsin–Agarose column. The elution step with 0.1 M KCl-HCl pH 2.0 can be seen and fractions of 1.0 mL were collected. (B) Trypsin inhibitor activity (TIA) of MoFTI on bovine trypsin.

Figure 3

Effects of M. oleifera flower trypsin inhibitor (MoFTI) and benznidazole (Bz) on the viability of human peripheral blood mononuclear cells (PBMCs) after exposure by 24 h (A), 72 h (B), and 120 h (C). Different letters (a,b) indicate significant differences between the negative control and the other treatments by analysis of variance (ANOVA) followed by the Kruskal–Wallis test (p < 0.05). The asterisk (*) indicates significant differences between Bz and the other treatments. Control: negative control (untreated cells).

Figure 4

Effect of M. oleifera flower trypsin inhibitor (MoFTI) on the production of cytokines (A–F) and nitric oxide (G) by human peripheral blood mononuclear cells (PBMCs) infected or not with T. cruzi trypomastigotes after 48-h exposure. The treatments were: negative control (C = untreated and uninfected PBMCs); untreated PBMCs infected with trypomastigotes (C + T); infected PBMCs treated with MoFTI at 43.5 µg/mL (C + T + LC₅₀) and 87.0 µg/mL (C + T + 2 × LC₅₀); infected PBMCs treated with benznidazole (C + T + Bz); uninfected PBMCs treated with benznidazole (C + Bz); uninfected PBMCs treated with MoFTI at 43.5 µg/mL (C + LC₅₀) and 87.0 µg/mL (C+ 2 × LC₅₀). Different letters (a,b) indicate significant differences between treatments by analysis of variance (ANOVA) followed by the Tukey post-test (p < 0.05).

Figure 5

Effect of M. oleifera flower trypsin inhibitor (MoFTI) on the production of cytokines (A–F) and nitric oxide (G) by human peripheral blood mononuclear cells (PBMCs) infected or not with T. cruzi trypomastigotes after 120-h exposure. The treatments were: negative control (C = untreated and uninfected PBMCs); untreated PBMCs infected with trypomastigotes (C + T); infected PBMCs treated with MoFTI at 43.5 µg/mL (C + T + LC₅₀) and 87.0 µg/mL (C + T + 2 × LC₅₀); infected PBMCs treated with benznidazole (C + T + Bz); uninfected PBMCs treated with benznidazole (C + Bz); uninfected PBMCs treated with MoFTI at 43.5 µg/mL (C + LC₅₀) and 87.0 µg/mL (C+ 2 × LC₅₀). Different letters (a,b) indicate significant differences between treatments by analysis of variance (ANOVA) followed by the Tukey post-test (p < 0.05).