Lectins from Synadenium carinatum (ScLL) and Artocarpus heterophyllus (ArtinM) Are Able to Induce Beneficial Immunomodulatory Effects in a Murine Model for Treatment of Toxoplasma gondii Infection

Abstract

Infection by Toxoplasma gondii affects around one-third of world population and the treatment for patients presenting toxoplasmosis clinically manifested disease is mainly based by a combination of sulfadiazine, pyrimethamine, and folinic acid. However, this therapeutic protocol is significantly toxic, causing relevant dose-related bone marrow damage. Thus, it is necessary to improve new approaches to investigate the usefulness of more effective and non-toxic agents for treatment of patients with toxoplasmosis. It has been described that lectins from plants can control parasite infections, when used as immunological adjuvants in vaccination procedures. This type of lectins, such as ArtinM and ScLL is able to induce immunostimulatory activities, including efficient immune response against parasites. The present study aimed to evaluate the potential immunostimulatory effect of ScLL and ArtinM for treatment of T. gondii infection during acute phase, considering that there is no study in the literature accomplishing this issue. For this purpose, bone marrow-derived macrophages (BMDMs) were treated with different concentrations from each lectin to determine the maximum concentration without or with lowest cytotoxic effect. After, it was also measured the cytokine levels produced by these cells when stimulated by the selected concentrations of lectins. We found that ScLL showed high capacity to induce of pro-inflammatory cytokine production, while ArtinM was able to induce especially an anti-inflammatory cytokines production. Furthermore, both lectins were able to increase NO levels. Next, we evaluated the treatment effect of ScLL and ArtinM in C57BL/6 mice infected by ME49 strain from T. gondii. The animals were infected and treated with ScLL, ArtinM, ArtinM plus ScLL, or sulfadiazine, and the following parameters analyzed: Cytokines production, brain parasite burden and survival rates. Our results demonstrated that the ScLL or ScLL plus ArtinM treatment induced production of pro-inflammatory and anti-inflammatory cytokines, showing differential but complementary profiles. Moreover, when compared with non-treated mice, the parasite burden was significantly lower and survival rates higher in mice treated with ScLL or ScLL plus ArtinM, similarly with sulfadiazine treatment. In conclusion, the results demonstrated the suitable potential immunotherapeutic effect of ScLL and ArtinM lectins to control acute toxoplasmosis in this experimental murine model.

Keywords: ArtinM; ScLL; Toxoplasma gondii; lectins; therapeutic agents.

Figure 1

Cell viability determined by MTT assay in murine bone marrow-derived macrophages (BMDMs), treated with ScLL (A) or ArtinM (B) in several concentrations, ranging from 0.06 to 50 μg/ml or from 0.00004 to 1 μg/ml of ScLL or ArtinM, respectively. The cell suspensions were cultured in RPMI-1640 medium and the data are representative of one from three independent experiments. ^*P < 0.05.

Figure 2

Cytokine measurements in supernatants from bone marrow-derived macrophages (BMDMs) cultures after 48 h of stimulation with different concentrations of ScLL or ArtinM. Medium alone (RPMI) or LPS (1 μg/mL) were included in all experiments, as negative and positive controls, respectively. Levels of IL-12 (A) and (C) and IL-10 (B) and (D) were determined by immunoenzymatic assay ELISA. Results are expressed as mean ± SD of cytokine levels in pg/mL. ^*Statistically significant in relation to the control (RPMI-1640). (ANOVA and Tukey's multiple comparison post-test; P < 0.05).

Figure 3

Nitric oxide production in supernatants from bone marrow-derived macrophages (BMDMs) cultures after 48 h of stimulation with different concentrations of ScLL or ArtinM and LPS (1 μg/mL). Cells were stimulated with ScLL (50; 16.6; 5.5; 1.8; 0.61; 0.20; and 0.06 μg/mL) or ArtinM (1; 0.33 and 0.11 μg/mL). Nitrite concentration measured by Griess Reaction. The data are representative of one from three independent experiments in triplicate for each condition. ^*Statistically significant in relation to the control (RPMI-1640). (ANOVA and Tukey's multiple comparison post-test; P < 0.05).

Figure 4

Effects of sulfadiazine and lectin treatments in the levels of IL-2 (A), IL-4 (B), IFN-γ (C), IL-10 (D), IL-6 (E), TNF (F), and IL-17 (G) cytokines in serum samples from C57BL/6 mice. Results are expressed as mean ± SD of the cytokine levels in pg/mL. Statistically significances were determined by comparison of the values obtained in the infected and treated groups with non-treated and non-infected mice. Statistical test was performed using One-way ANOVA and Tukey's multiple comparison post-test. ^*Indicates significant differences (P < 0.05).

Figure 5

Parasite burden in the brain tissues from treated mice after 30 days of infection with T. gondii ME-29 strain and analyzed by quantitative real-time PCR. Statistical significances were calculated by comparison the parasitic DNA values among the treated and non-treated groups of mice. Statistical test was performed using One-way ANOVA and Tukey's multiple comparison post-test. ^*Indicates significant differences (P < 0.05).

Figure 6

Survival curve of C57BL/6 mice after infection with T. gondii ME49 strain. Animals were treated with ScLL, ArtinM, ScLL plus ArtinM, or sulfadiazine during 6 days, one time a day. As control, animals were infected with the same strain, but treated with phosphate-buffered saline (PBS) only. The survival curves were compared using the Log-rank Mantel-Cox test. Values of P < 0.05 were considered statistically significant. ^*Indicates significant differences (P < 0.05).