Leaf hydroalcoholic extract and oleoresin from Copaifera multijuga control Toxoplasma gondii infection in human trophoblast cells and placental explants from third-trimester pregnancy

Abstract

The conventional treatment of congenital toxoplasmosis is mainly based on the combination of sulfadiazine and pyrimethamine. However, therapy with these drugs is associated with severe side effects and resistance, requiring the study of new therapeutic strategies. There are currently many studies with natural products, including Copaifera oleoresin, showing actions against some pathogens, as Trypanosoma cruzi and Leishmania. In the present study, we investigated the effects of the leaf hydroalcoholic extract and oleoresin from Copaifera multijuga against Toxoplasma gondii in human villous (BeWo) and extravillous (HTR8/SVneo) trophoblast cells, as well as in human villous explants from third-trimester pregnancy. For this purpose, both cells and villous explants were infected or not with T. gondii, treated with hydroalcoholic extract or oleoresin from C. multijuga and analyzed for toxicity, parasite proliferation, cytokine and ROS production. In parallel, both cells were infected by tachyzoites pretreated with hydroalcoholic extract or oleoresin, and adhesion, invasion and replication of the parasite were observed. Our results showed that the extract and oleoresin did not trigger toxicity in small concentrations and were able to reduce the T. gondii intracellular proliferation in cells previously infected. Also, the hydroalcoholic extract and oleoresin demonstrated an irreversible antiparasitic action in BeWo and HTR8/SVneo cells. Next, adhesion, invasion and replication of T. gondii were dampened when BeWo or HTR8/SVneo cells were infected with pretreated tachyzoites. Finally, infected and treated BeWo cells upregulated IL-6 and downmodulated IL-8, while HTR8/SVneo cells did not change significantly these cytokines when infected and treated. Finally, both the extract and oleoresin reduced the T. gondii proliferation in human explants, and no significant changes were observed in relation to cytokine production. Thus, compounds from C. multijuga presented different antiparasitic activities that were dependent on the experimental model, being the direct action on tachyzoites a common mechanism operating in both cells and villi. Considering all these parameters, the hydroalcoholic extract and oleoresin from C. multijuga can be a target for the establishment of new therapeutic strategy for congenital toxoplasmosis.

Keywords: Copaifera multijuga; Toxoplasma gondii; congenital toxoplasmosis (CT); hydroalcoholic extract; oleoresin; trophoblast.

Figure 1

Cell viability. BeWo (A, B) and HTR8/SVneo (C, D) cells were treated or not with several concentrations of oleoresin or hydroalcoholic extract from C. multijuga for 24 h, and MTT assay was performed. Cell viability was expressed as percentage (cell viability %), considering the absorbance of cells incubated only with the medium as 100% viability. The results are expressed as means ± standard deviation of three independent experiments performed with eight replicates. Significant differences detected by One-Way ANOVA with Bonferroni’s multiple comparison post-test, or Kruskal-Wallis when appropriate. ^* P < 0.05, ^** P < 0.001, ^*** P < 0.0001, and ^**** P < 0.00001 in relation to the medium.

Figure 2

T. gondii intracellular proliferation. BeWo (A) and HTR8/SVneo (B) cells were infected by T. gondii and treated or not with non-toxic concentrations of hydroalcoholic extract or oleoresin from C. multijuga for 24 h. Untreated and infected cells (medium) were considered as 100% parasite proliferation, and SDZ + PYR was used as positive control. T. gondii intracellular proliferation was analyzed using a β-galactosidase colorimetric assay and expressed as percentage (% of T. gondii proliferation) in comparison to untreated cells (medium). The results were expressed as means ± standard deviation of three experiments performed in eight replicates. Significant differences detected by One-Way ANOVA, Bonferroni’s multiple comparisons post-test. ^*** P < 0.0001 or ^**** P < 0.00001 in relation to the medium.

Figure 3

Reversibility test. BeWo (A) and HTR8/SVneo (B) cells were infected by T. gondii and treated or not with non-toxic concentrations of the hydroalcoholic extract or oleoresin from C. multijuga for 24 h. In parallel, both cells were infected, treated, washed to remove the treatments, and maintained for additional 24 h in the medium free of treatment. Untreated and infected cells (medium) were considered as 100% reversibility of treatment, and SDZ + PYR was used as positive control. T. gondii intracellular proliferation was analyzed using a β-galactosidase colorimetric assay and expressed as percentage (reversibility of treatment) in comparison to untreated cells (medium). The reversibility index measured the ability of parasites to recover from treatment and proliferate in new cells. Finally, parasites obtained directly from BeWo (C) and HTR8/SVneo (D) cells treated with the hydroalcoholic extract or oleoresin were collected and used to infect new cells for 3 h. The number of tachyzoites was determined using the β-galactosidase assay and expressed as % of T. gondii invasion. The results were expressed as means ± standard deviation of three experiments performed in eight replicates. Significant differences detected by the One-Way ANOVA, Bonferroni’s multiple comparisons post-test. ^* P < 0.05, ^** P < 0.001, ^*** P < 0.0001 or ^**** P < 0.00001 in relation to the medium. ^& P < 0.05 in relation to SDZ + PYR.

Figure 4

Adhesion assay in BeWo cells. T. gondii tachyzoites were preincubated for 1 h with the hydroalcoholic extract or oleoresin from C. multijuga, and then allowed to interact with previously fixed BeWo cells during 3 h. As control, tachyzoites were incubated with SDZ + PYR or only medium (medium). The number of cells with adhered parasites (A) and the total number of adhered parasites (B) were determined in a total of 20 fields examined randomly with 20X objective lens. Representative images highlighting the effect of treatments on the tachyzoite-host cell interaction: (C) untreated parasites, (D) 32 µg/mL hydroalcoholic extract, (E) 16 µg/mL oleoresin and (F) 32 µg/mL oleoresin. The results were expressed as means ± standard deviation of two experiment performed in three replicates. Significant differences detected by One-Way ANOVA and Bonferroni’s multiple comparisons post-test. ^* P < 0.05, **P < 0.001, ^*** P < 0.0001 or ^**** P < 0.00001 in relation to the medium. ^& P < 0.05 in relation to SDZ + PYR. White arrows indicate tachyzoites attached to BeWo cells. The cell nucleus is labeled with TOPRO-3 (blue). T. gondii tachyzoites labeled with Alexa Fluor 488-conjugated anti-mouse IgG (green). Phalloidin-TRITC labeled F-actin is shown in red. Scale bar: 50 µm.

Figure 5

Adhesion assay in HTR8/SVneo cells. T. gondii tachyzoites were preincubated for 1 h with the hydroalcoholic extract or oleoresin from C. multijuga, and then allowed to interact with previously fixed HTR8/SVneo cells during 3 h. As control, tachyzoites were incubated with SDZ + PYR or only medium (medium). The number of cells with adhered parasites (A) and the total number of adhered parasites (B) were determined in a total of 20 fields examined randomly with 20X objective lens. Representative images highlighting the effect of the treatments on the tachyzoite-host cell interaction: (C) untreated parasites, (D) 16 µg/mL hydroalcoholic extract, (E) 8 µg/mL oleoresin, and (F) 16 µg/mL oleoresin. The results were expressed as means ± standard deviation of two experiment performed in three replicates. Significant differences detected by One-Way ANOVA and Bonferroni’s multiple comparisons post-test. ^** P < 0.001, ^*** P < 0.0001 or ^**** P < 0.00001 in relation to the medium. ^& P < 0.05 in relation to SDZ + PYR. White arrows indicate tachyzoites attached to HTR8/SVneo cells. The cell nucleus is labeled with TOPRO-3 (blue). T. gondii tachyzoites labeled with Alexa Fluor 488-conjugated anti-mouse IgG (green). Phalloidin-TRITC labeled F-actin is shown in red. Scale bar: 50 µm.

Figure 6

Invasion and proliferation assay. T. gondii tachyzoites were preincubated for 1 h with the hydroalcoholic extract or oleoresin from C. multijuga, and then allowed to interact with BeWo (A, B) or HTR8/SVneo (C, D) cells during 3 or 24 h to verify invasion and intracellular proliferation, respectively. As control, tachyzoites were incubated with SDZ + PYR or only medium (medium). The % of T. gondii invasion and the % of T. gondii proliferation were determined using the β-galactosidase activity. Untreated parasites (medium) were considered as 100% invasion and proliferation. The results were expressed as means ± standard deviation of three experiments performed in eight replicates. Significant differences detected by One-Way ANOVA and Bonferroni’s multiple comparisons post-test. ^** P < 0.001, ^*** P < 0.0001 or ^**** P < 0.00001 in relation to the medium.

Figure 7

Cytokine and ROS production in BeWo cells. BeWo cells were infected and treated or not with the hydroalcoholic extract or oleoresin from C. multijuga for 24 h. Untreated and uninfected cells (medium), untreated and infected cells (medium Tg) and SDZ + PYR were used as controls. Then, supernatants were collected and used to measure IL-6 (A, B), IL-8 (C, D) and MIF (E, F). In parallel, BeWo cells were infected, treated or not with the hydroalcoholic extract or oleoresin from C. multijuga for 24 h, incubated with the probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), ROS production was measured by a plate reader and data expressed as mean fluorescence intensity (MFI) (G, H). The results were expressed as means ± standard deviation of three experiments performed in eight replicates. Significant differences detected by One-Way ANOVA, Bonferroni’s multiple comparisons post-test. ^* P < 0.01, ^** P < 0.001, ^*** P < 0.0001 or ^**** P < 0.00001 in relation to medium (for uninfected cells) or to medium Tg (for infected cells). ^& P < 0.05 in relation to SDZ + PYR (for uninfected or infected cells). ^# P < 0.05 between medium and medium Tg.

Figure 8

Cytokine and ROS production in HTR8/SVneo cells. HTR8/SVneo cells were infected and treated or not with the hydroalcoholic extract or oleoresin from C. multijuga for 24 h. Untreated and uninfected cells (medium), untreated and infected cells (medium Tg) and SDZ + PYR were used as controls. Then, supernatants were collected and used to measure IL-6 (A, B), IL-8 (C, D) and MIF (E, F). In parallel, HTR8/SVneo cells were infected, treated or not with the hydroalcoholic extract or oleoresin from C. multijuga for 24 h, incubated with the probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), ROS production was measured by a plate reader and data were expressed as mean fluorescence intensity (MFI) (G, H). The results were expressed as means ± standard deviation of three experiments performed in eight replicates. Significant differences detected by One-Way ANOVA, Bonferroni’s multiple comparisons post-test. ^* P < 0.01, ^** P < 0.001, ^*** P < 0.0001 or ^**** P < 0.00001 in relation to the medium (for uninfected cells) or to the medium Tg (for infected cells). ^& P < 0.05 in relation to SDZ + PYR (for uninfected or infected cells). ^# P < 0.05 between medium and medium Tg.

Figure 9

Viability of human villous explants. The villous explants were treated for 24 h with hydroalcoholic extract or oleoresin from C. multijuga, SDZ+PYR or culture medium alone (medium). (A) Tissue viability is shown in percentages (% viability by incorporation of MTT). (B) Supernatants were collected and used to measure LDH levels (U/L). Representative images of villi incubated with (C) culture medium alone, (D) SDZ+PYR, 256 µg/mL hydroalcoholic extract (E), 256 µg/mL oleoresin (F). Data are expressed as means ± standard deviation of two experiments performed in six replicates. Significant differences detected by One-way ANOVA, Bonferroni’s multiple comparisons post-test (statistically significant when P < 0.05). Hematoxylin-eosin (HE) stained histological sections show syncytiotrophoblast cells (black arrows) and mesenchyme (*). Scale bar: 50 µm.

Figure 10

T. gondii intracellular proliferation. The human villous explants were infected with T. gondii followed by treatment for an additional 24 h with hydroalcoholic extract, oleoresin, SDZ+PYR or culture medium alone (medium). T. gondii intracellular proliferation was measured by β-galactosidase assay, and the number of tachyzoites was expressed as a percentage (% of T. gondii proliferation), and the untreated/infected (medium) was considered as 100% parasite proliferation. Data are expressed as means ± standard deviation of four experiments performed in six replicates. ^*Comparison with infected/untreated cells (^**** P < 0.00001). Significant differences detected by One-way ANOVA, Bonferroni’s multiple comparisons post-test differences were considered when P < 0.05.