Total Phenolic Fraction (TPF) from Extra Virgin Olive Oil: Induction of apoptotic-like cell death in Leishmania spp. promastigotes and in vivo potential of therapeutic immunomodulation

Abstract

Background: Leishmaniasis is a serious multifactorial parasitic disease with limited treatment options. Current chemotherapy is mainly consisted of drugs with serious drawbacks such as toxicity, variable efficacy and resistance. Alternative bioactive phytocompounds may provide a promising source for discovering new anti-leishmanial drugs. Extra Virgin Olive Oil (EVOO), a key-product in the Mediterranean diet, is rich in phenols which are associated with anti-inflammatory, anti-cancer and anti-microbial effects. In this study, we investigate the anti-leishmanial effect of Total Phenolic Fraction (TPF) derived from EVOO in both in vitro and in vivo systems by investigating the contributing mechanism of action.

Methodology/principal findings: We tested the ability of TPF to cause apoptotic-like programmed cell death in L. infantum and L. major exponential-phase promastigotes by evaluating several apoptotic indices, such as reduction of proliferation rate, sub-G0/G1 phase cell cycle arrest, phosphatidylserine externalization, mitochondrial transmembrane potential disruption and increased ROS production, by using flow cytometry and microscopy techniques. Moreover, we assessed the therapeutic effect of TPF in L. major-infected BALB/c mice by determining skin lesions, parasite burden in popliteal lymph nodes, Leishmania-specific antibodies and biomarkers of tissue site cellular immune response, five weeks post-treatment termination. Our results show that TPF triggers cell-cycle arrest and apoptotic-like changes in Leishmania spp. promastigotes. Moreover, TPF treatment induces significant reduction of parasite burden in draining lymph nodes together with an antibody profile indicative of the polarization of Th1/Th2 immune balance towards the protective Th1-type response, characterized by the presence of IFN-γ-producing CD4+ T-cells and increased Tbx21/GATA-3 gene expression ratio in splenocytes.

Conclusions/significance: TPF exhibits chemotherapeutic anti-leishmanial activity by inducing programmed cell death on cell-free promastigotes and immunomodulatory properties that induce in vivo T cell-mediated responses towards the protective Th1 response in experimental cutaneous leishmaniasis. These findings enable deeper understanding of TPF's dual mode of action that encourages further studies.

Fig 1. Effect of TPF on the viability of L. infantum and L. major promastigotes.

Fig 2. Growth kinetics of Leishmania spp. promastigotes exposed to TPF.

Fig 3. Leishmania spp. promastigote proliferation determined by CFSE staining.

L. infantum (A) and L. major (B) early exponential-phase promastigotes were treated with IC₅₀ and 2 x IC₅₀ concentrations of TPF and their proliferation rate was qualitatively monitored at 24 h intervals for 3 consecutive days by CFSE staining and subsequent analysis of fluorescence intensity in FACS. HePC (IC₅₀)-treated and untreated parasites were used as positive and negative control groups, respectively. The results are expressed as mean fluorescence intensity values ± SD of three independent experiments. Symbols of * and ^¥ indicate statistically significant differences compared to negative and positive control groups, respectively, while ^≠ indicates significant differences between TPF groups.

Fig 4. Microscopy images of Leishmania spp. promastigotes.

Fig 5. Analysis of cell cycle arrest in TPF-treated L. infantum and L. major promastigotes.

Fig 6. PS externalization in TPF-treated L. infantum and L. major promastigotes.

Exponential-phase L. infantum (A, C) and L. major (B, D) promastigotes were either left untreated (negative control) or were treated with IC₅₀ and 2 x IC₅₀ concentrations of TPF and HePC (IC₅₀, positive control) for 24, 48 and 72 h. At the end of the aforementioned time-points, parasites were double stained with annexin V-FITC and PI and were analyzed by FACS. The results are presented as mean values of % annexin V+ parasites ± SD in bar diagrams (A, B) representative of three independent experiments and as flow cytometric dot plots with respective quadrants (C, D), representative of one experiment. Symbols of * and ^¥ indicate statistically significant differences compared to negative and positive control groups, respectively while ^≠ indicates significant differences between TPF groups.

Fig 7. Evaluation of mitochondrial membrane potential in Leishmania spp. promastigotes treated with TPF.

Fig 8. ROS production in TPF-treated Leishmania spp. promastigotes.

Fig 9. Therapeutic effect of TPF in a murine experimental model of cutaneous leishmaniasis.

(A) Definition of treatment schedule and experimental groups. BALB/c mice were subcutaneously infected into the right hind footpad with 10⁶ L. major promastigotes and one week later, were either treated with TPF (20 mg/kg b.w., ip) or HePC (15 mg/kg b.w., oral gavage) for up to 28 days or left untreated (control group). (B) Time course of L. major infection. The increase in footpad swelling was monitored weekly by measuring the increase of footpad thickness (in mm) as compared with the uninfected contralateral footpad. Results are expressed as mean values ± SD. (C) Parasite load estimation in popliteal lymph nodes of TPF-treated mice. Parasite load was determined at five weeks post-treatment termination by a limiting dilution assay. Values represent the mean value ± SD of five animals per experimental group. (D) The IgG1 and IgG2a reciprocal end-point titers against L. major SLA were determined by ELISA at five weeks post-treatment termination (n = 5 mice per group) and represented as whisker (min to max) plots. * indicates the statistical differences between IgG1 and IgG2a anti-SLA titers among experimental groups. (E) Effect of TPF treatment on the percentage of IFN-γ-producing CD4+ T cells. Spleen cells from each experimental animal were isolated five weeks post-treatment termination and intracellular staining was performed. Flow cytometric analysis was conducted and total splenocytes were first gated on a forward scatter (FSC-H)/side scatter (SSC-H) plot and then subgated on the CD4+ population. Results of cytokine profiles are presented in bar diagram showing the percentage of IFN-γ-producing CD4+ T cells. Symbol of * indicates statistically significant differences among the experimental groups. (F) Quantitative analysis of Tbx21 and GATA-3 mRNA in spleen cells. The expression of Tbx21 and GATA-3 mRNA was evaluated by real-time PCR and the expression level of the GAPDH housekeeping gene from the same samples was used as normalizer. All expression levels were computed via the ΔΔCt method. Symbol of * indicates statistically significant differences among the experimental groups.