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

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-gamma-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. Author summary Leishmaniasis is an important protozoan parasitic disease and the currently active pharmaceutical compounds used for its treatment are limited with various drawbacks. Therefore, the need for new drug alternatives is evident and the development of novel anti-leishmanial agents based on natural products (NPs) is challenging. Olive oil (OO) is an integral part of the exquisite Mediterranean diet, constituting a high-value nutritional element associated with the lower incidence of coronary heart diseases and neurological disorders. Various studies conducted thus far, including human, animal, and in vitro models, attribute these intriguing biological properties to its adequate fatty acid profile and phenolic composition. Compositional studies have identified a plethora of phenolic compounds, including phenolic alcohols, secoiridoid derivatives, phenolic acids, lignans and flavonoids. Some of the most important biophenols found in OO are hydroxytyrosol, tyrosol, and oleuropein and its derivatives, oleocanthal and oleacein. Our recent studies concern the evaluation of the anti-leishmanial properties of NPs derived from Olea europaea L. (Oleaceae) in in vitro and in vivo models of experimental leishmaniasis. We have previously revealed Total Phenolic Fraction (TPF), as a promising product with anti-leishmanial properties in in vitro and in vivo systems. This study validates TPF as a potent inhibitory factor against Leishmania spp., driving parasites into an apoptotic-like cell death. Its leishmanicidal activity is further established in the in vivo murine experimental model of cutaneous leishmaniasis, evaluating also its ability to promote the host's protective Th1-type immune response. This study suggests the potential use of TPF against leishmaniasis because of its dual action as chemotherapeutic compound that eliminates parasite alone and also as immunostimulator of host's immune system.

Automated summary

Total Phenolic Fraction (TPF) derived from Extra Virgin Olive Oil (EVOO) exhibits anti-leishmanial activity by inducing apoptotic-like programmed cell death in Leishmania spp. promastigotes, and also enhances the protective Th1-type immune response in experimental cutaneous leishmaniasis in vivo. These findings suggest TPF has potential as both a chemotherapeutic agent and an immunomodulator against leishmaniasis.