Dissertation committee:
Ελένη Ντότσικα (επιβλέπουσα): Διευθύντρια Ερευνών, Τομέας Μικροβιολογίας, Εργαστήριο Κυτταρικής Aνοσολογίας, Ελληνικό Ινστιτούτο Παστέρ
Λέανδρος- Αλέξιος Σκαλτσούνης: Καθηγητής, Τμήμα Φαρμακευτικής, Τομέας Φαρμακογνωσίας και Χημείας Φυσικών Προϊόντων, Ε.Κ.Π.Α.
Νεκτάριος Αληγιάννης: Αναπληρωτής Καθηγητής, Τμήμα Φαρμακευτικής, Τομέας Φαρμακογνωσίας και Χημείας Φυσικών Προϊόντων, Ε.Κ.Π.Α.
Μαρία Χαλαμπαλάκη: Αναπληρώτρια Καθηγήτρια, Τμήμα Φαρμακευτικής, Τομέας Φαρμακογνωσίας και Χημείας Φυσικών Προϊόντων, Ε.Κ.Π.Α.
Σοφία Μητάκου: Καθηγήτρια, Τμήμα Φαρμακευτικής, Τομέας Φαρμακογνωσίας και Χημείας Φυσικών Προϊόντων, Ε.Κ.Π.Α.
Εμμανουήλ Αγγελάκης, Διευθυντής Ερευνών, Τομέας Μικροβιολογίας, Διαγνωστικό Τμήμα, Ελληνικό Ινστιτούτο Παστέρ
Δέσποινα Σμυρλή: Κύρια Ερευνήτρια, Τομέας Μικροβιολογίας, Εργαστήριο Μοριακής Παρασιτολογίας, Ελληνικό Ινστιτούτο Παστέρ
Summary:
Plants have always been a valuable source for the discovery of molecules with pharmacological activity. Their numerous secondary metabolites possess a wide range of biological effects including antioxidant, anti-inflammatory, anticancer and antimicrobial activities. Among plants whose constituents have been extensively studied as pharmacological compounds, is the Olive tree (Olea europaea). A large number of experimental and clinical studies have demonstrated the capability of extra virgin olive oil to promote human health through the reduction of inflammatory and oxidative processes that lead to chronic diseases associated with inflammation and oxidation. These properties of extra virgin olive oil have placed it high in the hierarchy as “functional food” and numerous studies have been devoted to analyze the bioactivity of its compounds. It has been shown that the benefits of olive oil are attributed mainly to its phenolic fraction that includes a variety of more than 50 phenolic compounds. The Mediterranean region is an area that the olive tree flourishes and olive oil is an irreplaceable ingredient of the so-called Mediterranean diet that has been linked with indisputable benefits for the human health.
In the Mediterranean region, leishmaniasis is an endemic disease, along with other tropical and sub-tropical regions worldwide, such as South America, Middle East and Central Asia, whose etiological agent is the protozoan parasites of the genus Leishmania spp. The parasite is transmitted through the bite of the intermediate host that is the phlebotomine sand fly. The main hosts of the parasite are humans, dogs and rodents, while dogs are the main domestic reservoirs. The outcome of the disease depends strongly on the host’s immune response, where a cellular immunity of the Th1 type is associated with control of the disease, whereas the Th2 type is associated with disease progression. Since a vaccine appropriate for human use does not exist, the battle against the disease relies on a limited number of chemotherapeutic drugs with various side effects, such as resistance and high toxicity. Consequently, the search for new antileishmanial molecules is mandatory in order to overcome the problems of the existing therapies and the field of natural products can be a promising source of new drugs.
Following this pave of research, the aim of the present PhD thesis was to investigate the antileishmanial and immunomodulatory properties of the total phenolic fraction (TPF) of extra virgin olive oil and its pure molecule oleocanthal that is included in the total phenolic fraction, in in vitro and in vivo systems.
TPF was found to be effective against two different species of Leishmania spp., L. infantum and L. major in its both developmental forms, promastigotes and amastigotes, and the half – maximal inhibitory concentration (IC50) was determined as a measure of its antileishmanial effectiveness. Moreover, in order to determine the mode of antileishmanial action of TPF, the study was focused on the investigation of the type of the induced cell death in the Leishmania spp. promastigotes upon their exposure to TPF by determining morphological features and biochemical markers of apoptosis. The morphological alterations were obtained by optical microscopy, and the data showed that TPF provoked cell shrinkage in Leishmania spp. promastigotes, while fluorescence microscopy pointed out significant damage of their DNA integrity. The study of the biochemical alterations revealed that TPF caused DNA fragmentation, disorder of the cytoplasmic membrane structure, disruption of the mitochondrial membrane potential (ΔΨm), and excessive ROS production. These findings indicated that TPF provoked apoptotic cell death in Leishmania spp. promastigotes. The aforementioned promising in vitro results prompted the extension of the study in an in vivo murine model of cutaneous leishmaniasis, in order to investigate the potential use of TPF as an antileishmanial drug. BALB/c mice were infected in the footpad with L. major promastigotes and were treated with TPF. The treatment with TPF caused reduction of the footpad swelling along with significant reduction in the parasitic load of the popliteal lymph nodes. Interestingly, TPF exerted an immunomodulatory effect in experimental mice by promoting the polarization of the immune response towards a protective regime and eventually the control of the disease. Clearly, the production of IgG1 and IgG2a antibody isotypes characterize the induction of a mixed Th1- and Th2-type immune response. However, the cytokine production of INF-γ and the gene expression of Tbx21 and GATA-3 transcription factors indicate a more prominent Th1- over Th2-type response.
Qualitative and quantitative analysis of TPF, revealed that it was rich in the phenolic molecule oleocanthal. Consequently, the next goal of the study was to test the pure molecule of oleocanthal for its antileishmanial effect against the promastigotes and amastigotes of the same Leishmania species, in order to determine its antileishmanial effect. Oleocanthal is a molecule that gathers a lot of scientific attention because of its anti-inflammatory properties that are comparable to those of the non-steroidal anti-inflammatory drug ibuprofen. It also posseses antioxidant capacity and antimicrobial properties. The present study revealed the in vitro antileishmanial activity of oleocanthal, as well as its therapeutic effect in in vivo murine experimental models of cutaneous and visceral leishmaniasis. In the murine model of cutaneous leishmaniasis, BALB/c mice were infected with L. major promastigotes in the footpad and were treated with oleocanthal in two experimental doses. Oleocanthal in both doses reduced significantly both the skin lesions that were measured as footpad swelling and the parasitic load of the popliteal lymph nodes. Feet of the mice were submitted to histopathological analysis which revealed a topical immune response that leads to retrenchment of the infection and the recruitment of cells of the innate and adaptive immunity, such as macrophages, mast cells, T- and B-lymphocytes that are recruited in order to control the infection. Oleocanthal caused alterations in the IgG1 and IgG2a antibodies isotype production and the obtained profile indicated the dominance of a Th1 rather immune response. Moreover, analysis of the immune response induced by oleocanthal treatment demonstrated enhanced production of IL-12 cytokine and elevated gene expression ratios of Tbx21/GATA-3 and IL-12/IL-4 that suggest a polarization of the immune responses towards the Th1- type that is strongly associated with the resolution of the disease. In the murine model of visceral leishmaniasis, BALB/c mice were intravenously infected with L. infantum promastigotes and were treated with two doses of oleocanthal. Oleocanthal in both experimental doses caused the significant reduction of parasitic load in both spleen and liver which are the target organs of visceral leishmaniasis. It was also found that oleocanthal provoked elevation of the production of nitric oxide (NO), an important leishmanicidal molecule, in spleen cells. Despite the low parasitic load and the induction of NO, the IgG1 and IgG2a antibodies isotype production, the cytokines production (IL-12, IFN-γ and IL-4) and the gene expression of certain transcription factors (Tbx-21, GATA-3, STAT-1 and STAT-4), did not show a clear dominance of a Th1- over a Th2-type immune response.
Overall, the results of this study suggest that TPF from extra virgin olive oil and its pure molecule oleocanthal, are able to act as antileishmanial and immunomodulatory agents. These promising data suggest that there is an open field for further research development on the phenols from extra virgin olive oil as potential drugs against infectious diseases and as immunomodulatory agents, that can be further exploited for the advantageous regulation of the host’s immunity, in order to confront serious health conditions.
Keywords:
Natural Products, Leishmania spp, Leishmaniasis, Promastigotes, Amastigotes, Mediterranean Diet, Extra Virgin Olive Oil, Total Phenolic Fraction, Oleocanthal, Apoptosis, Histopathology, Immune Response
