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-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.

Answer 1: The assessment of promastigote growth using the Trypan blue exclusion dye is indeed a crude approach and can be considered as redundant in the view of the CFSE staining method.Nevertheless, these methods for quantification of viable parasites using Trypan blue and the CFSE as a cell division marker, have been previously published in similar studies at yours and other scientific journals:  Christine Achiaa Antwi et al., In vitro 125,2010, 384-388, DOI: 10.1016/j.exppara.2010.03.006  Sarah W. Kamau et al., Flow cytometry  Moreover, the Trypan blue approach is considered as a quantitative approach while the CFSE assay as a more qualitative approach on parasite replication.Taking into consideration the Reviewer No. 1 reviewer's comment, in the revised manuscript, we answered the question of "what is the effect of TPF on parasite growth" by combining three methods.We firstly determined the effect of TPF on promastigote viability with the use of a resazurin-based assay and determined the effect of TPF on the proliferation rate of promastigotes using both Trypan blue approach and CFSE staining.
Answer 2: The incubation periods are now mentioned in lines 241 -242 of the revised manuscript.
The relevant incubation periods have also being clarified in line 256 and lines 274 -275 of the revised manuscript.
Comment 3: "A major problem in all assays is that it is stated that the concentration of the extract selected for this study is the IC50 and 2X1C50, and therefore the results section should begin with a viability assay that calculates this value.Nowhere could I find a numerical value for the IC50 which is a major drawback." Answer 3: We accept the reviewer's comment 3 that the numerical values for the inhibitory concentration were not obvious.To this purpose, we added Table 1 with the half-maximal inhibitory concentrations (IC50 values) of TPF against L. infantum and L. major promastigotes, in the revised manuscript.Moreover, we added one paragraph in Materials and Methods section describing the procedure for the determination of the IC50 values against Leishmania spp.promastigotes.Additionally, in Results section, we inserted one paragraph presenting the inhibitory effect of TPF on L. infantum and L. major promastigotes with the concomitant insertion of the relevant graph (Figure 1 in the revised manuscript).
Comment 4: "How was the cell size determined by Flow Cytometry?A reference is needed of this methodology.Composite results is not provided, only a representative profile." Answer 4: The relevant reference that is indeed needed to be added for the substantiated use of the methodology is: Nevertheless, we considered that the cell volume analysis of untreated and treated parasites by flow cytometry, is redundant in the view of the obtained microscopy images.For this reason, we withdrew the relevant methodology and figure, in the revised manuscript.
Comment 5: "In Fig. 2A and 2B, why is there no cell growth, cells appear to be in stationary phase.However, all experiments pertaining to cell growth should always be performed with log phase cells.If an IC50 concentration decreases cell viability to 57%, it is expected that with 2X IC50, there should be a sharper fall in cell viability, or is the drug cytostatic?" Answer 5: In the first version of the manuscript, the parasite growth experiments had been performed with late-exponential phase parasites.Taking into consideration the reviewer's comment, we repeated the parasite growth experiments (Trypan blue assay, CFSE staining) and we performed these assays with early-exponential phase (log phase) parasites and the relevant results are presented in Figures 2 and 3 of the revised manuscript.Comment 7: "For the cell cycle study, it has been stated that 'induced a significant increase in the number of parasites in the sub G0 peak region, consisting the 68.8% ± 8% for IC50 concentration and the 27.9% ± 4.1% 442 for 2 x IC50 concentration, compared to 10.4% ± 5.3% of untreated parasites (negative control group) (Fig 4A , 4B).Why should untreated promastigotes undergo apoptosis?Why should an 2XIC50 conc show a lesser degree of apoptosis than the IC50?
The data is very poorly presented." Answer 7: Taking into consideration the reviewer's comment, we re-analyzed all the data and the relevant results are presented in Figure 5 and in Figure in S2 Text, in the revised manuscript.Indeed, within the total cell population, the analysis of the percentage of untreated parasites in the sub-G0/G1 region consists of 0.7% and 1.2% of cells for L. infantum and L. major, respectively, at 24 h.presenting pictures with footpad lessions would be an added value to the data of parasite burden in the lymph nodes.Unfortunately, we did not take such photos during the in vivo experiment and a repeat of the in vivo experiment during the revision period (60 days) was not feasible.This consideration will be seriously taken for our future in vivo experimentations.
Comment of Editorial office: "We noticed that you used "data not shown" in the manuscript, we do not allow these references.The PLOS data access policy requires that all data be either Reviewer No. 2

Editorial Office
published with the manuscript or made available in a publicly accessible database.Please amend the supplementary material to include the referenced data or remove the references.Please make a note of this change in your response to reviewers." Answer: "Data not shown" has been removed and all data are within the Revised Manuscript and the Supplementary material.

Comment 6 :
"In Fig 3A & 3B, Leishmania spp.promastigote proliferation determined by CFSE staining, there are no values stated, just a representative profile."Answer 6: We accept the reviewer's comment and in the revised manuscript, all the CFSE relevant data are presented as 2-D line charts expressing the mean fluorescence intensity values ± SD of three independent experiments (Figure 3) and as single parameter histogram overlays representative of one experiment (Figure in S1 Text).

Comment 8 :Answer 8 :
"In the Annexin V data, I fail to understand why control cells have such a high % of Annexin V positivity, There are no values stated in the representative profiles."Taking into consideration the reviewer's comment, we re-analyzed all the data and the relevant results are presented in Figure6and in Figure in S3 Text, in the revised manuscript.The results are plotted as mean values of % annexin V+ parasites ± SD in bar diagrams representative of three independent experiments and as flow cytometric dot plots with respective quadrants, representative of one experiment.
Cynthia Mmalebna Amisigo et al., In vitro anti-trypanosomal effects of selected phenolic acids on Trypanosoma brucei, PlosOne, 2019, DOI: 10.1371/journal.pone.0216078 I. Messaritakis et al., Leishmania donovani s.l.: Evaluation of the proliferation potential of promastigotes using CFSE staining and flow cytometry, Experimental Parasitology activity and mode of action of phenolic compounds onLeishmania donovani, PLOS Neglected Tropical Diseases, 2019, DOI:  10.1371/journal.pntd.0007206 Comment 1: Results."The analysis one are in agreement with the objectives proposed, the results are presented clearly and the figures included in the manuscript are of sufficient quality for clarity.Indeed, some of the FACS images included in the figures can be eliminated for the article publication, keeping just the bar graphics.Such FACS images can be included in a supplementary data file."plus Comment 2: "As mentioned before, FACS images included in the figures together with the bar graphic are redundant.Such figures can be included in the supplementary data file."Taking into consideration the reviewer's comment, the majority of FACS images are included in Supplementary data files, in the revised manuscript.More specifically, single parameter histogram overlays obtained from CFSE assay are presented in Figure in S1 Text.Moreover, single parameter histograms obtained from cell cycle analysis and flow cytometric dot plots obtained from the Annexin-PI assay, for the time-points of 48 and 72 h, are presented in Figures in S2 and S3 Texts, respectively.We accepted the reviewer's comment and thus we inserted Fig 9B in the revised manuscript that illustrates the monitoring of the L. major-infection progress in BALB/c mice treated with TPF or HePC or left untreated.As it was correctly mentioned by the reviewer, a new figure