https://orcid.org/0000-0003-3367-5365
Figures
Abstract
Macrophages represent a fundamental component of the innate immune system that play a critical role in detecting and responding to pathogens as well as danger signals. Leishmania spp. infections lead to a notable alteration in macrophage metabolism, whereby infected cells display heightened energy metabolism that is linked to the integrity of host mitochondria. However, little is known about how different species of Leishmania manipulate host metabolism. Here, we demonstrate that despite differences in their mechanisms for evading host immune responses, L. amazonensis and L. braziliensis induce comparable disruptions in key metabolic pathways. We found that infected macrophages exhibited an overall elevation in energy metabolism regardless of the parasite strain, evidenced by the elevation in glycolysis and oxygen consumption rates, along with increased proton leak and decreased ATP production. We also analyzed the effects of both Leishmania spp. strain infection on mitochondria function, further revealing that infected cells display heightened mitochondrial mass and membrane potential. To investigate the metabolic pathways required for Leishmania amastigotes to persist in BMDMs, we pre-treated cells with small molecule drugs that target major metabolic pathways, revealing that perturbations in several metabolic processes affected parasite survival in a strain-independent manner. Treatments with inhibitors of the oxidative phosphorylation and glycolysis substantially reduced parasite loads. Collectively, our findings suggest that L.amazonensis and L.braziliensis exploit host cell metabolic pathways similarly to survive in macrophages.
Author summary
Our study delves into the intricate interplay between macrophages and Leishmania spp. infections, shedding light on how these parasites manipulate host cell metabolism. Despite variations in the mechanisms employed by different Leishmania species to evade immune responses, our findings reveal a striking similarity in their impact on host metabolic pathways. Infected macrophages consistently exhibit heightened energy metabolism, characterized by increased glycolysis and oxygen consumption rates, alongside alterations in mitochondrial function. Importantly, we demonstrate that targeting key metabolic pathways with drugs significantly affects parasite survival, offering promising avenues for therapeutic interventions against leishmaniasis. By elucidating these complex metabolic processes in a clear and accessible manner, our research not only deepens our understanding of host-parasite interactions but also holds significant implications for the development of novel treatment approaches. We aim to bridge the gap between scientific knowledge and its broader implications for both scientists and non-scientists alike, advancing the fight against neglected tropical diseases.
Citation: de Oliveira EC, Tibúrcio R, Duarte G, Lago A, de Melo L, Nunes S, et al. (2025) Pharmacological inhibition of key metabolic pathways attenuates Leishmania spp infection in macrophages. PLoS Negl Trop Dis 19(1): e0012763. https://doi.org/10.1371/journal.pntd.0012763
Editor: Mitali Chatterjee, Institute of Postgraduate Medical Education and Research, INDIA
Received: March 11, 2024; Accepted: December 6, 2024; Published: January 7, 2025
Copyright: © 2025 de Oliveira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are in the manuscript and/or supporting information files.
Funding: ECO, RT, GD, AL, received fellowships from CAPES, Funding Code 001.AJM received a fellowship from FAPESP (21/01147-1).This study was funded by FAPESB-Fundação de Amparo a Pesquisa do Estado da Bahia (APP0108/2016 for N.T), FAPESP -Fundação de Amparo a Pesquisa do Estado de São Paulo (2016/16030-0; 2022/04576-3 for P.M.M.V),the National Council for Scientific and Technological Development (CNPq) (Process 476926/2011-4 for C.I.B.), FIOCRUZ (Grant INOVA, FIOCRUZ 920285408 for C.I.B) and D.M, P.V., N.T., P.M.M.V., and C.I.B. acknowledge CNPq for the research productivity scholarships (processes, 309283/2022-1, 309901/2023-5, 17021/2021-4, 1, 304280/2020-8 and 304876/2019-4, respectively). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: All the authors have declared that no competing interests exist.
Introduction
Macrophages comprise a set of innate immune cells that are instrumental in the maintenance of homeostatic processes and the integration of bodily systems [1]. These cells respond to pathogens and danger cues by coordinating both cell-intrinsic and -extrinsic signals [2]. In the settings of infection and inflammation, macrophages undergo a dynamic remodeling of their enzymatic activity to acquire energy and synthesize new building blocks, processes essential for the effective immune responses of these cells [2]. Not surprisingly, intracellular pathogens have developed means to manipulate macrophage metabolic machinery to favor its survival and proliferation [3]. The mounting evidence emphasizing the pivotal role of metabolic processes in shaping the outcome of intracellular pathogen infections has elevated energy metabolism to a central aspect of immune cell biology.
Under resting conditions, macrophages typically rely on oxidative phosphorylation (OXPHOS) of molecules (such as glucose, amino acid, and fatty acids) to meet their energy demands [4]. However, upon inflammatory activation, these cells shift their metabolism from OXPHOS to aerobic glycolysis, which is distinguished by the conversion of glucose into lactate in the presence of oxygen to generate ATP. The process by which macrophages undergo metabolic rewiring from a quiescent state, which relies on OXPHOS-dependent pathways, to an activated phenotype, dependent on glycolysis, is commonly referred to as the Warburg effect [4]. Shreds of evidence suggest that eliminating intracellular pathogens, including viruses, bacteria, fungi, and protozoa depends on this metabolic shift [5,6,7,8]. We, therefore, reasoned that the reorganization of the macrophage metabolic landscape is paramount for elimination of the intracellular trypanosomatids. To further expand on this hypothesis, we leveraged a model of trypanosomatid infection in which bone-marrow-derived macrophages (BMDMs) harvested from C57/Bl6 mice were exposed to either L. amazonensis or L. braziliensis. Subsequently, we dissected the metabolic intricacies within these in vitro infection models.
Notably, Leishmania spp. is the causative agent of leishmaniasis, a significant health concern affecting millions of individuals, particularly in tropical regions worldwide [9,10]. Abundant evidence supports the notion that L. amazonensis and L. braziliensis employ distinct strategies to evade host immune responses, which is evidenced by differences in immunopathological processes. Infections with L. amazonensis are characterized by a high parasite burden at site of infection, elevated IL-10 production, and an impaired cellular immune response [11]. Conversely, L. braziliensis infections elicit an intense inflammatory response with the presence of fewer parasites, heightened IFN-γ production, and robust cellular immune responses [11]. Thus, understanding the immunological and metabolic nuances in Leishmania spp. infection is imperative for enhancing our knowledge surrounding leishmaniasis and open avenues for better treatment options and patient care. Here, we elucidate key metabolic variances in macrophages infected with L. braziliensis and L. amazonensis.
Materials and methods
Ethics statement
Female C57Bl/6 mice (Mus musculus), aged 6 to 12 weeks, were kept in the Animal care facility at Gonçalo Moniz Institute / Fiocruz (IGM-FIOCRUZ) in Salvador, Brazil, under controlled conditions and pathogen-free settings. All mouse experiments were performed in accordance with procedures approved by the Ethical Animal Use in experimentation Committee, under protocol number (CEUA/N°006/2018/ IGM / Fiocruz-Bahia).
Parasite culture
L. amazonensis (MHOM/Br/00/BA125) and L. braziliensis (MHOM/BR/01/BA788) strains were used to infect bone marrow-derived macrophages (BMDMs). The promastigotes were maintained in Schneider’s culture medium (Sigma Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Sigma), 2 mM L-glutamine, 100 μg/mL penicillin, and 100 μg/mL streptomycin (Sigma) at 24°C. Throughout the culture period, promastigote counts were performed using a hemocytometer, with a 100x dilution in saline solution (0.9% sodium chloride) to monitor growth dynamics. For all experiments, stationary-phase parasites, enriched in the infective metacyclic promastigotes, were used. The percentage of metacyclic promastigotes in the stationary phase is approximately 70–90% for L. braziliensis and 60–70% for L. amazonensis [12].
In vitro bone marrow-derived macrophages differentiation
Bone marrow precursors were harvested by flushing the femurs and tibias of C57BL/6 mouse rear legs with RPMI-1640 medium (Gibco Invitrogen Corporation, Carlsbad, CA, USA) using a 10 mL syringe and a 21 G needle. The collected cells were centrifuged at 1500 RPM for 10 minutes at 4°C. Bone marrow cells were then resuspended in RPMI culture medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 2 mM L-glutamine, 100 μg/mL penicillin, 100 μg/mL streptomycin (Gibco), and 30% L929 cell-conditioned medium. The cells were seeded into Petri dishes and incubated for 7 days at 37°C with 5% CO₂ to promote differentiation into macrophages. On the fourth day of culture, the cells were supplemented with complete RPMI medium containing 30% L929 cell-conditioned medium. After differentiation, the adherent BMDMs were harvested using a cell scraper, washed with ice-cold saline solution (0.9% sodium chloride), and centrifuged at 1500 RPM for 10 minutes at 4°C. The cell pellet was resuspended in complete RPMI medium, and the cells were counted using a Neubauer chamber. The cells were then plated at 3×105 cells per well in 24-well plates and incubated for 24 hours to allow macrophage adherence.
In vitro macrophage infection and pharmacological modulation of metabolic pathways
Bone marrow-derived macrophages (BMDMs) were pre-treated with specific metabolic pathway inhibitors for 1 hour before infection, as detailed in S1 Table, and incubated at 37°C with 5% CO₂. Following treatment, the cells were washed with room temperature saline solution (0.9% sodium chloride) to remove any residual inhibitors from the supernatant. Subsequently, the BMDMs were exposed to L. amazonensis and L. braziliensis promastigotes at ratios of 5:1 and 10:1, respectively, to ensure comparable infection rates for both Leishmania species. After 6 hours of co-incubation, non-internalized parasites were removed by washing with saline solution (0.9% sodium chloride), and the cells were incubated for an additional 12, 18 or 42 hours (corresponding to 18, 24 and 48 hours post infection) at 37°C with 5% CO₂. The cells were then fixed with methanol and stained using hematoxylin eosin kit (LB Laborclin, Brazil). Coverslips were examined under a light microscope (Nikon), and a minimum of 100 cells per experimental condition were counted. The infection index was calculated by multiplying the number of parasites by the percentage of infected cells.
To assess parasite viability, after 24 hours of infection, the cells were incubated in Schneider’s Insect Medium (Sigma Aldrich, St. Louis, MO) at 24°C for 5 days to promote the growth of non-eliminated parasites, which were then counted using a Neubauer chamber.
Seahorse metabolic analysis
The bioenergetic profile of L. amazonensis and L. braziliensis-infected BMDM was analyzed using an XFe-96 extracellular flux analyzer (Seahorse Bioscience). The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured at the indicated time points post-infection. BMDMs were seeded at 3 × 104 cells/well in 100 μL of RPMI in XFe-96 cell culture plates. After an overnight incubation, the cells were infected with L. amazonensis and L. braziliensis metacyclic promastigotes. One hour before the designated infection times, the cells were washed, and the medium was replaced with XF medium (XF RPMI Medium pH 7.4 containing 1 mM HEPES and supplemented with L-glutamine). The bioenergetic profile was measured in real-time under basal conditions and in response to glucose (10 mM), oligomycin (1 μM) and and 2-deoxyglucose (2-DG, 50 mM), for the ECAR evaluation and oligomycin (1 μM), fluoro-carbonyl cyanide phenylhydrazone (FCCP, 2 μM), rotenone (100 nM) and antimycin A (1 μM) for OCR evaluation. Non-glycolytic acidification was determined by selecting the lowest positive value after 2-DG injection. Glycolysis rates were calculated by subtracting non-glycolytic acidification from the highest value post-glucose injection. Glycolytic capacity was determined by subtracting non-glycolytic acidification from the highest value after oligomycin injection, and glycolytic reserve was calculated as the glycolytic capacity minus glycolysis. Non-mitochondrial respiration was assessed by selecting the lowest positive value after rotenone and antimycin A injections. Basal respiration was determined by subtracting non-mitochondrial respiration from basal values. Maximal respiration was assessed by subtracting non-mitochondrial respiration from the values obtained after FCCP injection. Proton leak was calculated by subtracting non-mitochondrial respiration from the values obtained after oligomycin injection. ATP production was assessed by subtracting proton leak from basal respiration rates. The spare respiratory capacity (SRC) was obtained by subtracting the FCCP values from basal OCR values. The procedures used in these experiments followed the manufacturer’s instructions (Seahorse). After the readings on the XFe96, the cells underwent additional protein quantification for data normalization using the Pierce BCA Protein Assay kit (Thermo Scientific).
Quantification of proteins by BCA kit Pierce reaction
After the XF assays, the cell culture plate was washed once with PBS, and 30 μL of lysis buffer containing 1% NP-40, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA (pH 8), 10 mM 1,10-phenanthroline, and phosphatase and protease inhibitors (Roche) was added to each well. The plate was then incubated at 4°C under agitation for 20 minutes then frozen and unfrozen. Afterwards, 10 μL of the lysate was transferred to a new plate for protein quantification using the Pierce BCA kit assay. Following the addition of the reagent A+B mix to the samples, the plate was incubated for 30 minutes at 37°C and then read by spectrophotometry at 562 nm, according to the manufacturer’s instructions. The quantification results were subsequently uploaded into the Wave Desktop software, where they were normalized by the software.
Mitochondrial markers and reactive oxygen species content assessment
Mitochondrial function was assessed by analyzing membrane potential and mitochondrial mass. BMDMs were seeded in polypropylene round-bottom tube (FALCON) at a density of 1 × 106 cells per tube. After pre-treating the cells with metabolic inhibitors, the BMDMs were washed and infected with L. amazonensis and L. braziliensis. Then, the BMDMs were washed with 1x PBS, and the cells were transferred to a 96-well plate. They were then incubated with a mix (50 μL per well) of MitoTracker Green (50 nM) (Invitrogen), MitoTracker Red (125 nM) (Invitrogen), anti-mouse F4/80 (BioLegend), and Live/Dead (eBioscience) dye for 30 minutes at 37°C in the dark. After staining, the cells were washed with 100 μL of 1x PBS + 2% FBS and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in 200 μL of 1x PBS + 2% FBS for flow cytometry analysis. Samples were acquired using FACSDIVA software (BD Biosciences) and analyzed with FLOWJO software (BD).
Reactive oxygen species (ROS) production was measured using the CellROX probe. BMDMs were seeded in 24-well plates at a density of 5 × 105 cells per well, pre-treated with metabolic inhibitors, and infected with L. amazonensis and L. braziliensis. Afterwards, cells were incubated with CellROX Green reagent (5 μM) for 30 minutes at 37°C with 5% CO₂, then washed three times with 1x PBS (Gibco), fixed with 3% paraformaldehyde (PFA), and stained with DAPI antifade ProLong Gold (Thermo Fisher Scientific, IL, USA). Fluorescence microscopy (Leica DMi8) with 485/520 nm excitation was used to capture images. Approximately 30 cells per experimental condition were selected and analyzed, measuring area, integrated density, mean gray value, and median. Measurements were also taken in non-fluorescent areas adjacent to the cells. The obtained values were analyzed to yield the Corrected Total Cell Fluorescence (CTCF), calculated as: CTCF = Integrated density—(selected cell area × mean fluorescence of background), using ImageJ software (National Institutes of Health, USA).
Determination of the intracellular parasite load
At 18, 24 and 48 hours post-infection, slides containing pre-treated and infected macrophages were fixed with methanol and stained using the hematoxylin-eosin kit (LB Laborclin, Brazil). Coverslips were examined under a light microscope (Nikon). To evaluate the infection rate, a minimum of 100 cells per experimental condition were counted. The parasite load within the macrophages was determined by counting the amastigotes present in each infected cell out of the total number of cells counted across the various experimental conditions. The infection index was calculated by multiplying the number of parasites by the percentage of infected cells.
Determination of parasite binding and phagocytosis activity in infected Bone marrow- derived macrophages
Bone marrow-derived macrophages (BMDMs) were pre-treated with specific metabolic pathway inhibitors for 1 hour prior to infection and incubated at 37°C with 5% CO₂. After treatment, the cells were washed with room-temperature saline solution (0.9% sodium chloride) to remove any residual inhibitors. BMDMs were then exposed to L. amazonensis and L. braziliensis promastigotes at ratios of 5:1 and 10:1, respectively, and centrifuged at 300g for 5 minutes at 4°C. For binding analysis, the plate remained in the centrifuge for an additional 25 minutes, after which the cells were washed with cold saline, fixed with methanol, and stained with eosin and hematoxylin. For phagocytosis analysis, following the initial 5-minute centrifugation, the plate remained in the centrifuge for another 25 minutes. Afterward, the cells were washed with cold saline, the medium was replaced with RPMI at 37°C, and incubation continued for 2 additional hours at 37°C with 5% CO₂. The cells were then fixed with methanol and stained using a hematoxylin-eosin kit (LB Laborclin, Brazil). Coverslips were examined under a Nikon light microscope, and a minimum of 100 cells were counted for each experimental condition
Parasite growth curve and parasite viability after infection
For growth curve analysis, metabolic pathway inhibitors were added individually to the cultures of L. amazonensis and L. braziliensis for 1 hour. After this period, the culture was centrifuged with cold saline at 3000 rpm for 10 minutes at 4°C and resuspended in Schneider’s medium. Daily counts of the parasite cultures were performed for 4 days. For the viability of the parasites, BMDMs were treated with inhibitors for 1 hour either before or after infection, and the infection lasted 24 hours with L. amazonensis or L. braziliensis. After the infection period, the RPMI medium was replaced with Schneider’s medium, and cultivation continued for an additional 5 days to count the viable parasites.
Statistical analysis
Statistical analysis was conducted using GraphPad Prism v.10 (GraphPad Software, La Jolla, CA) and R statistical software, version 4.31. Packages utilized for the generation of plots include ggplot2 and tidyverse. The analyzed data followed a non-parametric distribution pattern. The analyses were performed using the Kruskal-Wallis test, followed by the Dunn post-test, and the values were expressed as the median with interquartile deviation. Differences were considered significant when p < 0.05, using a 95% confidence interval.
Results
01- Leishmania spp. infection promotes metabolic rewiring in bone-marrow derived macrophages
To assess the impact of Leishmania spp. infection on macrophage metabolism, we exposed BMDMs to either L. braziliensis or L. amazonensis promastigotes for 6 hours and subsequently incubated the infected BMDMs for more 18 and 42 hours, corresponding to a total time of 24 and 48 hours. Subsequently, we quantified metabolic changes by measuring extracellular acidification (ECAR) and oxygen consumption rates (OCR) after 6, 24 and 48 hours of infection. On one hand, both Leishmania-infected groups exhibited a notable increase in both ECAR and OCR at 6 and 24 hours after infection when compared to uninfected controls (Fig 1A, 1B, 1C and 1D). The increase in OCR in infected macrophages may be attributed to oxygen consumption by both the macrophages and Leishmania. However, distinguishing between the two bioenergetic contributions is not possible due to limitations in the Seahorse extracellular flux analyzer system. On the other hand, at 48 hours after infection, it was detected a metabolic profile similar to uninfected macrophages (Fig 1A, 1B, 1C and 1D).
A) Real-time extracellular acidification rate (ECAR) analysis of bone marrow-derived macrophages from uninfected (mock) C57BL/6 mice, infected with La (Leishmania amazonensis) for 6 hours, and then incubated for an additional 18- and 42-hours post-infection. B) Real-time oxygen consumption rate (OCR) analysis of bone marrow-derived macrophages from uninfected (mock) C57BL/6 mice, infected with La (Leishmania amazonensis) for 6 hours, and then incubated for an additional 18- and 42-hours post-infection. C)Real-time extracellular acidification rate (ECAR) analysis of bone marrow-derived macrophages from uninfected (mock) C57BL/6 mice, infected with Lb (Leishmania braziliensis) for 6 hours, and then incubated for an additional 18- and 42-hours post-infection. D) Real-time oxygen consumption rate (OCR) analysis of bone marrow-derived macrophages from uninfected (mock) C57BL/6 mice, infected with Lb (Leishmania braziliensis) for 6 hours, and then incubated for an additional 18- and 42-hours post-infection. E) Non-glycolytic acidification, F) Glycolysis, G) Glicolytic capacity, H) Glicolytic reverse, I) Non- mitochondrial respiration, J) Basal respiration, K) Maximal respiration, L) ATP-production, M) Proton leak, N) Spare respiratory capacity, O) Principal component analysis (PCA) and Heatmap of observed alterations in metabolic reprogramming, P) Energy map.Two independent experiments were conducted, each with five replicates, using two different cell lots. Statistical analysis was performed using the 2-way ANOVA test with Dunn’s post-test. ***<0,0001 **p < 0.01, *p < 0.05.
This metabolic alteration was comparable between parasite strains. Expanding our investigation into the modulation of macrophage metabolic capacities by Leishmania spp., we further characterized the metabolic landscape in our cell cultures. Our data indicate that Leishmania infection led to a significant elevation in basal glycolysis, glycolytic capacity, maximal respiration rates and proton leak (Fig 1F, 1G, 1K and 1M). Conversely, Leishmania spp. infection resulted in reduced ATP production (Fig 1L). To gain a comprehensive perspective on the influence of parasite infection on key metabolic parameters measured by ECAR and OCR, we applied principal component analysis (PCA)and a heat map of the results obtained in the SeaHorse analysis This analytical approach consistently confirmed a parasite-induced metabolic reprogramming in BMDMs, irrespective of the parasite strain (Fig 1O). Furthermore, our energy map illustrates that both L. amazonensis and L. braziliensis can shift the metabolic profile of infected cells toward a higher-energy state, as indicated by the concurrent enhancements in both ECAR and OCR, when compared to metabolically quiescent uninfected macrophages (Fig 1P). In summary, these findings support that infection with both Leishmania parasite strains results in the metabolic rewiring of infected BMDMs.
02- Impact of Leishmania spp. infection on mitochondrial function
Recognizing the pivotal role of mitochondria in meeting cellular energy demands and orchestrating responses to disruptions in cellular homeostasis, we speculated that Leishmania spp. infection alters mitochondria function. We evaluated the mitochondria mass (MM) content and membrane potential (MP) using commercially available mitochondrial probes (MitoTracker green and red, respectively). Our findings indicate a substantial increase in both mitochondria mass (Fig 2A) and membrane potential (Fig 2B) in BMDMs infected with Leishmania parasites. Notably, we observed a strong positive correlation between these markers, with infected macrophages displaying a notably elevated profile compared to their non-infected counterparts (Fig 2C). These results underscore the significant influence of Leishmania spp. infection on mitochondria functionality within the host cells.
After the infection, BMDMs were incubated for 30 minutes with 50nM of MitoTracker Green probe (A) and 125 nM of MitoTracker Red probe (B), for analysis of mitochondrial mass (MM) and mitochondrial membrane potential (MMP), respectively. Samples were acquired using the FACSDIVA software (BDBiosciences) and analyzed using FLOWJO Software (BD). (C) correlation between MitoTracker green and MitoTracker Red with infected macrophages. The experiments were performed in triplicate with five replicates per experiment. Statistical analysis was conducted using the Kruskal-Wallis test with Dunn’s post-test, with a significance threshold of *p < 0.05.
03- Pharmacological targeting of macrophage metabolic reprogramming mitigates Leishmania spp. infection
Next, we sought to understand which metabolic pathways are required to support Leishmania amastigote persistence in BMDMs. Therefore, we pre-treated cells with modulators of metabolic pathways for 1 hour prior to exposure to parasites. Notably, the drugs used in our assay targeted key metabolic pathways in host cells, including glycolysis, OXPHOS, and fatty acid metabolism (detailed in S1 Table). The doses of the inhibitors of metabolic pathways were chosen considering the maintenance of cellular viability of the macrophages, as shown in S2 Fig. Subsequently, as a proxy for the metabolic influence on both susceptibility and permissibility to Leishmania spp. infection, we assessed the percentages of infected cells and the number of viable amastigotes within pre-treated BMDMs.
Overall, perturbations in several metabolic processes affected the survival of both parasite strains (Fig 3A and 3B). Treatment with 2DG, which inhibits the glucose flux, resulted in a decreased percentage of macrophages infected with parasites compared to untreated infected macrophages. Additionally, we observed a decrease in the percentage of L. amazonensis-infected cells treated with PFK15, a small molecule inhibitor targeting PFKFB3 an enzyme pivotal in glycolysis though this effect was not observed in L. braziliensis-infected cells. Moreover, PFK15 treatment led to a reduction in the number of amastigotes for both parasite strains.
Infection rate and parasite load of BMDMs treated with metabolic inhibitors. BMDMs were treated with the inhibitors 1 hour before infection for 6 hours and then incubated for a further 12 hours. a) Infection rate and parasite load of BMDMs by L. amazonensis under the influence of metabolic pathway inhibitors. b) Infection rate and parasite load of BMDMs infected with L.braziliensis under the influence of metabolic pathway inhibitors representing the number of internalized amastigotes in 100 cells. 2DG, 2-deoxyglucose; PFK15, Phosphofructokinase; FCCP, Phenylhydrazone; OLG, Oligomycin; Ant A, Antimycin, ROT; Rotenone, ETMX, Etomoxir, C75; MDVI. c) Longitudinal Analysis of the Impact of Metabolic Inhibitors on Leishmania spp. Infection in BMDMs. 2DG, 2-deoxyglucose; C75, ETMX, Etomoxir, OLG, Oligomycin. The experiments were performed in triplicate with five replicates per experiment. Statistical analysis was conducted using the Kruskal-Wallis test with Dunn’s post-test. ****p <0.0001, ***p <0. 001, **p < 0.01, *p < 0.05.
We noted that the mitochondria complex III inhibitor antimycin had the most significant impact on parasite load in both infection models. Also, inhibition of mitochondrial fission (MDVI treatment) resulted in a marked reduction in parasite load only in cells infected with L. amazonensis. Additionally, we found that uncoupling of mitochondrial membrane potential (treatment with FCCP) only reduced the number of L. braziliensis amastigotes. These observations may point to differences in how these Leishmania species exploit host mitochondrial metabolism to support parasite survival. Blocked mitochondrial complexes I with rotenone did not attenuate parasite loads. Interfering with fatty acid metabolism substantially impacted the survival of both species, as indicated in cells treated with low doses of etomoxir and C75 (inhibitors of fatty acid oxidation and synthesis, respectively). We observed that both the infection rate and the parasitic load in infected macrophages slightly increased at 6, 24, and 48 hours post-infection in BMDMs infected with any of both species of Leishmania. The addition of 2DG, C75, Etomoxir, and oligomycin significantly reduced these parameters (Fig 3C). In conclusion, our findings reveal intricate metabolic nuances underlying Leishmania spp. infection and shed light on how different strains exploit host cell energy metabolism to survive within macrophages.
04- Assessment of most effective metabolic inhibitor to mitigate in vitro Leishmania spp. infection
To assess the overall extent of infection in our experimental model of L. amazonensis and L. braziliensis infection, we calculated the infection index. This index is determined by multiplying the percentages of infected cells by the number of parasites encountered in each well, thereby assigning equal weight to both infection rates and parasite load. In our analysis, treatment with 2DG and antimycin A consistently reduced the infection index in both parasite strains (as shown in Fig 4A and 4B). Intriguingly, we observed that PFK15 and MDVI treatments selectively lowered the infection index in L. amazonensis-infected macrophages, while FCCP and oligomycin had the same effect in L. braziliensis-infected cells. These findings collectively suggest strain-specific dependencies on distinct metabolic pathways for the survival of these parasites within host cells.
The infection index is determined by multiplying the percentages of infected cells by the number of parasites encountered in each well, thereby assigning equal weight to both infection rates and parasite load. a) L. amazonensis infection and b) L. braziliensis infection using different metabolic inhibitors 2DG, 2—Deoxyglucose; PFK15, Phosphofructokinase; FCCP, Phenylhydrazone; OLG, Oligomycin; Ant A, Antimycin, ROT; Rotenone, ETMX, Etomoxir, C75; MDVI. The experiments were performed in triplicate with five replicates per experiment. Statistical analysis was conducted using the Kruskal-Wallis test with Dunn’s post-test. ****p <0.0001, ***p <0. 001,**p < 0.01, *p < 0.05.
To further validate our findings, we used metabolic inhibitors to assess their impact on the growth of L. amazonensis and L. braziliensis in axenic cultures, as well as on the viability of intracellular parasites. S2A Fig shows that 2DG does not affect parasitic growth in axenic cultures of either Leishmania species. In contrast, oligomycin and antimycin significantly reduced parasite numbers after 3 days of culture, which is expected given Leishmania’s reliance on mitochondrial metabolism for proliferation [13]. These two compounds also decreased the number of viable parasites, although this effect was observed after 5 days of culture, following the replacement of RPMI with Schneider medium (S3A Fig). S3A Fig illustrates the different treatment periods with metabolic inhibitors before and after infection, respectively. Despite these treatments, a considerable number of parasites remained viable, and it is important to note that all of our analyses were conducted within 48 hours, a time frame in which the inhibitors would likely affect the metabolism of the infected macrophages rather than directly impact Leishmania.
05-Impact of metabolic reprogramming on the killing of parasites during phagocytosis
To determine whether metabolic pathway inhibitors influence parasite death during phagocytosis, we pre-treated macrophages with these inhibitors before infection. We then assessed binding and phagocytosis rates to evaluate any potential alterations caused by these compounds. As shown in Fig 5, no differences were observed among the experimental conditions, suggesting that the metabolic pathway inhibitors neither induced parasite killing at early post-infection time points nor interfered with the phagocytosis process.
BMDMs were treated with the inhibitors 1 hour before infection and then infected for 30 minutes for binding and 2 hours for phagocytosis. A) Infection rate and parasite load of BMDMs by La—L. amazonensis. B) Infection rate and parasite load of BMDMs infected with Lb—L.braziliensis. 2DG, 2-deoxyglucose; OLG, Oligomycin; Ant, Antimycin. The experiments were performed in triplicate. Statistical analysis was conducted using the Kruskal-Wallis test with Dunn’s post-test. *p < 0.05.
06- Treatment with 2-Deoxy-6-glucose and Antimycin A amplifies superoxide production in infected macrophages
It is widely recognized that both pathogen-associated molecular patterns (PAMPs) and the immunometabolic status play pivotal roles in shaping the redox environment within immune cells [12]. Moreover, there is evidence suggesting that the augmentation of reactive oxygen species (ROS) levels could serve as a potential mechanism for eliminating Leishmania spp. in infected macrophages [14]. In our study, we aimed to elucidate the mechanistic underpinnings of Leishmania elimination in macrophages treated with metabolic inhibitors. To achieve this, we assessed the levels of hydrogen peroxide in mitochondria utilizing the commercially available probe MitoSox. Our findings revealed that treatment with 2DG and Antimycin A, known metabolic inhibitors with a high potential for reducing parasite loads, led to increased levels of mitochondria-derived ROS in both L. amazonensis- and L. braziliensis-infected macrophages (Fig 6A, 6B, 6D, and 6E). Furthermore, we observed a negative correlation between ROS levels and the number of amastigotes present in macrophages, suggesting that the elevation of ROS induced by metabolic perturbation impairs parasite survival (Fig 6C and 6F). Taken together, these findings indicate a potential connection between immunometabolic perturbation, remodeling of the redox landscape, and the persistence of parasites in infected macrophages.
To investigate the hypothesis that metabolic inhibitor treatment induces an increase in ROS levels, we evaluated superoxide production in infected BMDS treated with 2DG and Antimycin A using the MitoSOX probe. Total Cellular Corrected Fluorescences (CTCF) were analyzed and quantified for approximately 30 cells per experimental condition. a) Confocal microscopy images depict the experimental groups. b) Scatterplots illustrate the levels of ROS detected via the MitoSOX probe. c) Spearman correlations were performed to analyze the relationship between the number of amastigotes in 100 macrophages (parasite load) and ROS levels. Kruskal-Wallis test with Dunn post-test. ***p <0.001.
Discussion
When encountering pathogens, quiescent macrophages rapidly adjust their metabolic processes to orchestrate immune functions and respond to infections [4]. However, over the course of evolution, intracellular pathogens have developed strategies to manipulate host metabolism, forming a crucial component of their tactics to evade immune responses and, consequently, to persist within the host [14,15,16,17]. Observations from various studies suggest that macrophages serve as a preferred niche for intracellular protozoa such as Leishmania spp., facilitating their differentiation and proliferation [18]. This notion finds support through multiple approaches grounded in metabolic and RNA-sequencing analyses, which consistently highlight macrophages as primary sources of essential metabolites for Leishmania spp. [19].Nevertheless, the extent to which distinct Leishmania species vary in their impact on macrophage metabolic programs remains a topic that lacks comprehensive understanding.
The present study reveals that both L. amazonensis and L. braziliensis infections enhance the energy metabolism of BMDMs, as indicated by increased ECAR and OCR (after 24 and 48 hours of promastigote to amastigote differentiation) In light of this, we conducted experiments at 6, 24, and 48 hours post-infection to observe the metabolic profiles of macrophages infected with Leishmania amazonensis or Leishmania braziliensis. The metabolic profiles at 6 and 24 hours post-infection were found to be very similar. However, at 48 hours post-infection, we observed a reduction in metabolic activity, as measured by ECAR and OCR, in infected macrophages for both Leishmania species. Notably, at 48 hours, L. amazonensis-infected macrophages exhibited metabolic activity levels comparable to those of non-infected macrophages in the ECAR evaluation. These results suggest that following differentiation into amastigotes, the host cell behaves similarly to a non-infected macrophage, at least concerning L. amazonensis infection. We can hypothesize that the metabolic shift occurs during the early events of infection, specifically during the differentiation of parasites from promastigotes to amastigotes, as previously demonstrated by Moreira et al. and Ospina et al. with L. infantum and L. donovani [20,21].
These findings align with previous observations regarding the influence of other Leishmania species on macrophage metabolism. For instance, a study by Ty and colleagues demonstrated that L. amazonensis and L. donovani upregulate OXPHOS in human monocyte-derived macrophages [22]. Similarly, Moreira et al showed that L. infantum-infected macrophages shift from glycolysis to OXPHOS in a SIRT1-AMPK dependent manner [20].
In our study, Leishmania infection resulted in decreased ATP production related to oxidative phosphorylation (OXPHOS) and an increase in maximum respiration. We propose that L. amazonensis may cause this ATP reduction by redirecting or depleting host cell energy sources, thereby compromising energy production capacity in host cells. It is important to note that ATP also functions as a signaling molecule [23], and the decrease in ATP levels might disrupt various intracellular signaling pathways, potentially impacting macrophage immune functions. However, it is worth considering that the intracellular ATP pool may be maintained at a stable level due to compensatory ATP production from glycolysis. Further investigations are necessary to accurately measure the ATP pool size in Leishmania infection and qualitatively assess its role in influencing resistance to the infection.
Mitochondria plays a pivotal role in diverse cellular processes. In our study, we observed that infection with L. amazonensis and L. braziliensis led to a significant increase in mitochondrial mass and membrane potential. Previous studies have reported that L. donovani-infected macrophages experience a substantial loss of mitochondrial potential and a reduced proximity of mitochondrial-endoplasmic reticulum contact sites [24]. Notably, Ospina et al demonstrated that the upregulation of oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis during L. donovani infection is contingent on the virulence factor glycolipid lipophosphoglycan (LPG), which induces the expression of key transcription factors related to mitochondrial characteristics [21]. These same researchers also found that enhanced mitochondrial biogenesis is associated with a more pronounced infection in macrophages. In our experiments, targeted inhibition of mitochondrial fission resulted in a reduction in L. amazonensis infection. This suggests the parasite reliance on a well-maintained mitochondrial environment for its successful survival and proliferation.
The reduced infection rate and parasite load in macrophages treated with inhibitors targeting metabolic pathways shed light on the intricacies of how the studied Leishmania species persist within host cells.
It is evident that the heightened glycolysis in macrophages significantly contributes to their capacity for increased pro-inflammatory cytokine production, including interleukin-1 beta (IL-1β), thus reinforcing their effectiveness in eradicating intracellular pathogens [4]. Furthermore, research has shown that the ability of neutrophils to curtail the persistence of L. donovani depends on their enhanced glycolytic activity, suggesting that the induction of glycolysis is a shared antileishmanial mechanism among myeloid cells [25]. Once glycolysis was inhibited with PFK15, the number of amastigotes found in macrophages was significantly reduced. This prompts the hypothesis that the accumulation of glycolytic intermediates plays a central role in Leishmania replication within macrophages, which could explain the decrease in parasite load, even if it coincides with a reduction in pro-inflammatory cytokine release by macrophages.
Inhibition of fatty acid synthesis and oxidation with C75 and etomoxir, respectively, also led to a decrease in parasite load and the infection rate. Fatty acid metabolism is a crucial intracellular process that contributes to energy production, cell structure, cell signaling, and various physiological functions [26]. Rabhi et al demonstrated that L. major-infected macrophages can accumulate lipid droplets, which are subsequently utilized by L. major as high-energy sources [27]. Mitochondrial complex III, also known as the cytochrome bc1 complex or the ubiquinol-cytochrome c oxidoreductase, is a vital component of the mitochondrial electron transport chain [28] and the inhibition of complex III with Antimycin A decreased parasite loads without affecting macrophage viability. However, it is important to recognize that this effect may also be attributed to a direct impact on the mitochondrial fitness of Leishmania, as Smilkstein et al. demonstrated that Antimycin A treatment eliminated Plasmodium spp. parasites in in vitro assays [29]. Indeed, both oligomycin and antimycin resulted in a reduction in the number of promastigotes in axenic cultures, as well as a decline in parasite viability. Notably, these decreases were observed after 72 hours and 5 days, respectively, which supports the notion that mitochondria play a crucial role in the growth of the parasite within infected cells. The decrease in parasites observed in our assays does not correlate with destruction or reduction of binding, as demonstrated in Fig 5, supporting the idea that the metabolism of the infected macrophage contributes to the proliferation of Leishmania spp.
Significantly, treatment with 2DG and Antimycin A not only increased ROS levels but also showed a negative correlation with parasite load in both infection models. This suggests that the treatment-induced alteration of the redox landscape in infected macrophages may play a pivotal role in reshaping the intracellular microenvironment, thereby potentially impacting Leishmania survival dynamics.
To the best of our knowledge, this study is important since it investigated the application of a library of small molecules that target key metabolic pathways in an in vitro model of L. amazonensis and L. braziliensis infection. Nevertheless, a notable limitation in our research lies in the absence of functional assays that could establish a clear link between the metabolic states of macrophages and their effector functions responsible for Leishmania elimination. Put together, our findings have identified crucial metabolic pathways essential for Leishmania persistence within macrophages. This highlights the intricate interplay between host cell metabolic processes and Leishmania survival strategies within macrophages, underscoring the significance of comprehending these dynamics for the development of effective interventions against Leishmania infections.
Supporting information
S1 Fig. Assessment of metabolic alterations in Leishmania-infected bone marrow-derived macrophages comparing different infection time points.
Real-time extracellular acidification rate (ECAR) analysis of bone marrow-derived macrophages from uninfected (mock) C57BL/6 mice and those infected with La (Leishmania amazonensis) or Lb (Leishmania braziliensis) for 6 hours, followed by incubation for an additional 18 or 42 hours post-infection. Real-time oxygen consumption rate (OCR) analysis was also performed under the same conditions. This data highlights metabolic differences in macrophages at distinct infection time points. A)OCR/ECAR ratio, B) Non- mitochondrial respiration, C) Basal respiration, D) Proton leak, E) ATP-production, F) Spare respiratory capacity, G) Maximal respiration, H) non-glicolitic acidification, I) Glycolysis, J) Glicolytic capacity, K) Glycolytic reserve. Two independent experiments were conducted, each with five replicates, using two different cell lots. Statistical analysis was performed using the 2-way ANOVA test with Dunn’s post-test. ***<0,0001 **p < 0.01, *p < 0.05.
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S2 Fig. Growth curve of L. amazonensis and L. braziliensis parasites over 4 days.
The parasites were treated with metabolic inhibitors for 1 hour and cultured for 4 days to determine the growth curve. The experiments were performed in triplicate.A) Growth curve of L. amazonensis B) Growth curve of L. braziliensis. Statistical analysis was conducted using the 2-way ANOVA test with Dunn’s post-test. *p < 0.05.
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S3 Fig. Parasite viability 5 days after the end of the infection.
To assess parasite viability, BMDMs were treated with inhibitors for 1 hour either before or after infection, and the infection lasted 24 hours with L. amazonensis or L. braziliensis. After the infection period, the RPMI medium was replaced with Schneider’s medium, and cultivation continued for an additional 5 days to count the viable parasites. 2DG, 2—Deoxyglucose; OLG, Oligomycin; Ant A, Antimycin; DMSO, Dimethyl sulfoxide. The experiments were performed in triplicate. A) BMDMs were treated with inhibitors for 1 hour before the infection B) BMDMs were treated with inhibitors for 1 hour after infection. Statistical analysis was conducted using the 2-way ANOVA test with Dunn’s post-test. *p < 0.05.
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S4 Fig. Cellular viability after treatment with metabolic inhibitors.
The cultures were treated with different concentrations of each metabolic inhibitor for 1 hour, washed, stained with AlamarBlue (a viability indicator dye), incubated for 72 hours, and subjected to spectrophotometry. For our dead control, we cultivated the cells in sterile water for injection. 2DG, 2—Deoxyglucose; PFK15, Phosphofructokinase; C75; ETMX, Etomoxir; OLG, Oligomycin; Ant A, Antimycin; ROT, Rotenone; FCCP, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; MDVI, 3-(2,4-Dichloro-5-methoxyphenyl) -2,3-dihydro-2-thioxo-4(1H)-quinazolinone3-(2,4-Dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone; DMSO, Dimethyl sulfoxide. The experiments were performed in triplicate. Statistical analysis was conducted using the Kruskal-Wallis test and Dunn’s post-test.
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S5 Fig. Images of macrophages at binding and phagocytosis.
The BMDMs were treated with metabolic inhibitors for 1 hour before infection and then infected for 30 minutes for binding and 2 hours for phagocytosis. A) La, L. amazonensis; 2DG, 2-deoxyglucose; OLG, Oligomycin; Ant, Antimycin B) Lb, L.braziliensis; 2DG, 2-deoxyglucose; OLG, Oligomycin; Ant, Antimycin.
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S1 Table. Concentration of Metabolic Inhibitors utilized in macrophage infection with Leishmania spp.
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S1 Data. FIG1A- FIG1N.
This excel table outlines the experimental setup for evaluating metabolic parameters in macrophages (MO) and those infected with Leishmania species (Leishmania amazonensis [LA] and Leishmania braziliensis [LB]). These results concern Fig 1A to 1N –Assessment of metabolic alterations in Leishmania-infected bone marrow-derived macrophages. FIG2A-FIG2B. MitoTracker Green columns A-C includes three groups: (A) Control group (Mock),(B) Leishmania amazonensis (La) infection,(C) Leishmania braziliensis (Lb) infection. MitoTracker Red columns E-G) shows the same groups: (E) Control group (Mock) (F) Leishmania amazonensis (La) infection (G) Leishmania braziliensis (Lb) infection. Five replicates were used for each experimental condition. These results concern Fig2A and Fig2B- Fig 2. Leishmania spp promote increased mitochondrial mass and membrane potential. FIG3A- FIG3B the results concern the infection rate and parasite load of macrophages infected with Leishmania amazonensis and Leishmania braziliensis. Evaluation of infection rate and parasite load in macrophages treated with metabolic inhibitors during L. amazonensis or L. braziliensis infections. Fig 4. Infection Index: concern the results shown in Fig 4 Infection index is calculated by multiplying the infection rate by the number of amastigotes. The values represent the average of parasite load and infection rate data.—Determination of infection index following metabolic inhibitor treatment. FIG 5. Results associated to the Fig 5 Inhibitor metabolic pathways did not affect binding and phagocytosis of the parasites Binding and phagocytosis in macrophages infected with Leishmania amazonensis and Leishmania braziliensis: FIG6B-Fig6F Results associated to the graphs shown in Fig 6 that evaluates mitochondrial reactive oxygen species (ROS) production: Effects of 2DG and Antimycin A treatment on ROS levels and Leishmania parasite load in infected macrophages.
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