Data from: Unraveling the biochemical aspects of the interaction between ticks and Leishmania using a tick cell line
Data files
Oct 02, 2025 version files 204.41 KB
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Figure_2_A.xlsx
9.62 KB
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Figure_2_B.xlsx
9.67 KB
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Figure_2_C.xlsx
9.38 KB
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Figure_3_C.xlsx
16.47 KB
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Figure_3A.xlsx
9.12 KB
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Figure_3B.xlsx
9.41 KB
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Figure_4_A.xlsx
16.50 KB
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Figure_4_B.xlsx
16.41 KB
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Figure_4_C.xlsx
16.41 KB
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Figure_4_D.xlsx
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Figure_4_E.xlsx
16.34 KB
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Figure_4_F.xlsx
16.36 KB
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Figure_4_G.xlsx
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README.md
25.96 KB
Abstract
Leishmaniasis comprises a group of vector-borne neglected tropical diseases caused by species of the obligatory intracellular parasite Leishmania, transmitted by the bite of dipteran sand flies. Infected dogs serve as the primary domestic reservoir of Leishmania parasites and are often found in close association with various arthropods, such as fleas and ticks. There have been recent reports of Leishmania infections occurring in areas non-endemic for sand fly species, leading to reconsideration of the hypothesis that other arthropods, such as ticks, may also play a significant role in the natural history and epidemiology of leishmaniasis. Here, we used a tick cell line as a tool to study Leishmania infantum and tick interaction. The results showed that L. infantum can bind and proliferate inside Ixodes scapularis IDE8 tick cells. The infection reduced tick cell viability and induced ROS production. Lipid profile analysis showed that the presence of L. infantum increased oxysterol in tick cells and influenced tick cell lipid biosynthesis, since an increase in glycerolipids and esterified cholesterol was observed in infected cells at 48. Further experiments are necessary to elucidate whether Leishmania can overcome the various biochemical and tissue barriers within ticks and be transmitted to the host.
Dataset DOI: 10.5061/dryad.dv41ns2bb
Description of the data and file structure
This dataset contains experimental data from Filgueiras et al (2025), testing the hypothesis that tick cell could interact with the Leishmania parasite and that the presence of the parasite cause biochemical alterations in tick cells.
Each file corresponds to each figure of the paper, containing : (1) tick cell and Leishmania interaction in different time points, Figures 2 (A, B ,C), (2) Biochemical results, Figure 3 (A-C), (3) Lipid metabolism (Figures 4 A-G)
Files and variables
File: Figure_2_A.xlsx
Description: Tick cells were incubated with Leishmania parasite. The percentage of infected- tick cell was determined at 2, 24 and 48 hours by counting at least 200 fixed and stained cells under an optical microscope
Variables
- The blank cells are not applicable
File: Figure_2_B.xlsx
Description: Tick cells were incubated with Leishmania parasite. The number of parasite per tick cells was determined at 2, 24 and 48 hours by counting at least 200 fixed and stained cells under an optical microscope
Variables
- The blank cells are not applicable
File: Figure_2_C.xlsx
Description: Tick cells were incubated with Leishmania parasite. The Association Index (AI) was determined at 2, 24 and 48 hours. The AI was determined by counting at least 200 fixed and stained cells under an optical microscope. IA is calculated multiplying the % infected cells x number of parasites/cell
Variables
- The blank cells are not applicable
File: Figure_3A.xlsx
Description: Tick cell viability was analysed through LDH activity using a CytoTox96 Non-Radioactive Cytotoxicity Assay kit (Promega) at 48 hours. Tick cells were incubated with Leishmania parasite and the supernatant was collected, and lactate dehydrogenase (LDH) activity read using a spectrophotometer.
Variables
- The blank cells are not applicable
- The percentage of viable cells were calculated considering optical density of uninfected cells as control (100%)
File: Figure_3B.xlsx
Description: ROS production was determined through extracellular hydrogen peroxide (H2O2) that was quantified by the Amplex Red oxidation method (Invitrogen). Reactions without cells were considered as blanks, and uninfected cells were used as controls.
Variables
- The blank cells are not applicable
- pico mol H2O2 x 2 x 105 cells: (numeric)
File: Figure_3_C.xlsx
Description: This experiment was used to obtain a semi-quantitative quantification of the relative abundance of different lipid classes present in the cell.
The objective was to evaluate the steady-state lipid profile by quantifying changes in the relative abundance of neutral lipid classes in IDE8 cells at a specific time point during infection (48 hours) with L. infantum. This approach quantifies the relative mass of neutral lipids accumulated in the cells. The samples obtained in lipid composition assays (without lipid precursors) had their lipids extracted and resuspended. They were analyzed by high-performance thin layer chromatography (HPTLC) on a 20 x 10 cm silica plate with an aluminum support.
For the analysis of neutral lipid profiles, a solvent system composed was used (VOGEL, ZIEVE and CARLETON, 1962). The lipids were identified by comparison with commercial standards , such as: 1-oleoyl-rac-glycerol (monoacylglycerol - MAG); 1,2-dioleoyl-sn-glycerol (diacylglycerol 1,2 - DAG 1,2); 1,3-diolein (diacylglycerol 1,3 - DAG 1,3); glycerol trioleate (triacylglycerol - TAG); 3β-cholest-5-en-3-ol (cholesterol - CHO); cholesteryl palmitate (esterified cholesterol - CHOE); 24-hydroxycholesterol (oxysterol - OXI); and oleic acid (free fatty acid - FA). 15 μL of each of these standards was used on each silica plate. After drying, the silica plates were sprayed with Charring's reagent (RUIZ and OCHOA, 1997). The HPTLCs were dried, and digitized (scanned) and the pixel densitometry associated with each lipid band was analyzed in the Image Master Total Lab version 1.11 program and the pixel volume or arbitrary units (A.U.) were used to compose the graphs, which represents the pixel intensity relative to each lipid band. This value allows a comparison of the relative abundance of a lipid between different samples
Variables
- The blank cells are not applicable
- The data refer to arbitrary number determined by densitometry associated with each lipid
- The variables are IDE8 alone and IDE8 incubated with Leishmania
File: Figure_4_C.xlsx
Description: The methodology used above refered to Figure 4 (A, B, C, D, E, F, G).
This experiment was designed to quantitatively measure metabolic activity, specifically the conversion of palmitate into more complex lipids. to quantify the activity of lipid synthesis pathways that use palmitate as a substrate. This was achieved by measuring the incorporation of the radioactive precursor, ³H-palmitate, into the different classes of lipids over time (24 and 48 hours). To determine fatty acid incorporation, at the time of infection, 5μCi of the ³H-FA-BSA complex was incubated individually/well in 500 μL of culture medium. Samples were collected after 24 and 48 hours of parasite interaction. After incubation, the cells were washed twice and Lipids were extracted as previously described.
To characterize neutral lipid metabolism, the same solvent system described previously (for the lipid profile) was used. After the run, the dried plates were stained with iodine vapor, and the selected spots were marked with a pencil. The iodine was evaporated from the HPTLC in a fume hood, and the previously marked spots were scraped off. The silica was collected, allocated into vials, and the radioactivity associated with each spot was determined by liquid scintillation. The liquid scintillation counter quantifies radiation by estimating how much the sample scintillates compared to its internal scintillation standards—whether for alpha, beta, or alpha and beta radiation—emitted by isotopes such as carbon-14 (14C), phosphorus-32 (³²P), and the one used in this work, tritium (³H). Therefore, it generates the result in Counts per Minute (CPMA) of radiation that composes a given sample. Since the amount of CPMA depends on the precision of the device used, a conversion to a universal radiation unit is necessary. Therefore, the results in CPMA were converted to Disintegrations per Minute (DPM) with the aid of an external counting standard, the Quench Curve for Tritium (Packard/PerkinElmer). With the CPMA results in hand, the transformed Spectral Index of the External Standard (tSIE) column of the samples was interpolated to the tSIE of the Quench Curve, which was paired with the curve's efficiency percentage, using GraphPad Prism (v. 6.1, GraphPad Inc., CA, USA). The program generated the sample's efficiency percentage (%Efficiency). The sample was then corrected:
The unit used is Disintegrations per Minute (DPM), as DPM represents an absolute and quantitative measure of radioactive activity, corrected for detector efficiency.
Unlike raw counts (CPM - Counts per Minute), which can vary depending on the equipment, DPM provides a true and standardized value of the amount of precursor incorporated. The use of DPM is the gold standard for metabolic flux studies, as it allows for direct and rigorous comparison of biosynthetic activity across different conditions, reflecting metabolic flux accurately and quantitatively.
Variables
- The blank cells are not applicable
- The variables are IDE8 alone and IDE8 infected with Leishmania at 24 and 48 h
- The data (number) refer to desintegration per minutes
File: Figure_4_A.xlsx
Description: Phospholipid
Variables
- The blank cells are not applicable
- The variables are IDE8 alone and IDE8 infected with Leishmania at 24 and 48 h
- The data (number) refer to desintegration per minutes
File: Figure_4_B.xlsx
Description: 1.2 DAG
Variables
- The blank cells are not applicable
- The variables are IDE8 alone and IDE8 infected with Leishmania at 24 and 48 h
- The data (number) refer to desintegration per minutes
File: Figure_4_F.xlsx
Description: CHOE
Variables
- The blank cells are not applicable
- The variables are IDE8 alone and IDE8 infected with Leishmania at 24 and 48 h
- The data (number) refer to desintegration per minutes
File: Figure_4_E.xlsx
Description: TAG
Variables
- The blank cells are not applicable
- The variables are IDE8 alone and IDE8 infected with Leishmania at 24 and 48 h
- The data (number) refer to desintegration per minutes
File: Figure_4_G.xlsx
Description: FA
Variables
- The blank cells are not applicable
- The variables are IDE8 alone and IDE8 infected with Leishmania at 24 and 48 h
- The data (number) refer to desintegration per minutes
File: Figure_4_D.xlsx
Description: 1.3 DAG
Variables
- The blank cells are not applicable
- The variables are IDE8 alone and IDE8 infected with Leishmania at 24 and 48 h
- The data (number) refer to desintegration per minutes
Parasite
Leishmania infantum (MCAN/BR/2008/1112), originally isolated in 2008 from a dog in Brazil, was maintained in Schneider’s insect medium supplemented with 10% fetal bovine serum (FBS) and 50 µg/mL gentamicin (Sigma) at 26 °C or Schneider’s insect medium supplemented with 10% FBS, 2% human urine, and 50 µg /mL gentamicin (Sigma) at 26 °C, in sealed T25 tissue culture flasks with weekly subculture.
Tick Cell Line
The I. scapularis tick cell line IDE8 was cultured in sealed T25 flasks in 5 mL of complete L15B medium as described previously (Marotta et al., 2018), at 32 °C.
Leishmania** - Tick Cells Interaction In Vitro**
IDE8 tick cells (2 x 105 per well) were seeded onto round glass coverslips in 24-well plates and maintained at 34 °C overnight. After this period, the cells were incubated with L. infantum promastigotes at a multiplicity of infection of 5 parasites to 1 cell (MOI 5) for 2 h. Following the incubation, free parasites were removed by washing with PBS, and the interaction was assessed at 2 h, 24 h, and 48 h. At the end of each time period, the coverslips were fixed in methanol and stained with Giemsa or Diff-Quick. The association index (% infected cells x number of parasites/cell) was determined by counting at least 200 cells per coverslip.
At 48h post, *Leishmania *infected-IDE8 tick cells were incubated with Schneider´s insect medium and incubated to 27 ºC for more 48h, to allow viable parasites inside the cells to grow. Then, the number of promastigotes recovered was counted in Neubauer chamber.
Lactate Dehydrogenase (LDH) Activity
To evaluate tick cell viability during Leishmania interaction, IDE8 tick cells (2 x 105/ well of a 24-well plate) were incubated with or without L. infantum promastigotes (MOI 5) for 2 h, 24 h or 48 h at 34 °C, as described above. Then, 50 µL of supernatant was used to evaluate LDH activity using a CytoTox96 Non-Radioactive Cytotoxicity Assay kit (Promega). LDH activity was read at 490 nm at 23°C using a SpectraMax spectrophotometer (Molecular Devices).
Reactive Oxygen Species (ROS) Production by Tick Cells
To measure ROS production of tick cells during Leishmania interaction, IDE8 tick cells (2 x 105 per well of a 24-well plate) were incubated with or without L. infantum promastigotes (MOI 5) at 34 °C for 2 h, non-adherent parasites were removed by washing and the cells were incubated at 34°C. After 48 h, tick cells were washed with PBS, counted, and assayed for ROS production. Extracellular hydrogen peroxide (H2O2) production was quantified by the Amplex Red oxidation method (Invitrogen^®^). Tick cells were added to a reaction medium containing PBS, 10 µM Amplex Red, and 0.1 U/mL horseradish peroxidase (HRP) in a final volume of 0.2 mL at room temperature. Reactions without cells were considered as blanks, and uninfected cells were used as controls. After 1 h of reaction, resorufin formation was measured by the change in absorbance at 540 nm (Rocco-Machado et al., 2019).
Lipid Extraction
IDE8 tick cells (1x106/mL in T25 flasks) were incubated with or without L. infantum promastigotes (MOI 5) for 48 h at 34 °C, as described above. After this period, tick cells were washed three times with PBS and used for lipid extraction, which was performed according to Bligh and Dyer (1959). A mixture of methanol:chloroform:H2O (2:1:0.8 v/v) was added to the samples. After intermittent agitation for 2 h, the solution was centrifuged for 20 min at 3300 x g in a clinical centrifuge and the supernatant was collected. The precipitate was subjected to a second extraction with the same mixture, followed by intermittent agitation for 1 h, and centrifugation for 20 min at 2000 x g. The supernatants were pooled, and 1.0 ml of water and 1.0 ml of chloroform were added thereto. After stirring and verifying the presence of two phases, the material was centrifuged again for 30 min at 3300 x g. The organic phase (lower), containing the lipids, was then removed with the aid of a Pasteur pipette and stored at -4oC.
High Performance Thin Layer Chromatography (HPTLC)
Extracted lipids were analyzed by HPTLC, as described previously for neutral lipids (Kawooya et al., 1988) and phospholipids (Horwitz and Perlman, 1987). Each lipid spot was identified by comparison with lipid standards run in parallel. Aliquots of 5 μg each of 1-oleoyl-rac-glycerol (MG), 1,3-diolein (DAG), glycerol trioleate (TAG), cholesterol (CHO), cholesteryl palmitate (CHOE), oleic acid (FA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC) were used as the lipid standards, purchased from Sigma-Aldrich^®^ (St Louis, Missouri, USA). To visualize the lipids, plates were immersed in a carbonization solution consisting of 8% CuSO4 and 10% H3PO4 for 10 s and heated at 110 °C for 20 min. Plates were analyzed by densitometry using ImageMaster TotalLab sofware (TotalLab, Newcastle, UK).
Measurement of uptake of 3H-Palmitic Acid Precursor
IDE8 tick cells (1x106/mL in T25 flasks) were incubated with or without L. infantum promastigotes (MOI 5) at 34 °C as described above. Cells were incubated with 100 µCi of ³H-palmitate (³H-palmitic acid 16:0 [9.10-3H(N)] (PerkinElmer, Boston, MA) complexed with 0.01 g fatty acid-free albumin (BSA-FFA, Sigma-Aldrich^®^, St Louis, Missouri, USA). After 48 h of interaction cells were subjected to lipid extraction and HPTLC. The lipid spots were scraped off the silica sheet, and radioactivity associated with each lipid was determined by scintillation counting using a PerkinElmer TriCarb scintillator.
Statistical Analysis
The data were analyzed using Student’s t-test to compare two groups and ANOVA for more than two groups. Analyses were performed using GraphPad Prism 8.0 software. Statistical differences were considered significant when *p *≤ 0.05.