Unraveling the biochemical aspects of the interaction between ticks and <em>Leishmania</em> using a tick cell line

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. Methods 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.