This study was reviewed by the University of Nottingham (UK) School of Veterinary Medicine and Science (SVMS) Ethical Review Committee. The Committee reviews all research studies involving School personnel and is chaired by Professor David Haig. The committee passed this in vitro study as good to proceed, not requiring any further ethical review as it doesn’t involve vertebrate or invertebrate animals.
Human brain microvascular endothelial cells (HBMECs) were maintained as described previously , in complete RPMI-1640 (cRPMI) medium supplemented with 20% heat inactivated fetal calf serum (FBS), 2 mM L-glutamine, 1 mM Sodium Pyruvate, 1 mM MEM non-essential amino acids, 1% MEM vitamins and 100 units/mL penicillin/streptomycin at 37°C under humidified 5% CO2 conditions. When cells were confluent they were harvested with trypsin-EDTA and passaged at a sub-cultivation ratio of 1:3 into new culture flasks with fresh medium. Cells were considered confluent when their expansion had reached a point where cells touched each other on all sides, leaving no intercellular spaces. To exclude if cell viability could be regarded as a factor affecting response of the host cell to parasite infection and hence any subsequent metabolic analysis, the number of viable cells was determined on a minimum of 100 cells by hemocytometer under a light microscope after staining with 0.15% trypan blue solution. Cells used in the experiments had a viability not less than 99% at all times.
Neospora caninum (Nc-Liverpool) strain was propagated in Vero cells as described . Infected host cell monolayers were scraped, parasites were isolated from host cells by passage through 25- and 27-gauge needles and purified by using PD-10 Desalting Columns prepacked with Sephadex G-25 medium as described previously . Purified parasites were centrifuged at 800 × g, washed twice with fresh cRPMI, re-suspended in fresh medium and quantified using a hemacytometer. The final volume of suspension was adjusted with cRPMI medium to achieve a ratio of 2:1 parasite/host cell for subsequent infection experiments. Parasite viability was checked by using trypan blue staining assay and parasite with more than 97% viability were used.
In vitro infection protocol
Cells (3 × 105 cells/mL) were seeded at the bottom of 6-well culture plates with a volume of 2 mL cRPMI medium/well. Cells were allowed to grow overnight by incubation at 37°C in a humidified atmosphere with 5% CO2 in air. Before infection, cell growth medium was removed and cells were washed three times with sterile PBS (8 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH2PO4, 1.15 g/L Na2HPO4). Then, in each 6-well plate, three wells were infected with parasites at a MOI of 2 in 2-ml fresh medium, and the remaining three wells received only 2-ml fresh medium (mock-infected) and considered controls. Culture plates were then incubated to allow infection to progress within cells. Culture media were sampled at different time point post infection (PI) starting from 0 h, and then, at 1, 2, 3, 6, 12, 18, 24, 48 h PI. At each sampling time six wells (three infected and three controls) were collected and centrifuged at 1000 × g for 3 min, and the supernatants collected and kept at −80°C until analysis of extracellular metabolites.
The nonradioactive metabolic assay MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromidin ) was used to assess the effect of N. caninum infection on the viability of host cells. HBME cells were trypsinized from T-75 culture flasks, seeded into 96-well tissue culture microtiter plates (Nunc) at 1 × 104 cells per well in 100 μl of culture medium, and incubated for 18 h in a humidified incubator (37°C, 5% CO2) until become confluent. N. caninum tachyzoites were added to the cells at 2 MOI for 2 h, followed by removal of the medium and 2x washing with fresh medium to remove unbound parasites and cellular debris. Each well was then filled with 100 μl of fresh culture medium and plates were incubated at the above culture conditions. As a positive control, cells were treated with 1 μM staurosporine, an apoptotic agent. Cell viability was measured at 3, 6, 12, and 24 h PI by the reduction of MTT in a colorimetric assay. Briefly, MTT (Sigma Chemical, St. Louis, MO, USA) was added to each well (to a final concentration of 0.5 mg/ml), and incubation was continued for 4 h in the dark at 37°C. The cells were then incubated for 1 h in solubilization solution (50% sodium dodecyl sulfate in 0.1 mM/L HCl). The spectrophotometric absorbance of the samples was subsequently measured with a microtiter enzyme-linked immunosorbent assay (ELISA) plate reader using a 570-nm filter. The level of MTT reduction was expressed as a percentage of that of the non-infected control cells. The assay was performed in triplicate.
Lactate dehydrogenase assay
Lactate dehydrogenase (LDH) activity released into the culture medium (a measure of cell membrane lysis due to necrotic cell death) was assayed using a CytoTox 96 Kit (Promega, Madison, Wis.) according to the manufacturer’s instructions. Briefly, 1 × 104 HBMECs were seeded onto sterile 96-well plates and grown until 90% confluence and subsequently infected with N. caninum tachyzoites using different multiplicities of infection (MOIs) ranging from 0.5 to 4. After 3, 6, 9, 12, 18, and 24 h of incubation, the supernatants were collected, centrifuged to obtain cell-free supernatants. Of each sample, 50 μl per well was transferred to new 96-well plates. LDH activity was detected by the addition of freshly prepared reagents followed by incubation for 30 min in the dark at ambient temperature. LDH activity was measured by a redox reaction that couples the oxidation of lactate iodotetrazolium chloride to a colored formazan salt, using NADH as the electron transfer agent and NADH diaphorase as the catalyst. The absorbance at 490 nm was read using a Bio-Tek Instruments EL311SX plate reader. The cytotoxicity was expressed as a percentage of maximum LDH release, i.e., 100 × (optical density at 490 nm [OD490] of infected cells − OD490 of uninfected cells)/(OD490 of 2% Triton X-100-lysed uninfected cells − OD490 of uninfected cells). This assay was performed in triplicate wells, and the data represent the mean ± standard error of the mean (SEM) from at least three separate experiments. Statistical analysis was calculated by Student’s t-test using the Graph Pad Prism 3.0 statistical program (GraphPad Software Inc., San Diego, CA). P < 0.05 was taken to indicate statistical significance.
Biochemical analysis of extracellular metabolites
The level of the 20 metabolites was determined colorimetrically in culture medium obtained from infected and control wells at different time points PI using commercially available kits and a Randox RX Imola clinical chemistry analyzer (Randox Laboratories Ltd., Belfast, UK) according to the manufacturer’s specifications. Biochemical parameters measured included albumin (AB3800), glucose hexokinase (GL3816), calcium (CA3871), magnesium (MG3880), phosphorus (PH3820), NEFA (FA115), BHB (RB1007), cholesterol (CH3810), TGA (TR3823), total protein (TP3869), urea (UR3825), lactate (LC3980), chloride (CL1645), sodium (NA3851), potassium (PT3852), iron (SI3821), HDL (CH3811), and LDL (CH3841). Additionally, pyruvate and ATP were measured. All reagents used in the experiment were of analytical grade, or better. All of the 20 metabolites were quantified at each sampling time in order to assess the fluctuation in their concentration in culture medium in response to the progression of infection within cells. In all parameters, the data are the means of at least three independent experiments ± SEM. Statistical difference in metabolite concentrations were examined by ANOVA for repeated measurements (Genstat 12, VSN International, Hemstead, UK). The main effect tested was exposure to infection. A one way ANOVA was used to determine significant differences between control and infected cell cultures over a range of pre-determined periods of time PI. Probability values P < 0.05 were considered to be significant.
Raman micro-spectroscopic analysis
The level of the culture metabolites was determined by Raman microspectroscopy imaging. Raman spectra were recorded using a custom built Raman micro-spectrometer based on an inverted optical microscope (Eclipse-Ti, Nikon) with a Leica 50×/0.55 objective, 785 nm wavelength laser (Starbright XM, Torsana), spectrometer (77200, Oriel), back-illuminated deep-depletion CCD (DU401-A-BR-DD, Andor Technology) and automated sample stage (H107 Proscan II, Prior Scientific). The acquisition time for the Raman measurements was 1 second per position and the laser power on the sample surfaces was 200 mW. The spectrometer was calibrated using naphthalene and 1,4-bis(2-methylstyryl) benzene samples (both from Sigma-Aldrich, UK) to an accuracy of 0.5 cm−1. From each sample of culture medium a drop of ~50 μl was placed on a MgF2 coverslip fitted in a titanium chamber. About 225 Raman spectra were uniformly measured over 50 × 50 μm2 area. These were then averaged, and Raman spectra were subtracted from ‘control’. Spectra from all samples were analysed using Matlab R2013a software. The experiment was conducted in triplicates and was repeated twice. Raman spectra were assigned by comparison of their chemical shift to published values. Due to the qualitative similarity and complexity of the spectra from the infected and control cultures, a visible inspection of differences would be very difficult and so an independent multivariate analysis was carried out with PCA, to investigate basic biological differences between the medium of infected versus non-infected cell cultures.