Diethylcarbamazine elicits calcium signals in HEK293 cells by activation of heterologously expressed <em>Brugia malayi</em> TRP-2b channels

Diethylcarbamazine is a classic anthelmintic that is used for the prevention and treatment of lymphatic filariasis. The mode of action of diethylcarbamazine is still not well understood, with the consensus that it acts on the host immune system, rather than directly acting on the adult parasite. Recent studies have found that diethylcarbamazine acts on the muscle of adult female Brugia malayi, generating temporary spastic paralysis mainly through the Transient Potential Receptor C (TRPC) orthologue TRP-2. Activation of TRP-2 leads to inward currents in the muscle, an increase in intracellular calcium, and subsequent muscle contraction. These studies have demonstrated that Brugia malayi TRP-2 is activated by diethylcarbamazine. In this study, we heterologously expressed the Brugia malayi TRP-2b channel in the Human Embryonic Kidney (HEK) 293 cell line. Application of diethylcarbamazine to Bma-trp-2 b-transfected HEK293 cells leads to larger and more frequent increases in intracellular calcium compared to non-transfected cells. This increase can be inhibited using the TRPC-specific antagonist SKF96365. Our study shows that diethylcarbamazine’s action is dependent upon the Brugia malayi TRP-2 channel and may also, in addition, activate endogenous mammalian TRP channels. Methods Materials and Methods HEK283 maintenance: The Human Embryonic Kidney 293 (HEK293) cell line was used for all experiments in this study. HEK293 cells were maintained in a humidified growth incubator set to 37 °C in 5% CO2 following previous published methods. Cells were cultured in 10 mL Dulbecco’s Modified Eagle Medium (DMEM 1X) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) and 1% (v/v) Penicillin-Streptomycin (Pen/Strep). Media was exchanged every 3 days. Cells were passaged for a maximum of 14 generations. Passaging was achieved by discarding the previous media and washing cells with 1X PBS warmed to 37 °C to remove cellular debris. Cells were treated with 2.5 mL of 0.25% trypsin-EDTA warmed to 37 °C, and incubated at 37 °C in 5% CO2 for 2-3 minutes to detach cells. After incubation, 10 mL of warmed DMEM + 10% FBS + 1% Pen/Strep was added as the serum contains deactivators of trypsin. Cells were gently pipetted up and down to break clumps and to help detach cells remaining on the plate. 1 mL of cells were added to 37 °C warmed 10 mL fresh DMEM + 10% FBS + 1% Pen/Strep in new plates and placed in the incubator. Cells were frozen during every passage by adding 950 µL cell media to 50 µL 5% (v/v) DMSO and mixing gently before being stored at -80 °C. Brugia supply and maintenance: Live adult female Brugia malayi were shipped overnight from the NIH/NIAID Filariasis Research Reagent Resource Centre (FR3; College of Veterinary Medicine, University of Georgia, Athens, USA). Brugia malayi were maintained in non-phenol red Hyclone Roswell Park Memorial Institute (RPMI) 1640 media containing 10% heat-inactivated FBS and 1% penicillin-streptomycin. Parasites were separated individually into a 24 well microtiter plate containing 2 mL of the RPMI media. Parasites were held in an incubator set to 37 °C in 5% CO2. Cloning and maintenance of Bma-trp-2b: Adult Brugia malayi worms were snap-frozen in liquid nitrogen and crushed into fine powder in a 1.5 mL micro-centrifuge tube using Kimble Kontes Pellet Pestle (Fisher Scientific, Waltham, MA, USA). Total RNA was extracted using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instructions. cDNA was synthesized using SuperScript VILO Master Mix (Life Technologies, Carlsbad, CA, USA) from approximately 1 µg RNA as template. Full-length Bma-trp-2b (Bm5246b.1) was amplified using Platinum SuperFi Polymerase Master Mix (Thermo Fisher, Waltham, MA, USA) using the primers SSK160F and SSK160R (Table 1). Primers were made with the sequences flanking the expression vector pTarget (P2A::mCardinal), with SSK160F flanking the BamHI site and SSK160R flanking KpnI and the P2A site. Sequencing of the pTarget (P2A::mCardinal)::Bma-trp-2b plasmid is available in supplementary information (see Supplementary data online). The amplicon was analyzed using agarose gel electrophoresis and was purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany). The purified trp-2b cDNA was cloned into the pTarget vector using Infusion HD Cloning Kit (Takara Bio, San Jose, CA, USA) under the manufacturer’s guidelines. Upon cloning, the plasmids were sequence-verified and ready for transfection into HEK293 cells. The plasmid was added to Mix and Go! Competent JM109 Escherichia coli bacteria (Zymo Research, Irvine, CA, USA) which were grown in LB media with 25 µg/ml ampicillin at 37 °C for 16-18 hours. The plasmid was purified from bacterial lysates using Monarch® plasmid miniprep kit (New England Biolabs, Ipswich, MA, USA) and diluted in nuclease free water. The extracted plasmid concentrations were analyzed using an Implen NanoPhotometer® N120 (Implen, Munich, Germany). All plasmid stocks were stored at -20 °C. Transfection of HEK293 cells: For transfection, we followed techniques as previously described. Briefly, 500 µL of HEK293 cells were cultured in 5 ml of DMEM + 10% FBS and 1% Pen/Strep for 72 hours or until 90% confluence was achieved in a humidified growth incubator set to 37°C and 5% CO2. Transfection was achieved using cationic lipid-based reagent Lipofectamine® 3000 transfection kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions. Before transfection, old media was discarded, and cells were washed with 5 mL warm 1X PBS. Cells were incubated in OPTI-MEM® I (1X) Reduced Serum Medium + GlutaMAX TM-I for 40 minutes at 37°C in 5% CO2. Whilst cells were incubating, the transfection media was prepared by adding 5-8 µg of plasmid to a ratio of 3.75 µL lipofectamine per µL plasmid in 125 µL OPTI-MEM and by adding 2 µL P3000 per µL of plasmid to 125 µL OPTI-MEM. All components were mixed and incubated for 15 minutes at room temperature in the dark to improve transfection quality. When added, the transfection solution was applied over the area of the cell plate and the sample was incubated for a minimum of 6 hours at 37 °C in 5% CO2. After the transfection period the OPTI-MEM solution was discarded and replaced with DMEM supplemented with 10% FBS and cells were placed in a humidified growth incubator set to 30 °C at 5% CO2 for 48 hours to promote expression but slow cell growth. Before experiments, cell health was assessed via light microscope. HEK293 cDNA synthesis and RT-PCR detection of TRP channels: To obtain cDNA we cultured HEK293 cells either transfected with 5-8 µg of the Bma-trp-2b expression vector or without the vector at 37 °C at 5% CO2 for 7 days. HEK293 cells were homogenized in 1 mL of TRIzol by gentle pipetting, followed by total RNA extraction according to the TRIzol Reagent protocol. One microgram (1 µg) of total RNA from each culture was used to generate cDNA by reverse transcription (RT) using SuperScript VILOTM Master Mix following the manufacturer's protocol. PCR was conducted to detect the presence of the known endogenous human TRPC channels TRPC1, TRPC3, TRPC4 and TRPC6 by targeting the cDNA of htrp-1, htrp-3, htrp-4 and htrp-6 using previously published primers that target the encoding regions of each gene (Table 1). To measure the expression of Bma-trp-2b cDNA, we screened using primers targeting the encoding region of trp-2b that we have previously used 7 (Table 1). hβ-actin was used as a reference gene. Negative controls included enzyme, water, and both forward and reverse primers for hβ-actin with no cDNA template. The cycling conditions for PCR were an initial denaturation for 2 mins at 95 °C, followed by 35 cycles of 95 °C for 30 secs, 61 °C for 30 secs, 72 °C for 20 secs, and a final extension at 72 °C for 10 mins using GoTaq ® G2 Hot Start Green Master Mix (Promega, Madison, WI, USA). PCR products were then separated on a 2% Agarose gel containing SYBR® Safe DNA Gel Stain, followed by visualization and images were captured using an Azure 600 imaging system set to SYBR Safe (Excitation 472nm, Emission 595nm). Capturing cell images: White and red fluorescent images were captured using Occular 2.0.1.496 imaging software (Digital Optics, Auckland, New Zealand). For white light images the exposure settings were 10 ms with no binning Figs. 2A & B (left panels). Red fluorescence imaging was achieved using a Lambda LS Xenon bulb lightbox which delivered light via a fiber optic cable to the microscope (Sutter Instruments, Novato, CA, USA) which passed through a green/red filter box (EF-4 AT TRITC/CY3 LP; Excitation: 527.5 - 552.5nm, Emission 575nm Long Pass, Dichroic Mirror) (Chroma Technology, Bellows Falls, VT, USA). Fluorescent light emission was controlled by using the shutter. The exposure settings for the mCardinal fluorescent images were 500 ms with 2x binning Figs. 2 A & B (middle panels). For Fluo-3 fluorescence, images were captured using MetaFluor 7.10.2 (MDS Analytical Technologies, Sunnyvale, CA) with exposure settings at 250 ms with 2x binning under pseudo-color settings to illustrate calcium levels Figs. 1 A & B (right panels). All images were captured using a Photometrics Retiga R1 Camera (Teledyne Photometrics, Tucson, AZ, USA). Preparation and loading Fluo-3AM: Before recording, DMEM + 10% FBS media is discarded, and cells are washed with warmed 1X PBS to remove cellular debris. Cell detachment was achieved by adding 500 µL of 0.25% trypsin-EDTA and incubating at 37 °C in 5% CO2 for 2-3 minutes. Next, 5 mL of DMEM + 10% FBS was added to deactivate the trypsin, and cells were resuspended by gentle pipetting. Clean 12 mm circle coverslips coated with poly-D-lysine following methods previously described were placed in a clean Petri dish and 20 µL of cell media is added to each disc. To promote cell adhesion, DMEM + 10% FBS was added to cover the coverslip, and samples are incubated for a minimum of 2 hours before recording at 37 °C in 5% CO2~. After incubation the 12 mm glass cover slip was placed in a Warner RC26G laminar flow chamber (Warner Instruments, Holliston, MA, USA), immersed in HEK293 cell buffer (150mM NaCl, 5 mM KCl, 5 mM CaCl2, 1 mM MgCl2, 15 mM HEPES and 10 mM glucose, pH 7.3) and cells were visualized under white light on a Nikon Eclipse TE3000 microscope fitted with a 20X/0.45 Nikon PlanFluor objective to ensure that they appeared healthy before Fluo-3AM loading (Figs.2A & B). Fluo-3AM (Sigma-Aldrich, St. Louis, MO, USA) loading was achieved by incubating the cells in HEK293 cell buffer containing 5 µM Fluo-3AM and 10% Pluronic F-127 (10% v/v) for 20 minutes with the recording chamber connected to a Dual Automatic Temperature Controller (Warner Instruments, Holliston, MA, USA) maintained at 36-37 °C. After incubation, the Fluo-3AM solution was discarded, and the sample was incubated in HEK293 cell buffer for an additional 10 minutes at 36-37 °C to promote calcium loading. Before performing calcium imaging, cell transfection of the Bma-trp-2b plasmid was verified under green light using a long pass green/red filter box. Transfected cells that were fluorescing red were selected for recording Fig. 2A. We observed no red fluorescence in non-transfected cells Fig. 2B. Fluo-3AM loading was confirmed under blue light using a blue/green filter box (EF-4 AT EGFP/FITC/CY2/ALEXA FLUOR Band Pass; Excitation: 465-495nm, Emission: 525-545nm, Dichroic Mirror) (Chroma Technology, Bellows Falls, VT, USA) and visualized under pseudo-color settings using MetaFluor 7.10.2 (Figs. 2A & B). Any cell that did not show Fluo-3 fluorescence regardless of fluorescence under the red channel was discarded. We observed no significant difference in the levels of Fluo-3 fluorescence between Bma-trp-2b transfected and non-transfected cells (Fig. 2C). Measurement of Ca2+ fluorescence: All recordings were performed on a Nikon Eclipse TE3000 microscope fitted with a 20X/0.45 Nikon PlanFluor objective, using a Photometrics Retiga R1 Camera. Light control was achieved using a Lambda 10-2 with a shutter controller. Fluorescence was achieved using a Lambda LS Xenon bulb lightbox (Sutter Instruments, Novato, CA, USA) which delivered light via a fiber optic cable to the microscope which passed through a blue/green filter box. Fluorescent light emission was controlled by using the shutter. Minimal illumination exposure was used to prevent photobleaching. During recordings cells were continuously perfused with HEK293 cell buffer solutions. Cells were exposed to either 10 µM carbachol for one minute, 30 µM diethylcarbamazine (DEC) for five minutes, 30 µM arachidonic acid (AA) for five minutes, 10 µM SKF96365 for three minutes (SKF), 10 µM SKF96365 + 30 µM DEC (SKF + DEC) for 5 minutes or 10 mM CaCl2, which was used as a positive control to determine cell viability. If a cell failed to respond to the 10 mM CaCl2 control signal it was discarded from the data pool regardless of previous signals to our compounds. Every experiment in this study has a 100% cell response to 10 mM CaCl2. A change in fluorescence ≥3% was classified as a positive response to a compound as the average spontaneous change in cells continuously perfused with HEK293 buffer was ~2.5%. All solutions were delivered to the chamber under gravity feed through solenoid valves controlled using a VC-6 six-channel Valve Controller through an inline heater set at 37 °C (Warner Instruments, Holliston, MA, USA), at a rate of 1.5mL/min. At the start of all experiments, cell samples were left under blue light for a minimum of 1 minute to promote settling and equilibration of the fluorescent signal and to monitor any cells that may detach from the cover slip before the application of compounds. All calcium signal recordings were acquired and analyzed using MetaFluor 7.10.2 with exposure settings at 250 ms with 2x binning. Maximal percent calcium signal amplitudes (ΔF) were calculated using the equation F1-F0/F0 x 100, where F1 is the fluorescent value and F0 is the baseline value. All F0 values were taken at the time point each compound was applied for every cell analyzed. All representative response traces are presented as the average percent change in calcium fluorescence with the standard error of the mean (±SEM) of all cells from a single recording. Traces were generated by converting the calcium signal profiles of all the cells from a single recording to percentages using the previously described ΔF/F0 equation, with the time of stimulus application being F0 (0% for all cells) and 100% being the peak value for each cell. The mean percentage change in fluorescence and the SEM was calculated for each time point during the application of each compound. All baseline values were taken at the time point each compound was applied for every cell. Traces are represented as mean ±SEM. Statistical Analysis: Statistical analysis of all data was done using GraphPad Prism 9.0 (GraphPad Software, Inc., La Jolla, CA, USA). To ensure reproducibility, we repeated our experiments: the numbers of cells, number of preparations, the concentrations, and durations of applications are provided in the legends of the figures. Analysis of calcium amplitudes and percentage of cells responding was done using paired or unpaired student t-tests with P < 0.05 being considered significant. The data are presented as mean ± SEM for each treatment. Chemicals: Source of chemicals; SKF96365 was purchased from Tocris. Carbachol was purchased from EMD Millipore. All other chemicals were supplied by Sigma Aldrich.