Diethylcarbamazine elicits Ca²⁺ signals through TRP-2 channels that are potentiated by emodepside in Brugia malayi muscles

ABSTRACT

Filarial nematode infections are a major health concern in several countries. Lymphatic filariasis is caused by Wuchereria bancrofti and Brugia spp. affecting over 120 million people. Heavy infections can lead to elephantiasis, which has serious effects on individuals’ lives. Although current anthelmintics are effective at killing microfilariae in the bloodstream, they have little to no effect against adult parasites found in the lymphatic system. The anthelmintic diethylcarbamazine is one of the central pillars of lymphatic filariasis control. Recent studies have reported that diethylcarbamazine can open transient receptor potential (TRP) channels in the muscles of adult female Brugia malayi, leading to contraction and paralysis. Diethylcarbamazine has synergistic effects in combination with emodepside on Brugia, inhibiting motility: emodepside is an anthelmintic that has effects on filarial nematodes and is under trial for the treatment of river blindness. Here, we have studied the effects of diethylcarbamazine on single Brugia muscle cells by measuring the change in Ca²⁺ fluorescence in the muscle using Ca²⁺-imaging techniques. Diethylcarbamazine interacts with the transient receptor potential channel, C classification (TRPC) ortholog receptor TRP-2 to promote Ca²⁺ entry into the Brugia muscle cells, which can activate Slopoke (SLO-1) Ca²⁺-activated K⁺ channels, the putative target of emodepside. A combination of diethylcarbamazine and emodepside leads to a bigger Ca²⁺ signal than when either compound is applied alone. Our study shows that diethylcarbamazine targets TRP channels to promote Ca²⁺ entry that is increased by emodepside activation of SLO-1 K⁺ channels.

INTRODUCTION

Lymphatic filariasis (elephantiasis) is a neglected tropical disease caused by filarial nematode parasites that affects more than 120 million people worldwide, with 40 million people suffering from disfiguration or disability (1). Lymphatic filariasis is caused by the adults of the filarial parasites Wuchereria bancrofti, Brugia malayi, and Brugia timori. These parasites are transmitted between hosts through biting insects, and the adults live within the lymphatic vessels. In severe cases, the parasites block lymphatic ducts resulting in swelling of limbs and coarsening of the skin resulting in the condition known as elephantiasis. The swelling of limbs can result in disabilities, societal rejection, and prevent individuals from working. There are no effective vaccines against filarial parasites, nor have the measures to control the spread by their vectors been adequate.

Control of lymphatic filariasis relies on the use of chemotherapeutics to target and kill the microfilaria to prevent transmission between individuals by biting insects. The anthelmintics used to treat lymphatic filariasis include the following: benzimidazoles (albendazole) that bind to β-tubulin, inhibiting microtubule formation as well as metabolism (2); macrocyclic lactones (ivermectin), which target the glutamate-gated chloride channels (3); and diethylcarbamazine (DEC), a compound whose mode of action is not well characterized but have been reported to interact with transient receptor potential (TRP) channels (4, 5). Each of these anthelmintics may be administered alone or in combination in mass drug administration (MDA) programs (6, 7). However, the use of diethylcarbamazine in individuals who suffer from onchocerciasis is not recommended by the Centers for Disease Control and Prevention (CDC) or the World Health Organization (WHO) because of complications that could lead to aggravation of Onchocerca-induced eye disease. Although effective at killing and clearing microfilaria, none of these anthelmintics kill all of the adult worms; the surviving parasites live for 6–8 years producing microfilaria.

Historically, diethylcarbamazine had been understood to act on the host immune system rather than the parasite itself (8 – 10). However, recent studies have shown that diethylcarbamazine can stimulate nematode TRP channels, including the TRPC ortholog TRP-2 and the transient receptor potential channel, M subtype (TRPM) orthologs GON-2 and CED-11 (4, 5). In Brugia muscle cells, the effect of diethylcarbamazine opening TRP channels is an inward depolarizing current and contraction with spastic paralysis. The inward current produced by diethylcarbamazine is followed by an outward current produced by the activation of Ca²⁺-activated SLO-1 K⁺ channels. The anthelmintic emodepside (Emo), which activates nematode SLO-1 channels (11 – 14) is a broad-spectrum antiparasitic drug that has been reported to target and kill adult filaria. However, the in vivo potency of emodepside against some filarial parasites, like Brugia, is limited. Combinations of diethylcarbamazine and emodepside may promote increased efficacy: previous studies have highlighted the synergism between diethylcarbamazine and emodepside on muscle membrane potentials in the soil-transmitted helminth Ascaris suum (12); and the long-term muscle paralysis in Brugia malayi which is dependent on TRP-2 channels (13, 15).

In this study, we have utilized Ca²⁺ imaging to identify the role of Ca²⁺ in mediating diethylcarbamazine signaling and the role of the TRP-2 in promoting Ca²⁺ entry. We show that TRP-2 is a major source of diethylcarbamazine-stimulated Ca²⁺ entry as inhibition of the channels with SKF96365 and dsRNA knockdown inhibits the Ca²⁺ signal. We have tested the effects of arachidonic acid (AA) and miconazole (MIC), and have observed that they increase Ca²⁺ entry into the cytoplasm and TRP channel activation. Finally, we identify a synergistic relationship between diethylcarbamazine and emodepside in potentiating the Ca²⁺ signal that explains the enhanced paralysis observed in motility assays by disrupting the homeostasis of Ca²⁺ in the muscle cell. These results support previous observations that diethylcarbamazine interacts with TRP channels and that the combination of diethylcarbamazine and emodepside has potential for the treatment of individuals infected with Brugia malayi.

MATERIALS AND METHODS

Brugia supply and maintenance

Only female Brugia malayi worms were used for the study. Live adult female B. malayi were shipped overnight from the NIH/NIAID Filariasis Research Reagent Resource Center (FR3; College of Veterinary Medicine, University of Georgia, Athens, USA). B. malayi were maintained in non-phenol red Hyclone Roswell Park Memorial Institute (RPMI) 1640 media (Cytiva, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Fisher-Scientific) and 1% penicillin-streptomycin (Life Technologies, USA). Parasites were separated individually into a 24-well microtiter plate containing 2 mL of the RPMI media. Parasites were held in an incubator set at 37°C and 5% CO₂. All parasites were used up to 5 days post-delivery.

Dissection of B. malayi

Dissection of B. malayi was performed as previously described (16 – 18). All dissections were performed at room temperature. Briefly, worms were cut into 1 cm pieces from the anterior region and single sections placed into the recording chamber filled with B. malayi bath solution (23 mM NaCl, 110 mM NaAc, 5 mM KCl, 1 mM CaCl₂, 4 mM MgCl₂, 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 11 mM D-glucose, 10 mM sucrose, pH 7.2 using NaOH, ~320 mOsmol). The base of the chamber was a coverslip (24 × 50 mm) coated with a thin layer of Sylgard. The body piece was immobilized by gluing each end to the Sylgard pad using Glushield cyanoacrylate glue (Glustitch, BC, Canada) and immobilized by creating a wall down one side under the dissecting microscope. The body piece was cut longitudinally using a tungsten needle, and the “muscle flap” was glued to the coverslip along the cut edge exposing the muscle cells. The intestines and the uterus were removed using fine forceps and the prep was washed with bath solution to remove any eggs or debris.

Fluo-4 injection

To record Ca²⁺ signals, muscles were injected with 5 µM Fluo-4 penta-potassium salt (Thermo Fisher Scientific, USA) as previously described and viewed with DIC optics on a Nikon Eclipse TE300 inverted light microscope (400×) (19). Briefly, the dissected worms were treated with 2 mg/mL collagenase (Type 1A, Gibco) for 20–30 s and washed several times with buffer to remove excess collagenase. Patch pipettes were pulled from capillary glass and fire-polished. The pipettes were filled with pipette solution (120 mM KCl, 20 mM KOH, 4 mM MgCl₂, 5 mM Tris, 0.25 mM CaCl₂, 4 mM NaATP, 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and 36 mM sucrose, pH 7.2 with KOH, ~315–330 mOsmol). Fluo-4 was added to the pipette solution at the start of each recording day at a concentration of 5 µM and was kept in a dark environment to prevent degradation of the dye. Pipettes with a resistance of 1.5–3 MΩ were used. Giga ohm seals were formed before breaking the membrane by suction. After breaking in, cells were left to allow the Fluo-4 solution to diffuse the entire muscle cell (~5 to 10 minutes) (Fig. 1A).

Fig 1.

Fluo-4 induced Ca²⁺ signals in Brugia muscles. (A) Micrograph of a fluorescing dissected Brugia muscle injected with 5 µM Fluo-4 under blue light. Key structures of the body, nucleus, and muscle arms are indicated with the arrows. The red box indicates the region of interest (ROI) where we take the calcium recording. ROI is determined as the clearest part of the muscle cell with good Fluo-4 fluorescence. (B) Representative control trace of Fluo-4 fluorescence in a muscle being washed with bath solution containing 1 mM CaCl₂ followed by an increase to 10 mM CaCl₂ for 1 minute (gray box). (C) Total amplitudes of Fluo-4 fluorescence in response to constant 1 mM CaCl₂ bath solution perfusion (black bar) and to 10 mM CaCl₂ (gray bar). *** indicates significantly different to 1 mM CaCl₂ bath solution (bath solution vs CaCl₂). P < 0.001, t = 8.817, df = 5, paired t-test. n = 6, individual muscles from six individual Brugia females. All values are represented as means ± standard error of the mean (SEM).

Ca²⁺ imaging

All recordings were performed with a Nikon Eclipse TE300 microscope (20×/0.45 Nikon PlanFluor objective), fitted with a Photometrics Retiga R1 Camera (Photometrics, Surrey, BC, Canada). Light control was achieved using a Lambda 10-2 two-filter wheel system with a shutter controller (Lambda Instruments, Switzerland). Filter wheel one was set on a green filter (510–560 nM bandpass, Nikon, USA) between the microscope and camera. Filter wheel two was set on the blue filter (460–500 nM, bandpass, Nikon, USA) between a Lambda LS Xenon bulb light box which delivered light via a fiber optic cable to the microscope (Lambda Instruments, Switzerland). Blue light emission was controlled using a shutter. All Ca²⁺ signal recordings were acquired and analyzed using MetaFluor 7.10.2 (MDS Analytical Technologies, Sunnyvale, CA, USA). Exposure times were 150 ms with 2× binning. Maximal Ca²⁺ signal amplitudes (ΔF/F ₀, %) for all stimuli applied were calculated using the equation F − F ₀/F ₀ where F is the fluorescent value and F ₀ is the baseline fluorescent value, which was determined as the value immediately before the stimulus was applied to the sample for all recordings analyzed. Representative traces were generated using the same formula, with F ₀ being the value before the application of the first stimulus. For 1 mM CaCl₂ bath solution control experiments (Fig. 1B and C), F ₀ was determined to be the first value of the recording. Rise times were calculated by normalizing the trace during stimulus exposure until the application of the 10 mM CaCl₂ positive control, with the lowest fluorescence value being represented by 0% and the highest being 100%. The peak time was calculated by subtracting the time when the stimulus was applied from the time the signal reached 100%.

Application of compounds

The preparation was constantly perfused with all solutions being delivered to the chamber under gravity feed through solenoid valves controlled with a VC-6 six channel Valve Controller (Warner Instruments, Hamden, CT, USA) through an inline heater set at 37°C (Warner Instruments, Hamden, CT, USA) at a rate of 1.5 mL/min. New preparations were perfused with bath solution containing 1 mM CaCl₂ before being exposed to either 30 µM DEC for 5 minutes, 10 µM AA for 5 minutes, 10 µM MIC for 5 minutes or 1 µM Emo for 5 minutes. Exposure to diethylcarbamazine and SKF96365 was achieved by exposing muscle cells to 10 µM SKF96365 alone (SKF) for 5 minutes, followed by 10 µM SKF96365 and 30 µM diethylcarbamazine for 5 minutes (SKF + DEC), and finally to 30 µM DEC alone for 5 minutes giving a total exposure time of 15 minutes. For diethylcarbamazine and emodepside combination experiments, muscle cells were exposed to 30 µM DEC for 5 minutes and then 30 µM diethylcarbamazine + 1 µM emodepside (DEC + Emo) for 5 minutes, for a total of 10 minutes. To ensure that each of the preparations was viable after being exposed to any of the stimuli tested, all samples were subjected to 10 mM CaCl₂ for 1 minute to act as a positive control. Muscles that elicited Ca²⁺ signals to 10 mM CaCl₂ (>10%) were considered viable, while those that failed to elicit response (<10%) were classified as non-viable.

Synthesis and delivery of dsRNA

Trp-2 target T7 promoter labeled primers were amplified using the primers trp-2f2, trp-2f2t7, trp-2r2, and trp-2r2t7 previously described (Table S1) (4, 15). Non-specific T7 labeled LacZ dsRNA constructs were amplified using the primers LacZf, LacZr, LacZft7, and LacZrt7 (Table S1) (15). Amplification was performed from sequence-verified cDNA templates using a Techne PrimeG cycler (Bibby Scientific Limited, UK) with the following cycling conditions: 95°C × 5 minutes, 35 × (95°C × 30 s, 55°C × 30 s, 72°C × 1 minute), and 72°C × 10 minutes. dsRNA probes were synthesized (18, 20) using the T7 RiboMAX Express RNAi kit (Promega, USA) according to the manufacturer’s instructions (Table S2). The concentration of dsRNA was assessed using a spectrophotometer. Adult female B. malayi were soaked in 30 µg/mL of LacZ or trp-2 dsRNA for 4 days before being used for Ca²⁺-imaging experiments.

Analysis of transcript levels

cDNA from dsRNA-treated worms were amplified using the following target trp-2 and reference gene (Bma-gapdh) primers: trp-2f2, trp-2r2, SSK5F, and SSK5R (Table S1) (4, 15). These genes were amplified in triplicate by quantitative real-time PCR (qPCR) using the CFX96 Touch Real-Time PCR Detection System and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, USA). The cycling conditions used were 95°C × 10 minutes, 40 × (95°C × 10s, 55°C × 30 s). PCR efficiencies were calculated using the CFX96 Software Suite (Bio-Rad, USA). The relative quantification of target gene knockdown was estimated by the ΔΔCt method (21).

Chemicals

Emodepside was procured from Advanced ChemBlocks and SKF96365 was procured from Tocris; Sigma Aldrich supplied all other chemicals. All compounds were dissolved in either water or dimethyl sulfoxide (DMSO) and diluted in bath solution to obtain final concentrations. DMSO final concentration: 0.01%

Statistical analysis

Statistical analysis of all data was done using GraphPad Prism 9.0 (Graphpad Software, Inc., La Jolla, CA, USA). We repeated our experiments to ensure reproducibility. The number of experiments are provided in the results and figure legends. The concentrations and the duration of applications of diethylcarbamazine, arachidonic acid, miconazole, SKF96365, and emodepside are provided in the methods and legends of the figures. Analysis of Ca²⁺ amplitudes was done using either unpaired or paired Student’s t-tests with P values <0.05 considered as significant using Prism Graphpad version 9.0 software.

RESULTS

Diethylcarbamazine stimulates a detectable Ca²⁺ response in Brugia muscles

Ca²⁺ signals can be recorded in the muscles of Brugia malayi by directly injecting a fluorescent dye into the cell via a patch pipette (19). Here, we use Fluo-4, an analog of Fluo-3, which has increased fluorescence excitation and higher fluorescence signal levels according to the manufacturer. We loaded individual muscle cells with Fluo-4 until we were able to see key structures of the muscle cell including the muscle arms, nucleus, and the cell body (Fig. 1A). We exposed muscle cells to bath solutions containing 1 mM CaCl₂ and observed small (1.0% ± 0.3%, n = 6) fluctuations in the fluorescent signal (Fig. 1B and C, black bar) due to miniature end-plate potentials that are seen in the Brugia muscle cells. To verify that our muscles are physiologically active, we increased the CaCl₂ concentration by applying 10 mM CaCl₂ to the preparation. We observed rapid large reversible increases in fluorescence (27.4% ± 3.0%, n = 6) when 10 mM CaCl₂ was applied, and that fell when it was removed (Fig. 1B and C, gray bar).

We have reported previously that diethylcarbamazine inhibits motility and produces an initial spastic paralysis in B. malayi that is associated with inward currents on the muscle (4). This inward current was shown to be dependent on cation permeable TRP channels (4). We hypothesized that the activation of these channels would allow the entry of Ca²⁺ into the cell, which we could detect using our Ca²⁺-imaging protocol. We exposed muscles cells loaded with Fluo-4 to 30 µM diethylcarbamazine for 5 minutes and observed a characteristic increase in the Ca²⁺ signal which decreased when the diethylcarbamazine was removed (Fig. 2A). The average increase in fluorescence to diethylcarbamazine was 15% (15.4% ± 4.2%, n = 7; Fig. 2B, brown bar). The cells were then challenged with 10 mM CaCl₂ to ensure they were functioning physiologically, and we observed an increase in fluorescence (Fig. 2A and B, gray bar, 41.4% ± 11.9%, n = 7). We see that diethylcarbamazine simulates a reversible rise in cytoplasmic Ca²⁺ in Brugia muscle cells that is consistent with the opening of TRP channels that are permeable to Ca²⁺.

Fig 2.

Diethylcarbamazine stimulates increases in Ca²⁺ in adult Brugia muscles. (A) Representative Ca²⁺ trace in response to 30 µM diethylcarbamazine (brown box) for 5 minutes followed by 10 mM CaCl₂ control for 1 minute (gray box). (B) Total amplitudes of Fluo-4 fluorescence to 30 µM DEC (light brown bar) and 10 mM CaCl₂ (gray bar). * indicates significantly different to CaCl₂ (DEC vs CaCl₂). P < 0.017, t = 3.259, df = 6, paired t-test. n = 7, individual muscles from seven individual Brugia. All values are represented as means ± SEM.

Arachidonic acid and miconazole that stimulate TRP channels also promote Ca²⁺ entry

Nematodes produce poly-unsaturated fatty acids (PUFAs) by converting arachidonic acid into biologically active and inactive PUFAs via ω-hydroxylases and epoxygenases enzymes. Verma et al. (4) showed that arachidonic acid has a similar effect on the amplitude of the inward current on B. malayi muscles as diethylcarbamazine, but the signal was slower than diethylcarbamazine in onset and reaching a peak suggesting that arachidonic acid metabolites were responsible for the activation of the TRP channels. Here, we investigated the role of arachidonic acid on the Ca²⁺ signal by exposing the muscles to arachidonic acid. 10 µM arachidonic acid stimulated a Ca²⁺ signal that had a similar profile to that of diethylcarbamazine (Fig. 3A, compare blue trace to black trace) and a similar overall amplitude (13.3% ± 1.9%, n = 6 vs Fig. 3B, blue bar). Although similar in amplitude, the arachidonic acid signals had a significantly slower rise to the peak of the Ca²⁺ signal compared to diethylcarbamazine (5.6 minutes ± 1.0, n = 6 vs 2.6 minutes, ± 0.8, n = 7; Fig. 3C, blue bar), a phenotype that mimics the slower electrophysiology responses to arachidonic acid (4).

Fig 3.

Arachidonic acid and miconazole stimulate Ca²⁺ signals. (A) Representative individual traces of Ca²⁺ signals to 30 µM diethylcarbamazine (black line), 10 µM arachidonic acid (blue line), and 10 µM miconazole (green line). The bar represents the application of the compounds over 5 minutes. (B) Total amplitudes for DEC (light brown bar), AA (blue bar), and MIC (green bar). N.S. indicates not significantly different to diethylcarbamazine. DEC vs AA: P < 0.681, t = 0.422, df = 11 unpaired t-test and DEC vs MIC: P < 0.381, t = 0.909, df = 12, unpaired t-test. (C) Average time to maximum peak (100% of signal) for DEC (light brown bar), AA (blue bar), and MIC (green bar). * indicates significantly different to diethylcarbamazine. DEC vs AA: P < 0.044, t = 2.256, df = 12, unpaired t-test. N.S. indicates not significantly different to diethylcarbamazine. DEC vs MIC: P < 0.142, t = 1.573, df = 1, unpaired t-test. n = 7, individual muscles from seven individual Brugia for diethylcarbamazine responses; n = 7, individual muscles from seven individual Brugia for arachidonic acid; and n = 7, individual muscles from seven individual Brugia for miconazole treatments. All values are represented as means ± SEM.

Miconazole is an inhibitor of epoxygenase CYP450 enzymes that mimics the effects of arachidonic acid, which is explained by diverting the metabolism of arachidonic acid to active PUFAs that open the TRP channels in Brugia muscle; like arachidonic acid, miconazole produces a slow opening of TRP channels current during its application (4).

We applied 10 µM miconazole to Brugia muscles and recorded the Ca²⁺ signals, and observed Ca²⁺ signals with similar peak amplitudes to our responses to diethylcarbamazine and arachidonic acid (11.1% ± 2.1%, n = 7; Fig. 3A, green trace and Fig. 3B, green bar). The times to peak for the miconazole response were slower than our diethylcarbamazine responses (4.4 minutes ± 0.7, n = 7; Fig. 3C, green bar) although the difference did not reach statistical significance. Nonetheless, these results, taken together, further strengthen the idea that TRP channels play a key role in allowing the entry of Ca²⁺ into the muscle cells and that endogenous PUFAs can open TRP channels that yield similar Ca²⁺ amplitudes as the diethylcarbamazine-mediated responses.

TRP-2 mediates the diethylcarbamazine-induced Ca²⁺ signal

The TRPC nematode ortholog channel, TRP-2, has been identified as a key channel in mediating diethylcarbamazine effects, particularly effects on muscle membrane currents and motility (4, 15). To test if the observed diethylcarbamazine Ca²⁺ signal is mediated by Ca²⁺ entering through TRP-2 channels, we treated dissected muscles with the TRPC specific antagonist SKF96365, a compound which we have previously used to inhibit TRP channel activity in both Brugia and the gastrointestinal parasite Ascaris suum (4, 5). We exposed muscles to 10 µM SKF96365 for 5 minutes and observed no changes in the Ca²⁺ profile (Fig. 4A). We then exposed the muscle to 30 µM diethylcarbamazine in the presence of SKF96365 for an additional 5 minutes and observed that the Ca²⁺ signal was inhibited (1.9% ± 0.8%, n = 9; Fig. 4A and B, light brown bar). We also noticed that the inhibition was maintained after washing the preparation in the continued presence of 30 µM diethylcarbamazine for a final 5 minutes and that the effects of SKF96365 were not readily reversed (Fig. 4A). We observed no inhibition to the control CaCl₂ signal (33.2% ± 7.0%, n = 9; Fig. 4B, gray bar). This suggests that the TRPC channel TRP-2 is a major source of the diethylcarbamazine-stimulated Ca²⁺ signal in Brugia muscles.

Fig 4.

Inhibition of TRP-2 ablates diethylcarbamazine sensitivity: (A) Representative trace showing the application of 10 µM SKF96365 (light brown bar) for 5 minutes alone followed by the combination of SKF96365 and of 30 µM diethylcarbamazine (light brown box) for 5 minutes. SKF96365 is removed leaving diethylcarbamazine alone for a final 5-min exposure. Ten millimolar CaCl₂ control was applied for 1 minute (gray box) as a positive control. (B) Total Ca²⁺ amplitudes in response to SKF96365 + diethylcarbamazine (SKF + DEC) (light brown bar) and CaCl₂ control (gray bar). * indicates significantly different to SKF96365 + diethylcarbamazine. SKF96365 + diethylcarbamazine (SKF + DEC) vs CaCl₂: P < 0.002, t = 4.480, df = 8, paired t-test. n = 9 for diethylcarbamazine + SKF96365 responses and n = 9 for CaCl₂ responses from nine individual muscles from nine individual Brugia. All values are represented as means ± SEM.

While SKF-96365 is often used as a TRPC specific antagonist, it can affect other channels including the Ca²⁺ release-activated Ca²⁺ channel (Orai), stromal interacting molecule (STIM-1), voltage gated Ca_v channels, and K⁺ channels (22 – 25). To further test if the diethylcarbamazine-mediated Ca²⁺ signal was due to TRP-2 activation, we used dsRNA to selectively knockdown trp-2 channels in Brugia. Knockdown of trp-2 by RNAi has previously demonstrated the key importance of TRP-2 in mediated diethylcarbamazine effects on motility as dsRNA trp-2 animals showed reduced diethylcarbamazine sensitivity (4, 15). We exposed dsRNA trp-2 treated animals to 30 µM diethylcarbamazine and measured the Ca²⁺ signal. We observed that the Ca²⁺ was reduced but not inhibited in the trp-2 dsRNA treated worms (Fig. 5A, left panel, red trace and Fig. 5B, red bar), with an average amplitude of 3% (3.4% ± 0.5%, n = 3) compared to muscles not subjected to the dsRNA, 9% (9.4% ± 1.6%, n = 5; Fig. 5A, left panel, black line and Fig. 5B, brown bar). We observed no effect on the control CaCl₂ signal in the dsRNA-treated worms (26.8% ± 6.7%, n = 3; Fig. 5A, right panel, red bar and Fig. 5C, red bar) compared to our absent dsRNA samples (29.3% ± 8.8%, n = 5; Fig. 5A, right panel, black line and Fig. 5C, gray bar). Using qPCR, we found that the dsRNA construct knocked down the level of trp-2 by 83% (82.8% ± 2.3%, n = 3) compared to 5.5% (5.5% ± 3.0%, n = 3) in LacZ controls (Fig. 5D). Our data suggest that TRP-2 is the major channel in mediating the diethylcarbamazine signal. Taken together our data suggest that the TRP-2 channel (an ortholog of vertebrate TRPC) is one of the major targets of diethylcarbamazine in adult Brugia malayi that contributes to the entry of Ca²⁺ into the muscle and mediates its effects on motility.

Fig 5.

RNAi knockdown of trp-2 reduces diethylcarbamazine responses: (A) Left panel: representative traces showing diethylcarbamazine-induced Ca²⁺ responses in an untreated muscle (black line) and in a muscle treated with dsRNA for trp-2 (red line). Light brown box highlights diethylcarbamazine application for 5 minutes. Right panel: representative traces showing 10 mM CaCl₂-induced Ca²⁺ responses in an untreated muscle (black line) and in a muscle treated with dsRNA for trp-2 (red line). Gray box indicates 1-min exposure to 10 mM CaCl₂. All combined traces for untreated and dsRNA trp-2 treated responses to diethylcarbamazine and CaCl₂ are presented in Fig. S1. (B) Total amplitudes of Ca²⁺ fluorescence in response to 30 µM diethylcarbamazine in untreated muscles (light brown bar) and dsRNA trp-2 treated muscles (red bar). * indicates significantly different to untreated diethylcarbamazine. DEC vs dsRNA trp-2 diethylcarbamazine: P < 0.029, t = 2.864, df = 6, unpaired t-test. (C) Total amplitudes of Ca²⁺ fluorescence in response to 10 mM CaCl₂ in untreated muscles (gray bar) and dsRNA trp-2 treated muscles (red bar). N.S. indicates not significantly different to untreated muscle CaCl₂. CaCl₂ vs dsRNA trp-2 CaCl₂: P < 0.855, t = 0.191, df = 6, unpaired t-test. n = 5 for diethylcarbamazine responses, and n = 3 muscles from three individual Brugia for dsRNA trp-2 diethylcarbamazine treatments and corresponding 10 mM CaCl₂. (D) qPCR analysis for trp-2 expression in LacZ treated (control) and trp-2 dsRNA treated Brugia represented as percentage knockdown. *** indicates significantly different to LacZ. dsRNA trp-2 vs LacZ: P < 0.001, t = 20.260, df = 4, unpaired t-test. n = 3, individual Brugia treated with LacZ construct and n = 3, individual Brugia for trp-2 dsRNA treated. All values are represented as means ± SEM.

Emodepside potentiates diethylcarbamazine Ca²⁺ signals

Emodepside is a current anthelmintic used in veterinary medicine that targets different parasitic nematodes and has significant potential for human use (26, 27). Emodepside functions by targeting the Ca²⁺-activated K⁺ channel SLO-1 which causes paralysis of the nematode (11, 28). We have demonstrated that emodepside produces flaccid paralysis in Brugia, by opening SLO-1 potassium channels and hyperpolarizes the muscle membrane of Ascaris (12, 13, 15). Interestingly there is a synergistic relationship between emodepside and diethylcarbamazine resulting in potentiation of flaccid paralysis that is dependent on TRP-2 in Brugia (15) and which increases in the membrane potential hyperpolarization in the muscles of Ascaris suum (12).

We sought to determine if emodepside alone had any effect on the Ca²⁺ signal in our Brugia muscle cells. We predicted that there would be no effect of emodepside alone which by activating SLO-1 potassium channels would cause hyperpolarization (12) and thereby to close any voltage-sensitive Ca²⁺ channels. We applied 1 µM emodepside and observed no changes in the Ca²⁺ signal amplitude (0.9% ± 0.4%, n = 5), while there was no effect on the control 10 mM CaCl₂ Ca²⁺ signal (31.8% ± 8.1%, n = 5; Fig. 6A and B).

Fig 6.

Combination of emodepside elevates diethylcarbamazine-induced Ca²⁺. (A) Representative trace in response to 5-min exposure to 1 µM emodepside (pink box) followed by 1-min exposure to 10 mM CaCl₂ (gray box). (B) Total amplitudes for emodepside (pink bar) and control 10 mM CaCl₂ (gray bar) responses. * indicates significantly different to CaCl₂. Emo vs CaCl₂: P < 0.020, t = 3.745, df = 4, paired t-test, n = 5. (C) Representative trace highlighting responses to 30 µM diethylcarbamazine (light brown box) for 5 minutes before the addition of 1 µM emodepside (red bar) with 30 µM diethylcarbamazine for 5 minutes. Samples were exposed to 10 mM CaCl₂ (gray box) for 1 minute. (D) Total amplitudes in response to 30 µM diethylcarbamazine (light brown bar), 30 µM diethylcarbamazine with the addition of 1 µM emodepside (red bar), and control 10 mM CaCl₂ (gray bar). ** indicates significantly different to diethylcarbamazine. DEC vs DEC + Emo: P < 0.0066, t = 5.190, df = 4, paired t-test. DEC vs CaCl₂: P < 0.001, t = 10.72, df = 4, paired t-test. n = 5, individual muscles from five individual Brugia. All values are represented as means ± SEM.

Subsequently, we applied 30 µM diethylcarbamazine for 5 minutes and observed the characteristic rise in Ca²⁺ that plateaued with an average amplitude of 8% (8.0% ± 1.4%, n = 5; Fig. 6C and D, light brown bar) and then added 1 µM emodepside for 5 minutes on top of diethylcarbamazine; this generated a significant increase in the Ca²⁺ signal, 24% (24.2% ± 3.4%, n = 5) which failed to decline after emodepside and diethylcarbamazine were removed (Fig. 6C and D, red bar). This observation may explain the persistence of reduced motility in parasites treated with both diethylcarbamazine and emodepside (15). Our results illustrate a synergistic relationship between diethylcarbamazine and emodepside that together increases the entry of Ca²⁺ to disrupt the Ca²⁺ homeostasis within the muscles of Brugia malayi.

DISCUSSION

Use of diethylcarbamazine and emodepside

Diethylcarbamazine, ivermectin, and albendazole are recommended by WHO (1) for lymphatic filariasis MDA in areas without onchocerciasis. Diethylcarbamazine is a drug of choice for the treatment of lymphatic filariasis (7). It kills microfilaria and is active against adult worms, although its effects are less pronounced. Diethylcarbamazine is generally well tolerated but side effects can occur when there are higher numbers of microfilaria in the blood. Diethylcarbamazine should not be used when humans are infected with onchocerciasis because it can make eye disease worse due to the reactions of the larvae on the eye. There is also a concern of a possible severe reaction with individuals infected with Loa loa microfilaria and the development of encephalopathy.

Given that the existing registered antifilarial drugs (diethylcarbamazine, albendazole, and ivermectin) do not kill all adult worms, there are concerns that those that survive administration of therapeutic doses will enhance the rate of development of resistance. Also, if a therapeutic MDA program were able to kill all adults so that microfilariae are no longer produced, elimination would be speeded up. This is because adults can survive for 6–8 years producing millions of microfilariae maintaining the life cycle (29, 30) if they are not eliminated.

Emodepside is an effective veterinary anthelmintic for intestinal nematode parasites that is undergoing Phase II clinical trials for onchocerciasis by the Drugs for Neglected Diseases intiative (DNDi) that started in 2020. Emodepside opens nematode SLO-1 K⁺ channels (11, 13) and has inhibitory effects on the motility of different filariae (Acanthocheilonema viteae, Brugia pahangi, Litomosoides sigmodontis, Onchocerca gutturosa, Onchocerca lienalis, Brugia malayi, and Dirofilaria immitis) when tested in preclinical investigations (31 – 33). The effects of emodepside are dose-dependent, filarial species-specific-dependent, and life-stage-dependent (34 – 39). Adult Brugia malayi are a dose-limiting species because emodepside has the least potent effects on the adults of this filaria (31, 35). Also, concentrations of emodepside may not be sufficient, for pharmacokinetic reasons, in regions where filarial parasites are located. The potential for diethylcarbamazine to potentiate the anthelmintic effects of emodepside (4, 12, 15) may prove useful in combination therapies.

Interaction of diethylcarbamazine and emodepside involves TRP-2 and SLO-1 K⁺ channels

We have observed in Ascaris suum (12) and in adult Brugia malayi (4, 15) that diethylcarbamazine potentiates the effects of emodepside: 1 µM diethylcarbamazine shifts the effective concentration for 50% of maximum effect (EC50) of emodepside in female Brugia malayi from 395 nM to 114 nM an increase in potency of 3.5-fold (15). This potentiation was hypothesized to involve the activation of TRP-2 channels that allow entry of Ca²⁺ to increase cytosolic Ca²⁺ and thereby increase emodepside activation of the Ca²⁺-sensitive SLO-1 K⁺ channels. Here, we have seen that application of diethylcarbamazine increases cytosolic Ca²⁺ concentrations by activation of the TRP-2 channel. The involvement of TRP-2 channels mediating the effects of diethylcarbamazine on cytosolic Ca²⁺ is revealed by inhibition of TRP-2 by the TRP-C antagonist, SKF96365, and by RNAi knockdown of trp-2. Furthermore, we were able to observe that not only does diethylcarbamazine increase cytosolic Ca²⁺ directly but that activation of SLO-1 K⁺ channels by emodepside enhances the Ca²⁺ response to diethylcarbamazine (Fig. 6 and 7). The positive interaction between TRP channels and SLO-1 K⁺ channels has been described previously in mammalian systems (40). The opening by emodepside of potassium channels in the muscle membrane hyperpolarizes the muscle membrane and increases the driving potential for the entry of Ca²⁺ into the muscle cell through the TRP channels opened by diethylcarbamazine. Thus, there is a positive feed-back loop with diethylcarbamazine enhancing the effects of emodepside on SLO-1 K⁺ channels and emodepside enhancing the entry of Ca²⁺ produced by diethylcarbamazine (Fig. 7). This positive feed-back loop can explain the synergistic effect of diethylcarbamazine and emodepside on inhibition of motility which can be seen at 1 µM concentrations of diethylcarbamazine (15).

Fig 7.

Summary diagram of the positive feed-back loop produced by diethylcarbamazine and emodepside. Diethylcarbamazine stimulates Ca²⁺ entry into Brugia muscle cells via TRP-2 and enhances the activation of SLO-1 K⁺ channels by emodepside that by hyperpolarizing the muscle cell further increases Ca²⁺entry. There is a synergistic relationship between the actions of diethylcarbamazine and emodepside produced by a positive feed-back loop. Diethylcarbamazine activates the TRP-2 channel and emodepside activates the SLO-1 K⁺ channels.

Conclusion

We have seen that diethylcarbamazine has a rapid effect on increasing cytosolic Ca²⁺ that is maintained during its application. The increased cytosolic Ca²⁺ will activate the contractile machinery of the muscle cells and limit the normal vibrating muscle activity that is seen in healthy worms. Emodepside enhances the effects of diethylcarbamazine with a positive feed-back loop: diethylcarbamazine opens TRP-2 channels, increasing cytosolic Ca²⁺, and activating SLO-1 K⁺ channels; and emodepside hyperpolarizes the membrane potential of the muscle, increasing the driving force for the entry of Ca²⁺. The combination of diethylcarbamazine and emodepside may be useful where the potency of either drug is limited by species of parasite or location of the parasite.

DATA AVAILABILITY

All relevant data are within the manuscript and its Supporting Information files.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aac.00419-23.

Fig. S1 Legend. aac.00419-23-s0001.docx.

Legend for Fig. S1.

Fig. S1. aac.00419-23-s0002.tif.

Effect of RNAi of TRP-2 inhibiting DEC responses.

Tables S1 AND S2. aac.00419-23-s0003.docx.

Table S1: List of Primers used in the study Table S2: dsRNA probe sequences.

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