Cannabidiol enhances microglial phagocytosis via transient receptor potential (TRP) channel activation

Abstract

Background and purpose: Microglial cells are important mediators of the immune response in the CNS. The phytocannabinoid, cannabidiol (CBD), has been shown to have central anti-inflammatory properties, and the purpose of the present study was to investigate the effects of CBD and other phytocannabinoids on microglial phagocytosis.

Experimental approach: Phagocytosis was assessed by measuring ingestion of fluorescently labelled latex beads by cultured microglial cells. Drug effects were probed using single-cell Ca²⁺ imaging and expression of mediator proteins by immunoblotting and immunocytochemistry.

Key results: CBD (10 μM) enhanced bead phagocytosis to 175 ± 7% control. Other phytocannabinoids, synthetic and endogenous cannabinoids were without effect. The enhancement was dependent upon Ca²⁺ influx and was abolished in the presence of EGTA, the Ca²⁺ channel inhibitor SKF96365, the transient receptor potential (TRP) channel blocker ruthenium red, and the TRPV1 antagonists capsazepine and AMG9810. CBD produced a sustained increase in intracellular Ca²⁺ concentration in BV-2 microglia and this was abolished by ruthenium red. CBD rapidly increased the expression of TRPV2 and TRPV1 proteins and caused a translocation of TRPV2 to the cell membrane. Wortmannin blocked CBD enhancement of BV-2 cell phagocytosis, suggesting that it is mediated by PI3K signalling downstream of the Ca²⁺ influx.

Conclusions and implications: The TRPV-dependent phagocytosis-enhancing effect of CBD suggests that pharmacological modification of TRPV channel activity could be a rational approach to treating neuroinflammatory disorders involving changes in microglial function and that CBD is a potential starting point for future development of novel therapeutics acting on the TRPV receptor family.

Keywords: calcium influx; cannabidiol; cannabinoid; microglia; phagocytosis.

Figure 1

Images of latex bead phagocytosis in (A) BV-2 cells and (B) mouse primary microglia cell. BV-2 (5 × 10⁵ cells per well) were loaded with fluorescent beads in BSA for 2 h and then incubated at 37°C before staining with rhodamine phalloidin (a high-affinity F-actin probe conjugated with red fluorescent dye) and DAPI to label cell nuclei (blue). The arrows indicate examples of the ingested fluorescent beads (40× magnification). Scale bar = 5 μm.

Figure 2

The effect of cytochalasin D (a cytoskeleton inhibitor), oligomycin (a mitochondrial ATPase inhibitor) and FCCP (a mitochondrial uncoupler) on phagocytosis in BV-2 cells. 5 × 10⁵ BV-2 cells per well were pre-incubated with the indicated drugs (1 μM) for 1 h. Control wells (C) were incubated with culture medium alone. BV-2 cells were loaded with fluorescent beads (0.5 μL·mL⁻¹) for 2 h. The figure represents means ± SEM of triplicate determinations in three independent experiments. The data are presented as a percentage of control and were analysed using one-way anova, followed by post hoc Dunnett's test; **P < 0.01 cytochalasin compared with control.

Figure 3

(A) The effects of temperature, ethanol and minocycline on phagocytosis in BV-2 cells. (B) The effect of UDP, ATP, IFN-γ, LPS and dexamethasone (DEX) on phagocytosis. BV-2 cells (5 × 10⁵ cells per well) were loaded with fluorescent latex beads (0.5 μL·mL⁻¹) for 2 h then incubated at 4 or 37°C with or without ethanol. The figure represents means ± SEM of triplicate determinations from three independent experiments, presented as a percentage of control values. Data were analysed using one-way anova, followed by post hoc Dunnett's test; *P < 0.05 ethanol versus control; ***P < 0.001 UDP versus control; **P < 0.01 DEX versus control; *P < 0.05 IFN, LPS versus control.

Figure 4

The effect of cannabinoids and endocannabinoids on phagocytosis in BV-2 cells. JWH133 is a CB₂ receptor agonist; AM251 is a CB₁ receptor antagonist; CP55940 and WIN 55212 (synthetic) and anandamide (AEA) and 2-AG (endogenous) are agonists of CB₁ and CB₂ receptors; lysophosphatidylinositol (LPI) is a putative endogenous GPR55 agonist. Cells (5 × 10⁵ per well) were pretreated with the indicated drugs (all 10 μM) for 24 h and then incubated with fluorescent latex beads (0.5 μL·mL⁻¹ for 2 h). The figure represents means ± SEM of three independent experiments each conducted in triplicate presented as a percentage of control. Data were analysed using one-way anova, followed by post hoc Dunnett's test; *P < 0.05 versus control (C).

Figure 5

(A) The effects of phytocannabinoids (10 μM) in BV-2 cells. (B) The effect of CBD in BV-2 cells on phagocytosis. BV-2 cells were incubated with the indicated drugs for 24 h and then loaded with fluorescent beads (0.5 μL·mL⁻¹) for 2 h. The figure represents means ± SEM of triplicate determinations of three independent experiments, presented as a percentage of control. The vertical axis represents the number of beads ingested/number of the cells expressed as a percentage of no drug. Data were analysed using one-way anova, followed by post hoc Dunnett's test; *P < 0.05; **P < 0.01 CBD 10 μM versus control (C). CBDA, cannabidiolic acid; CBDV, cannabidivarin; THC, Δ⁹ tetrahydrocannabinol; THCV, tetrahydrocannabivarin.

Figure 6

The effects of BAPTA-AM (an intracellular Ca²⁺ chelator) on CBD-enhanced phagocytosis in BV-2. BV-2 cells were pretreated with BAPTA-AM (50 μM) or with EGTA (4 mM) for 60 min before CBD (10 μM) exposure for 24 h. Control wells were incubated in culture medium alone. Cells were then loaded with fluorescent latex beads (0.5 μL·mL⁻¹) for 2 h. The figure represents means ± SEM of three independent experiments, each conducted in triplicate, presented as a percentage of control. Data were analysed using one-way anova, followed by post hoc Bonferonni's multiple comparison; *P < 0.05; **P < 0.01 CBD versus control; CBD + EGTA versus CBD alone.

Figure 7

The effect of CBD on intracellular calcium ([Ca²⁺]_i) in BV-2 cells using fura-2 ratiometric calcium imaging. In (A), ATP was added to cells to evoke a rapid and transient increase in [Ca²⁺]_i. After a thorough wash with HBSS buffer, a second application of ATP was added to the cells. (B) 10 μM CBD was added after a wash with HBSS buffer. (C) CBD was added with ruthenium red (RR) to cells and changes in [Ca²⁺]_i summarized as the 340/380 ratio of fura-2 fluorescence in (D). Data were obtained from at least three independent experiments (n > 40 cells per experiment).

Figure 8

(A) The effect of ruthenium red (RR), SKF96365 (SK), capsazepine (CPZ) and AMG9810 on CBD-enhanced phagocytosis in BV-2 cells. BV-2 (5 × 10⁵ cells per well) were pre-incubated with indicated drugs (10 μM except AMG, 1 μM) for 1 h, and then treated with CBD alone or in combination with CBD for 24 h. Control wells (C) were incubated with culture medium alone. The cells were loaded with 0.5 μL·mL⁻¹ fluorescent latex beads for 2 h. The figure represents means ± SEM of three independent experiments each conducted in triplicate, presented as percentages of control. Data were analysed using one-way anova, followed by post hoc Bonferonni's multiple comparison; *P < 0.05 compared with CBD control; **P < 0.01; ***P < 0.001. (B) Enhancement of BV2 latex bead phagocytosis by the putative TRPV2 agonist probenecid. BV-2 (5 × 10⁵ cells per well) were pre-incubated with probenecid at the indicated concentrations for 24 h prior to loading with latex beads as in (A). *P < 0.05 compared with basal; **P < 0.01 compared with basal.

Figure 9

Localization of TRPV2 immunoreactivity in BV-2 cells by immunocytochemical analysis: (A) control; (B) cells incubated with CBD (10 μM) for 1 h; (C) cells incubated with cycloheximide (CHX, 1 μM) 1 h prior to treatment with CBD for 1 h; (D) cells incubated with non-fluorescent latex beads 0.5 μL·mL⁻¹ for 2 h; (E) cells incubated with wortmannin 10 μM 1 h before 10 μM CBD for 1 h; and (F) cells incubated with ruthenium red (RR, 10 μM) 1 h prior to CBD for 1 h. BV-2 cells were fixed with formaldehyde, permeabilized with 0.1% Triton X-100, incubated overnight with the TRPV2 antibody and for 60 min with a fluorescent secondary antibody. The cells were visualized by confocal microscopy under 63× magnification using a glycerine immersion objective lens.

Figure 10

Increase in TRPV2 protein immunoreactivity induced by CBD in whole BV2 cell lysates. Cells were incubated with CBD (10 μM) for 5, 15, 30 and 60 min and 24 h alone. The cycloheximide (CHX 1 μM) was incubated with the cell lysates for 6 h prior to CBD. Data shown are ratios of TRPV2 : GAPDH expression and represent the means ± SEM of three independent experiments. Immunoblot treatments (A) are in the same order as those in the corresponding histograms (B). Data were analysed using one-way anova, followed by post hoc Bonferonni's multiple comparison test; *P < 0.05 CBD 30 min versus control; ***P < 0.001 CBD 1 h versus control; **P < 0.01 CBD + CHX 30 min versus CBD 30 min; ***P < 0.001 CBD + CHX 1 h versus CBD 1 h. *P < 0.05 CBD + CHX 24 h versus CBD 24 h.

Figure 11

Increased expression of TRPV2 protein in BV2 cell membrane fraction due to CBD. Cells were incubated with no drug (C memb) or CBD (10 μM) for 24 h prior to preparation of membrane fractions. (A) Shows immunoblots and (B) quantification of the expression levels. Data shown are ratios of TRPV2 : GAPDH expression and represent the means ± SEM of three independent experiments. Data were analysed using Student's unpaired t-test (two-tailed). *P < 0.05 CBD 24 (memb) h versus C (control memb).

Figure 12

Western immunoblots showing mediation of the enhancing effect of CBD on TRPV2 immunoreactivity in whole cell lysates of BV-2 cells by channel activation and PI3K. Cells were pre-incubated (1 h) with or without ruthenium red (RR) or wortmannin (10 μM) prior to CBD (10 μM). Data shown are ratios of TRPV2: GAPDH expression and represent the means ± SEM of three independent experiments. Data were analysed using one-way ANOVA, followed by post hoc Bonferonni's multiple comparison; **P < 0.01 CBD 1 h versus control; ***P < CBD + RR 1 h versus CBD 1 h alone; **P < 0.01 CBD + wortmannin 1 h versus CBD 1 h alone.

Figure 13

TRPV2 mRNA expression in BV-2 cells and the effect of CBD. RNA was extracted using Trizole reagent and RNA samples were reverse transcribed using the superscript reverse transcription with mouse actin as a normalizing gene. Data are means ± SEM of triplicate determinations of TRPV2/actin mRNA ratios conducted in three separate experiments. Data were analysed using one-way anova, followed by post hoc comparison; ***P < 0.001, compared with control.

Figure 14

Effect of CBD on TRPV1 protein expression in BV2 whole cell lysates. BV-2 cells were incubated with CBD (10 μM) for 10, 15, 30, 60 min and 24 h. Ruthenium red (RR) (non-selective TRPV channel blocker) and wortmannin (a PI3K inhibitor) (10 μM) significantly inhibited CBD-enhanced TRPV1 expression after 1 h. The data represent the means ± SEM of three experiments, normalized to GAPDH from three independent cultures. Data were analysed using one-way ANOVA, followed by post hoc comparison; *P < 0.05 CBD 30 (min) versus control; *P < 0.05 CBD 1 h versus control; *P < 0.05 CBD 24 h versus control; **P < 0.01 CBD + RR compared with CBD 1 h; *P < CBD + wortmannin 1 h compare with CBD 1 h.