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Published in final edited form as: Cell Immunol. 2024 Jan 17;397-398:104812. doi: 10.1016/j.cellimm.2024.104812

The Mechanism by which Cannabidiol (CBD) Suppresses TNF-α Secretion Involves Inappropriate Localization of TNF-α Converting Enzyme (TACE)

Christa Frodella 2, Liyuan Liu 2, Wei Tan 2, Stephen B Pruett 1,2, Barbara L F Kaplan 1,2,*
PMCID: PMC10947891  NIHMSID: NIHMS1961905  PMID: 38245915

Abstract

Cannabidiol (CBD) is a phytocannabinoid derived from Cannabis sativa that exerts anti-inflammatory mechanisms. CBD is being examined for its putative effects on the neuroinflammatory disease, multiple sclerosis (MS). One of the major immune mediators that propagates MS and its mouse model experimental autoimmune encephalomyelitis (EAE) are macrophages. Macrophages can polarize into an inflammatory phenotype (M1) or an anti-inflammatory phenotype (M2a). Therefore, elucidating the impact on macrophage polarization with CBD pre-treatment is necessary to understand its anti-inflammatory mechanisms. To study this effect, murine macrophages (RAW 264.7) were pre-treated with CBD (10 μM) or vehicle (ethanol 0.1%) and left untreated (naive; cell media only), or stimulated under M1 (IFN-γ+ lipopolysaccharide, LPS), or M2a (IL-4) conditions for 24 hr. Cells were analyzed for macrophage polarization markers, and supernatants were analyzed for cytokines and chemokines. Immunofluorescence staining was performed on M1-polarized cells for the metalloprotease, tumor necrosis factor-α-converting enzyme (TACE), as this enzyme is responsible for the secretion of TNF-α. Overall results showed that CBD decreased several markers associated with the M1 phenotype exhibiting less effects on the M2a phenotype. Significantly, under M1 conditions, CBD increased the percentage of intracellular and surface TNF-α but decreased secreted TNF-α. This phenomenon might be mediated by TACE as staining showed that CBD sequestered TACE intracellularly. CBD also prevented RelA nuclear translocation. These results suggest that CBD may exert its anti-inflammatory effects by reducing M1 polarization and decreasing TNF-α secretion via inappropriate localization of TACE and RelA.

Keywords: cannabidiol, macrophage polarization, innate immunity, TNF-α

INTRODUCTION

Cannabidiol (CBD) exerts its anti-inflammatory mechanisms under various inflammatory conditions, including neuroinflammation [1]. Neuroinflammation is a complex concert of immune mediators that range from the innate to adaptive immune system, and CBD has been shown to impact each step of the inflammatory process [1]. For diseases that affect the brain, such as the murine multiple sclerosis (MS) model, experimental autoimmune encephalomyelitis (EAE), treatment with CBD decreased overall clinical symptoms and associated inflammation at the peak of the disease [2–7]. However, the CBD-associated anti-inflammatory mechanisms that contribute to the attenuation of the EAE remain to be elucidated.

Key innate immune cells of interest are macrophages, as they play a role in disease propagation and attenuation [8]. In pathologies such as MS and its murine model, EAE, macrophages not only make an essential contribution in pro-inflammatory cytokine secretion, such as tumor necrosis factor-alpha (TNF-α) [9, 10], but also phagocytose myelin, resulting in tissue damage [11, 12]. Both microglial cells and infiltrating macrophages contribute to demyelination, although there is evidence that microglial cells and meningeal macrophages specifically are responsible for antigen presentation and T cell activation initially in EAE disease [13].

Given the critical role of macrophages early in EAE disease initiation [13], and the fact that CBD administered for only the first 5 days after EAE disease initiation resulted in attenuation of clinical signs and inflammation [4], it is important to further characterize the effects of CBD on the anti-inflammatory effects of macrophages. It is established that macrophages (and microglial cells) can polarize into at least two phenotypes: the inflammatory phenotype (M1) and an anti-inflammatory, pro-tissue resolving phenotype (M2a) [14]. Thus, the purpose of this study was to examine the effects of CBD on M1/M2 polarization of mouse RAW264.7 macrophages and investigate mechanisms by which CBD was anti-inflammatory. We observed differential effects of CBD on TNF-α, depending on whether it was expressed intracellularly, extracellularly, or secreted, which prompted us to examine the mechanisms by which CBD altered TNF-α secretion. The tumor necrosis factor-α converting enzyme (TACE) is the enzyme responsible for cleaving membrane-bound TNF-α, facilitating its cellular release [15]. Thus, we hypothesized that the mechanism by which CBD suppressed secreted TNF-α is due in part to inappropriate localization of TACE.

METHODS

Cells

RAW 264.7 cells (ATCC®, TIB71™, Lot:70012232)) were maintained in RPMI containing 10% Bovine Calf Serum (BCS), 1% Penicillin Streptomycin, and 1% Glutamax. In addition, a separate transfected cell line, RAW 264.7 G9 cells expressing Green Fluorescent Protein (GFP)-RelA that were gifted from Dr. Iain Fraser (NIH/NIAID), were grown in DMEM supplemented with 10% BCS, 1% sodium pyruvate, 1% penicillin-streptomycin, and 1% Glutamax (Gibco) [16]. All cells were grown in a T-75 flask and maintained at 37 °C in a humidified incubator with 5% CO2.

CBD

CBD was provided by the National Institute on Drug Abuse (NIH/NIDA) and was >98% pure.

Macrophage Polarization and Flow Cytometry

Macrophage polarization and characterization of cells with flow cytometry were replicated with our recently published protocol [17]. Briefly, RAW 264.7 cells were seeded at 2×105 cells per well in a 12-well plate overnight. Cells were left untreated (control) or pre-treated with vehicle (VH, 0.1% ethanol), or CBD (10 μM) for 30 min. Cells were then unstimulated (naive) or M1- or M2a-polarized for 24 hr. M1 polarization was induced using IFN-γ (10 ng/mL) for 23 hr, and LPS (100 ng/mL) at hour 23 for one hr (IFN-γ+LPS). M2a polarization was induced using IL-4 (20 ng/mL) for 24 hr. Cells were stained for various markers for M1 versus M2a as commonly defined in other studies (a priori, M1 markers were defined as TLR4, CD14, CD86, MHCII, and TNF-α, while M2 markers were defined as CD206, Arginase-1 and VEGF) [17]. Antibodies are outlined in Table 1. Raw numbers used to generate graphs are provided in Supplemental Table 1. Cells were gated on fluidic stability, singlets, and live cells prior to gating for individual markers.

Table 1.

Antibodies

Flow Cytometry
Protein Fluorochrome Clone Location Source
TNF-α BV421 MP6-XT22 Intracellular Biolegend
CD14 BV510 SA14–2 Surface Biolegend
CD206 BV605 C068C2 Surface Biolegend
F4/80 BV711 T45–2342 Surface BD Biosciences
CD86 BV786 GL1 Surface BD Biosciences
Arginase-1 AF488 A1exF5 Intracellular Invitrogen/Thermo Fisher
MHCII PerCP-Vio700 REA813 Surface Miltenyi Biotec
TLR4 PE SA15–21 Surface Biolegend
VEGF AF594 VG1 Intracellular Novus Bio
TNF-α PE/Cy7 MP6-XT22 Surface Biolegend
Immunofluorescence
Protein Fluorochrome Source
WGA PE Texas Red Invitrogen/Thermo Fisher
TACE N/A Novus Bio
donkey anti-rabbit IgG FITC Biolegend
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Cytokine and Chemokine Quantification

TNF-α was quantified with an ELISA kit (Invitrogen). Thirty-two cytokines/chemokines were quantified with the following Luminex kit: MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel Premixed 32 Plex (Millipore). The data were generated by Milliplex Analyst software (Millipore). Raw numbers used to calculate percent vehicle, which were used to generate graphs, are provided in Supplemental Table 1.

Immunofluorescence Assay

M1-polarized RAW 264.7 cells were grown on a coverslip at 3×105 cells per well in a 6-well plate. Cells were washed with PBS and fixed with Cytofix for 10 min. Cells were then stained with wheat germ agglutinin (WGA) conjugated with Texas Red (Invitrogen) to define the extracellular membrane at a 1:200 dilution in PBS. Next, cells were permeabilized with 0.1% Tween-20 and 0.5% bovine serum albumin (BSA) in PBS and stained with TACE antibody (Novusbio) at a 1:50 dilution overnight. Cells were stained with the secondary, FITC donkey anti-rabbit IgG (Biolegend), for one hr in the permeabilization solution in a 1:25 dilution. Subsequently, the nucleus was stained with Hoechst 33258 (Invitrogen) at a 1:2500 dilution in PBS for 10 min. Finally, cells were washed with PBS and mounted on slides. Slides were visualized on a Cytation 5 instrument (Biotek).

Live Cell Experiment

RAW 264.7 G9 cells were seeded in a 48-well plate at 500,000 cells per well in 500 μL of phenol red-free DMEM and incubated overnight. On the second day, cells were stained with Hoechst 33342 (10 μM) in 500 μL of Fluorobright/DMEM (Gibco) for 20 minutes at 37° C. Subsequently, cells were washed with 1 mL of Fluorobright/DMEM media. Cells were then pre-treated with 200 μL of VH (0.1% ethanol) or CBD (10 μM) for 30 min. Lastly, 50 μL of LPS was added to achieve 100 ng/mL. Each group had an n=6. Imaging exposure settings were then adjusted for the DAPI and GFP channels, and the time course was set to take images approximately every 10 min for 100 min with Cytation 5 (Biotek) from which percent positive was calculated. The percent positive (i.e., percentage of cells that were positive for RelA translocation) was calculated with a pre-defined protocol from Biotek from their Image+ software.

Statistics

All statistical analyses were performed with GraphPad Prism 7 (San Diego, CA). Flow cytometry percent, cytokine/chemokine values, and RelA kinetics percent positive data were presented as mean ± standard error mean (SEM). Flow cytometry percentages were transformed using the logit function. A two-way ANOVA was then used to determine the significance of the flow cytometry data and TNF-α secretion that was quantified from the ELISA kit. Cytokines and chemokines quantified with the Luminex kit were analyzed with a t-test for M1: VH vs. CBD and M2a: VH vs. CBD. For the live cell, RelA kinetics experiment, the area under the curve (AUC) was calculated for each well, and a t-test was performed for VH compared to CBD and VH+LPS compared to CBD+LPS. For all experiments, a p-value ≤ 0.05 was deemed significant.

RESULTS

Effect of CBD on M1- or M2a-polarized Phenotype

Based on our previously published method for examining macrophage polarization [17], M1 markers were defined as TLR4, CD14, CD86, MHCII, iNOS and TNF-α, and M2 markers were defined as CD206, Arginase-1, and VEGF. M1 cells were polarized with IFN-γ+LPS while M2 cells were polarized using IL-4. First, the effects of CBD on viability and F4/80 were assessed and it was determined that CBD suppressed viability of M1-polarized cells (Figure 1A). CBD did not alter the percentage of cells expressing F4/80 but decreased the mean fluorescence intensity (MFI) of F4/80 in both M1 and M2a-polarized cells (Figure 1B, C). In looking at M1-associated markers, CBD suppressed percent of cells expressing TLR4, CD14 and CD86 regardless of polarization conditions (Figure 2A, B and Figure 3A). Only CD86 MFI was suppressed by CBD in response to both polarization conditions. CBD suppressed the percent of double positive TLR4 and CD14 (Figure 3A). CBD did not affect percent of cells expressing MHCII but suppressed MHCII MFI in response to M2a polarization (Figure 3B). Finally, with iNOS, there was only a slight increase in percent cells expressing iNOS in response to M1 polarizing conditions (Figure 3C). It was interesting to note that not all the a priori-assigned M1 markers were induced preferentially using the M1 conditions, which likely reflects the plasticity of macrophages. Next, the effect of CBD on M2a-associated markers was evaluated. CBD did not affect CD206 (Figure 4A) but decreased Arginase-1 under naive and M1-polarized conditions, both in percent cells and MFI (Figure 4B). CBD increased the MFI of VEGF in response to M2a polarization. Again we noted that not all the a priori-assigned M2a markers were induced preferentially using the M2a conditions.

Figure 1.

Figure 1.

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CBD pre-treatment suppressed expression of F4/80. Murine macrophages (RAW 264.7) were pre-treated with VH (0.1% ethanol) or CBD (10 μM) for 30 min and left unstimulated (naive) or polarized with M1 (IFN-γ+LPS) or M2a (IL-4) stimuli. Viability (A) and F4/80+ murine macrophage marker (B, C) were assessed by percent expression and MFI. The values were mean ± SEM with n=5/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 2.

Figure 2.

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CBD effect on M1-associated markers. Murine macrophages (RAW 264.7) were pre-treated with VH (0.1% ethanol) or CBD (10 μM) for 30 min and left unstimulated (naive) or polarized with M1 (IFN-γ+LPS) or M2a (IL-4) stimuli. TLR4 (A), CD14 (B), TLR4+CD14+ (C) were assessed by percent expression and MFI. The values were mean ± SEM with n=5/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3.

Figure 3.

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CBD effect on M1-associated markers. Murine macrophages (RAW 264.7) were pre-treated with VH (0.1% ethanol) or CBD (10 μM) for 30 min and left unstimulated (naive) or polarized with M1 (IFN-γ+LPS) or M2a (IL-4) stimuli. CD86 (A), MHCII (B), and iNOS (C) were assessed by percent expression and MFI. The values were mean ± SEM with n=5/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4.

Figure 4.

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CBD effect on M2a-associated markers. Murine macrophages (RAW 264.7) were pre-treated with VH (0.1% ethanol) or CBD (10 μM) for 30 min and left unstimulated (naive) or polarized with M1 (IFN-γ+LPS) or M2a (IL-4) stimuli. CD206 (A), Arginase-1 (B), and VEGF (C) were assessed by percent expression and MFI. The values were mean ± SEM with n=5/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Effect of CBD on TNF-α

TNF-α, an M1-associated cytokine, was pointedly impacted by CBD. CBD pre-treatment significantly increased percent cells expressing TNF-α intracellularly and at the surface (Figure 5A, B) under M1 polarizing conditions. Interestingly, CBD suppressed surface TNF-α MFI (Figure 5B) and also robustly suppressed TNF-α secretion in response to M1 polarization (Figure 5C).

Figure 5.

Figure 5.

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CBD effect on TNF-α. Murine macrophages (RAW 264.7) were pre-treated with VH (0.1% ethanol) or CBD (10 μM) for 30 min and left unstimulated (naive) or polarized with M1 (IFN-γ+LPS) or M2a (IL-4) stimuli. Intracellular or surface TNF-α (A or B, respectively) were assessed by percent expression and MFI. Secreted TNF-α was assessed in the supernatant using ELISA (C). The values were mean ± SEM with n=5/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Effect of CBD on M1- and M2a-polarized Cytokines and Chemokines

The significant effect of CBD on TNF-α during M1 polarization provided a basis to assess how CBD may impact the secretion of other cytokines and chemokines. Additionally, many cytokines and chemokines are implicated in both MS [18] and EAE [19]. A 32 cytokine/chemokine Luminex kit was utilized to quantify supernatants from M1 and M2a cells. The graphs presented for M1 (Figure 6A) and M2a (Figure 6B) were generated by calculating CBD pre-treatment compared to VH. Briefly, for each mediator, VH values were averaged from five replicate wells then the associated CBD values from individual wells were divided by the average VH and multiplied by 100%. Since IFN-γ and IL-4 were part of the stimulation cocktails for M1 and M2a polarization, they were removed from the respective data analysis. As a result, the total number of mediators analyzed was 31 cytokines/chemokines for each phenotype. For the M1 polarization, CBD significantly decreased the secretion of 20 cytokines and chemokines. In comparison, CBD treatment with the M2a phenotype significantly decreased 12 of the 31 mediators and substantially increased VEGF.

Figure 6.

Figure 6.

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Pre-treatment with CBD significantly affected the secretion of various cytokines and chemokines in response to M1 or M2a polarization. Murine macrophages (RAW 264.7) were pre-treated with VH (0.1% ethanol) or CBD (10 μM) for 30 min and polarized with either M1 (IFN-γ+LPS) or M2a (IL-4) stimuli. After 24 hr, supernatants were harvested and analyzed with a 32 cytokine/chemokine Luminex kit. A calculation generated the graph to determine the percent secretion under CBD pre-treatment compared to VH. Briefly, for each mediator, VH values were averaged. Associated CBD values were divided by the average VH and multiplied by 100%. The resulting values were mean percentages ± SEM with n=5/group. A t-test was performed for each mediator where VH was compared to CBD. Since M1 and M2a were polarized by cytokines IFN-γ and IL-4, respectively, they were removed from the M1 and M2a data analysis. This resulted in 31 cytokines/chemokines analyzed for each phenotype. M1 polarization (A), M2a polarization (B). *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

CBD Induced Intracellular TACE Staining in Naive and M1-polarized Macrophages

CBD treatment significantly increased TNF-α surface retention during M1 polarization but decreased TNF-α secretion. Therefore, it was necessary to determine the effect of CBD on TACE. For this analysis, localization studies were developed to determine whether TACE was present on the inside or outside of the cell since extracellular TACE would be necessary to cleave TNF-α from the cell surface. We included WGA to define the extracellular matrix. The immunofluorescence assay was performed on naive (Figure 7) and M1-polarized (Figure 8) cells treated with VH or CBD. Macrophages treated with CBD exhibited intense TACE staining in both naive and M1 phenotypes compared to VH-treated naive and M1 macrophages. In the VH-treated M1 macrophages, there was evidence of colocalization with the extracellular matrix stain (Texas Red WGA), as observed in Figures 7B and 8B. In contrast, upon CBD treatment in both naive and M1-polarized cells,TACE was concentrated in the cytoplasm and around the nucleus.

Figure 7.

Figure 7.

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Pre-treatment with CBD increased the intensity of TACE staining in naive RAW264.7 macrophages. RAW264.7 macrophages were pre-treated with VH (0.1% ethanol) or CBD (10 μM) for 30 min and left unstimulated (naive). Cells were stained with Hoechst (nucleus viewed under DAPI filter), WGA (extracellular matrix viewed under Texas Red filter), and TACE (viewed under GFP filter). Figures show DAPI+TACE stains (A), WGA+TACE stains (B), and DAPI+WGA+TACE (C). Bar, 30 μm. Results are representative of ≥ 3 wells.

Figure 8.

Figure 8.

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Pre-treatment with CBD increased the intensity of TACE staining in M1 polarized RAW 264.7 macrophages. RAW264.7 macrophages were pre-treated with VH (0.1% ethanol) or CBD (10 μM) for 30 min and polarized under M1 (IFN-γ+LPS) conditions. Cells were stained with Hoechst (nucleus viewed under DAPI filter), WGA (extracellular matrix viewed under Texas Red filter), and TACE (viewed under GFP filter). Figures show DAPI+TACE stains (A), WGA+TACE stains (B), and DAPI+WGA+TACE (C). Bar, 30 μm. Results are representative of ≥3 wells.

CBD Suppressed LPS-Induced RelA Translocation to the Nucleus

Activation of NF-κB plays a substantial role in inducing TNF-α [20] and other cytokines involved in EAE and MS [21, 22]. In fact, of the 32 cytokines and chemokines assessed using Luminex above, all but IL-3, IL-4, IL-5, IL-7 and LIF have been shown to be controlled by NF- κB [23]. In LPS-treated microglial cells, CBD interfered with NF-κB by reducing phosphorylated-p65 (RelA), a subunit of NF-κB, below control levels [24]. However, the kinetics of RelA (p65) translocating to the nucleus due to CBD pre-treatment has not been observed. In this experiment, G9 RAW 264.7 cells transfected with GFP-RelA were assessed under naive and LPS stimulation and treated with either VH or CBD. The percent positive (percentage of cells with RelA translocation) were calculated over a 100-min stimulation time with LPS. Results show that the percent positive for VH+LPS was significantly greater than CBD+LPS (Figure 9).

Figure 9.

Figure 9.

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CBD decreased LPS-induced RelA translocation to the nucleus. RAW 264.7 cells transfected with GFP-RelA (G9 cells) were pre-treated with VH (0.1% ethanol) or CBD (10 μM) for 30 min. Each group had n=6 wells that were then divided into naive or LPS (100 ng/mL). The percent positive was calculated every ten min for 100 min, and the time points were plotted to reflect mean ± SEM. Statistical significance was determined by calculating AUC and performing a t-test between VH vs. CBD and VH+LPS vs. VH+CBD. The VH+LPS group had significantly increased percent positive as compared to CBD+LPS at a significance of p<0.0001.

DISCUSSION

Macrophage polarization towards the M1 phenotype has become a prominent indicator of inflammation. This disturbance is evident in various neuroinflammatory diseases, including MS and EAE [25, 26]. As CBD has shown some efficacy in EAE disease models [2–7, 27], it was of interest to examine the effects of CBD on macrophage polarization. The data here show that CBD significantly impacted macrophage polarization by predominantly dampening responses under M1 polarizing conditions while only modestly affecting responses under M2a polarizing conditions. The data also showed that CBD significantly suppressed TNF-α secretion but increased the expression of intracellular and surface TNF-α in response to M1 polarization. This decreased TNF-α secretion could be due to delayed nuclear translocation of the RelA protein of the NF-κB complex as NF-κB drives TNF-α transcription [20] and/or suppression of extracellularly-expressed TACE, which is required to generate soluble TNF-α.

Together these data suggest that CBD might exhibit benefits in (neuro)inflammatory diseases through M1/M2 balance and inhibition of TNF-α. Specifically, for EAE and MS, however, the benefit of inhibiting TNF-α is not clear. The injectable that blocks the TNF receptor (via an immunoglobulin fusion protein) has been shown to worsen MS [28] but improve EAE [29]. Thus, if CBD is to be considered as a putative therapeutic for MS, it is imperative to understand the relationship between CBD and TNF-α and if CBD could be a viable alternative for TNF-α reduction. The locations of TNF-α may provide additional insights into potential pro-inflammatory and anti-inflammatory mechanisms. Soluble TNF-α (solTNF-α) has a preferential affinity for the TNF-α receptor, TNFR1 [30], which activates the NF-κB pathway [31] and is proapoptotic as it possesses an intracellular death domain [32]. In contrast, transmembrane TNF-α (tmTNF-α) has a higher affinity for the TNFR2 receptor [33], which lacks a death domain and is pro-survival [32]. In EAE, TNFR2 is a vital receptor as it is increased in spinal cord macrophage/microglia [34]. Additionally, remyelination might depend on the interaction between tmTNF-α and TNFR2 as receptor ablation in oligodendrocytes and treatment with a solTNF-α inhibitor was associated with disease exacerbation and reduced remyelination [35]. Therefore, CBD-associated increases in tmTNF-α and decreased solTNF-α may benefit EAE, and possibly MS, disease.

In addition, the augmented surface TNF-α by CBD might be mediated by increased intracellular TACE, which was observed in both naive and M1 conditions. Although it is unclear how CBD is contributing to this effect, there are several intracellular mechanisms that CBD might influence as TACE undergoes maturation to gain proteolytic activity and is trafficked from the endoplasmic reticulum to the cell membrane [36]. Interestingly, it has been shown that NF-κB also plays a role in TNF-α-mediated induction of TACE promoter activity in synovial cells suggesting that TNF-α regulates itself [37]. Given this phenomenon, CBD might inhibit TACE activity, which would be a potential treatment for various autoimmune diseases, including MS [38].

Although CBD primarily exerted anti-inflammatory effects, a potential adverse effect was the slight increase in CBD-induced percent iNOS in M1 macrophages, which appears controversial in MS and EAE. While both Arginase-1 and iNOS enzymes compete for the substrate, arginine, iNOS is incidental to inflammation and infection control, while Arginase-1 is correlated with tissue repair and anti-inflammatory mechanisms [39]. This evidence potentially presents an issue with CBD usage as arginase-1 diverts iNOS from utilizing arginine and increases the production of nitric oxide’s pro-inflammatory mediator. In MS, iNOS appears to have a pathological role as iNOS mRNA and the iNOS enzyme was present in MS brain extracts but absent in control tissue [40]. However, in EAE, iNOS inhibition with L-NAME [41] or knocking it out entirely with iNOS knock-out mice [42] increased the severity of the disease. Therefore, the long-term implication of CBD use and iNOS activity in these pathologies requires further investigation.

It was interesting that many of the markers associated with M1 or M2a polarization were not necessarily induced by their respective stimuli. For example, both TLR4 and iNOS are associated with inflammation and therefore were categorized as M1 markers, but were not preferentially induced using IFN-γ+LPS over IL-4. This likely reflects the plasticity in macrophages, which are susceptible to changes in the microenvironment [43]. It is important to appreciate the plasticity of macrophages and that their responses to drugs and chemicals will be influenced not only by the type of drug or chemical, but the concentration, timing of exposure relative to stimulation, type and magnitude of stimulation, and metabolic state of the cell. Thus, all of these considerations will be important in establishing CBD’s potential for immune-mediated disease states.

This is not the first investigation of CBD on macrophages. For instance, CBD (5–25 μM) treatment of human THP-1 macrophages suppressed several pro-inflammatory cytokines including TNF-α, and the mechanism was determined to involve autophagy [44]. Importantly, the authors found that CBD did not exhibit cytotoxicity in these cells at concentrations below 25 μM [44]. CBD and other cannabinoids (5 μg/ml; for CBD ~15 μM) were also found to suppress cytokine production in LPS-stimulated peritoneal macrophages [45]. CBD (3 μM) suppressed LPS-stimulated cytokines, again including TNF-α, in mouse MH-S alveolar macrophages [46]. In the latter study it was demonstrated that the CBD-mediated inhibition was due to inhibition of NF-κB [46].

Overall, these data demonstrated that the mechanism by which CBD suppressed inflammation, specifically that produced by TNF-α, was due to inappropriate subcellular localization of TACE. It is also likely that the altered kinetics of RelA nuclear localization contributed to CBD’s anti-inflammatory effects. Together these data suggest that a generalized mechanism for CBD’s immune suppression is altered subcellular localization of two critical components for TNF-α secretion, TACE and RelA. Perhaps these mechanisms that contribute to suppression of TNF-α by CBD will allow it to be used therapeutically for MS or other inflammatory diseases.

Supplementary Material

1

HIGHLIGHTS.

CBD preferentially suppressed markers and proteins in response to M1 polarization.

CBD induced intracellular localization of TACE, preventing release of surface TNF-α.

CBD prevented the NF-κB protein, RelA, from translocating to the nucleus.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health P20GM103646. The authors also acknowledge Dr. Nogi Park at MSU CVM for his help with RAW G9 cell culture.

ABBREVIATIONS

CBD

cannabidiol

EAE

experimental autoimmune encephalomyelitis

MS

multiple sclerosis

TACE

tumor necrosis factor-alpha converting enzyme

VH

vehicle

WGA

wheat germ agglutinin

Footnotes

CRediT author statement

Conceptualization – CMF, LL, WT, SBP, BLFK

Investigation – CMF, LL, WT

Data curation—formal analysis - CMF

Project Administration/oversight - BLFK

Writing—original draft - CMF

Writing—review, editing, and revision – CMF, LL, WT, SBP, BLFK

CONFLICTS OF INTEREST DISCLOSURE

No conflicts of interest.

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