cbd oil for acne inflammation pubmed

The Therapeutic Potential of Cannabinoids in Dermatology

Cannabinoids have demonstrated utility in the management of cancer, obesity, and neurologic disease. More recently, their immunosuppressive and anti-inflammatory properties have been identified for the treatment of several dermatologic conditions. This review thus assesses the therapeutic potential of phytocannabinoids, endoocannabinoids, and chemically synthetic cannabinoids in the management of cutaneous disease. The PubMed® and Scopus® databases were subsequently reviewed in December 2017 using MeSH and keywords, such as cannabinoid, THC, dermatitis, pruritus, and skin cancer. The search yielded reports on the therapeutic role of cannabinoids in the management of skin cancer, acne vulgaris, pruritus, atopic and allergic contact dermatitis, and systemic sclerosis. While cannabinoids have exhibited efficacy in the treatment of inflammatory and neoplastic skin conditions, several reports suggest pro-inflammatory and pro-neoplastic properties. Further investigation is necessary to understand the complexities of cannabinoids and their therapeutic potential in dermatology.

Keywords: acne; cannabinoid; cannabis; dermatitis; endocannabinoid; fibrosis; palmitoylethanolamide; inflammatory skin disease; pruritus; skin cancer; sclerosis; THC; tetrahydrocannabinol.

Conflict of interest statement

Adam Friedman is currently developing a nanoparticle encapsulated cannabinoid with Zylo Therapeutics – this work is not referenced in the manuscript. Dustin Marks has no conflicts of interest to report for this work. Funding: The George Washington Department of Dermatology received no funding in support of this manuscript.

The ameliorative effect of hemp seed hexane extracts on the Propionibacterium acnes-induced inflammation and lipogenesis in sebocytes

In this study, we investigated the anti-microbial, anti-inflammatory, and anti-lipogenic effects of hemp (Cannabis sativa L.) seed hexane extracts, focusing on the Propionibacterium acnes-triggered inflammation and lipogenesis. Hemp seed hexane extracts (HSHE) showed anti-microbial activity against P. acnes. The expression of iNOS, COX-2, and the subsequent production of nitric oxide and prostaglandin increased after infection of P. acnes in HaCaT cells, however, upon treating with HSHE, their expressions were reduced. P. acnes-induced expressions of IL-1β and IL-8 were also reduced. HSHE exerted anti-inflammatory effects by regulating NF-κB and MAPKs signaling and blunting the translocation of p-NF-κB to the nucleus in P. acnes-stimulated HaCaT cells. Moreover, P. acnes-induced phosphorylation of ERK and JNK, and their downstream targets c-Fos and c-Jun, was also inhibited by HSHE. In addition, the transactivation of AP-1 induced by P. acnes infection was also downregulated by HSHE. Notably, HSHE regulated inflammation and lipid biosynthesis via regulating AMPK and AKT/FoxO1 signaling in IGF-1-induced inflammation and lipogenesis of sebocytes. In addition, HSHE inhibited 5-lipoxygenase level and P. acnes-induced MMP-9 activity, and promoted collagen biosynthesis in vitro. Thus, HSHE could be utilized to treat acne vulgaris, through its anti-microbial, anti-inflammatory, anti-lipogenic, and collagen-promoting properties.

Introduction

Hemp (Cannabis sativa L.), also known as cannabis, has been an important plant used in dietary supplements, clothing, cosmetics, and medicines [1,2]. Hemp has also been used as a medicament for the treatment of medical conditions such as rheumatic pain, intestinal constipation, disorders of the female reproductive system, and malaria [3]. However, due to its hallucinogenic effects, the use of cannabis has been legally restricted in almost all countries of the world, except for some countries, such as the US that partially allow it for medicinal use. In US, with a doctor’s recommendation, the medical use of cannabis is legal in 29 states, the District of Columbia, and the territories of Guam and Puerto Rico. Seventeen other states allow the medical use of cannabis with low THC/high CBD. Tetrahydrocannabinol (THC) is the major psychoactive component of cannabis, even small amounts of which could be highly hallucinogenic, while cannabidiol (CBD) is the non-psychoactive component of cannabis. THC binds cannabinoid CB1 and CB2 receptors with high affinity and activates them, while CBD has a very low affinity for both CB1 and CB2 receptors, and acts as an indirect antagonist of these receptors [1,4]. THC is effective for relieving pain, inhibiting inflammation, and relaxing muscle tension. Thus, it can be used for patients with multiple sclerosis, neurodegenerative disorders, and severe pain. However, overdose of THC may cause serious mental side effects of hallucinations [5,6]. On the contrary, hemp seed does not have any psychotropic chemical components. Moreover, hemp is the only seed plant without any saturated fatty acids, containing mostly polyunsaturated fatty acids including linoleic acid and gamma-linolenic acid [7].

Acne vulgaris is observed in 85% of adolescents and refers to a disease that causes inflammation of sebaceous glands attached to hairfollicles. Propionibacterium acnes (P. acnes), present on normal skin, activates an immune response and changes the lipid composition of the sebaceous glands [8,9]. P. acnes stimulates the inflammatory cytokines such as interleukin (IL)-1β, IL-8, and leukotrienes by activating Toll-like receptors (TLRs) and induces the expression of enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), causing chronic inflammation [10–12]. It also activates the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signals, and upregulates related genes to carry out innate immunity.

Keratinocytes and dermal fibroblasts secrete matrix metalloproteinases (MMPs) which mediate the digestion of many components of extracellular matrix (ECM) in response to external stimuli such as bacterial infection, oxidative stress, and UV radiation. MMPs are classified into five main subgroups, such as collagenases, gelatinases, stromelysins, matrilysins, and 5 membrane-type MMPs, based on their structure and substrate specificity. MMP-1 is a collagenase that digests fibrillar collagen via recognition of a hemopexin-like domain, while MMP-2 and MMP-9 are gelatinases that degrade a number of ECM components, such as type I and IV collagen [13,14].

Human sebocytes are specialized sebum-producing epithelial cells that produce and release lipid droplets. Sebum is an integral component of the epidermal barrier and its formation is involved in the skin immune system. Excess sebum production causes acne by inducing an inflammatory reaction under the proliferating skin microflora [15]. Peroxisome proliferator-activated receptor gamma (PPARγ) present in sebaceous gland cells regulates the lipid production and lipid metabolism via modulation of the AMP-activated protein kinase (AMPK) signal. AMPK is a serine/threonine kinase that plays a regulatory role in glucose and lipid metabolism [16]. Activation of AMPK by phosphorylation down-regulates the expression of fatty acid synthase (FAS) and sterol regulatory element-binding protein 1 (SREBP-1), by inhibiting phosphorylation of mTOR [17,18]. Upon activation by insulin-like growth factor-1 (IGF-1), protein kinase B (AKT) phosphorylates fork head box protein O1 (FoxO1) to enhance lipogenesis. FoxO1 is reported to directly bind and modulate PPAR-γ function [19]. Additionally, 5-lipoxygenase induces the release of leukotriene (LT)-B4, a pro-inflammatory lipid, which promotes lipid synthesis in acne lesions. Thus, inhibition of 5-lipoxygenase may be an ideal target for downregulation of inflammation and lipid accumulation in sebocytes [20,21].

Hemp seeds have been primarily used in nutraceutical and pharmaceutical industries, utilizing their health-promoting properties and ideal nutrient content. However, information on its anti-acne activity and underlying mechanism is limited. In this study, the ameliorative effects of hemp seed hexane extracts on P. acnes-induced inflammation in HaCaT cells and IGF-1-stimulated lipogenesis in sebocytes were examined at the molecular and cellular level.

Materials and methods

Preparation of hemp seed hexane extracts

Hemp seeds imported from Canada were purchased from an online Korean store called Nooriwon. The dried seeds were ground and soaked with three volumes of hexane for 24 h while stirring. The extract was serially filtered using a No. 2 filter and a nylon filter (0.45 μm) (both from Whatman International Ltd., UK). The extraction procedure was repeated twice with the residue. The solvent was evaporated using a rotary vacuum evaporator (Eyela, Japan) at 35°C. Residual solvent was re-evaporated twice at 70°C for 10 min. To increase the solubility of HSHE in the cell culture media, same volume of dimethyl sulfoxide (DMSO) was added to the hexane-extracted hemp seed oil and mixed by vortexing at room temperature. All experiments were performed under conditions that did not show toxicity by hemp seed hexane extract and DMSO [22].

Microbial cultivation and anti-acne activity determination

Propionibacterium acnes (P. acnes, KCTC) was obtained from the Korean Culture Center of Microorganisms, Seoul, Korea. P. acnes was grown anaerobically in solid and liquid Reinforced Clostridial Medium (RCM) at 37°C for 72 h. P. acnes were harvested via centrifugation of the cultures at 4,500 rpm for 15 min at 4°C. The resulting bacterial pellets were pooled, washed in cold 1× PBS, and centrifuged again. Finally, the pooled bacterial pellets were resuspended in serum free medium (bacterial concentration was 1.2X10 9 CFU/mL). The suspension was heated at 80°C and the heat-killed bacteria were used for the stimulation experiments reported previously by us [9].

To determine anti-acne activity of the HSHE, the culture of P.acnes was standardized using microbiological No. 0.5 McFarland Standard’s solution according to the recommendations of the Clinical Laboratory Standards Institute (CLSI) [23]. The anti-microbial effect of HSHE was measured on agar medium. 200 μL cell suspension (10 5 cells/mL) was mixed with 100 μL of HSHE diluted in RCM medium at 37°C for 30 min. The control was mixed with 100 μL medium and reacted under the same conditions. Then, 100 μL of each mixture was spread on the surface of RCM medium on a petri dish. Both control and experimental groups were incubated at 37°C for 72 h.

Cell culture

The human keratinocyte (HaCaT) and human fibroblast cell lines (Hs68) were obtained from the American Type Culture Collection (ATCC). The cells were incubated in complete Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) containing 100 U/mL penicillin, streptomycin (100 μg/mL), and 10% fetal bovine serum (FBS) at 37°C [24,25]. Primary human sebocytes (Celprogen Inc., USA) were maintained in Human Sebocyte Complete Growth Media purchased from the same vendor. For HSHE treatment, cells were seeded and incubated overnight prior to the treatments. For the stimulation experiment, HaCaT and Hs68 cells were incubated with heat-killed P. acnes adjusted at the appropriate concentration in serum free media for 24 h at 37°C in 5% CO2. After stimulation, the HaCaT cells were treated with or without hemp seed hexane extracts for 24 h at 37°C in 5% CO2. Sebocytes were pre-treated with or without hemp seed hexane extracts for 2 h. Then, 120 ng/mL IGF-1 was added to each plate and the cells were incubated for 22 h [9].

NO production measurement

The nitrite concentration in conditioned medium was measured as an indicator of NO production according to the Griess reaction. Each supernatant (100 μL) was mixed with the same volume of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthyl ethylenediamine dihydrochloride in distilled water). The absorbance of the mixture was determined with an ELISA reader (Sunrise, Tecan, Switzerland) at 570 nm [9, 26].

Enzyme-Linked Immunosorbent Assay (ELISA)

The effects of HSHE on IL-8 and prostaglandin E2 (PGE2) productions in heat-killed P. acnes-infected HaCaT cells and on the 5-lipoxygenase production in sebocytes, stimulated with IGF-1, were measured with ELISA [27]. The IL-8 (BD Science, USA), PGE2 (LSBio, USA) and 5-lipoxygenase (MyBioSource, USA) concentrations were calculated according to the standard curve using standard in the ELISA kit.

Western blot analysis

Proteins were separated by 10% SDS-PAGE, and transferred onto polyvinylidene fluoride membrane (Bio-Rad Laboratories, CA) [28]. After blocking for 2 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against iNOS, IL-1β, COX-2, IKKα, IKKβ, p-IKKα/β, IκBα, p-IκBα, NF-κB p65, p-NF-κB p65, p38, p-p38, ERK, p-ERK, JNK, p-JNK, AKT, p-AKT, mTOR, p-mTOR, PPARγ, FAS, AMPKα, p-AMPKα, FoxO1, and p-FoxO1 (Cell Signaling Technology, USA), SREBP1 (Novus Biologicals, Canada) and β-actin (Santa Cruz Biotechnology, USA) which were diluted using manufacturers’ recommendations [25,29]. The membranes were then washed in 1× TBST and incubated with the appropriate secondary antibody HRP-conjugated (1:5000) at room temperature for 1 h. Protein bands were visualized using the Sensi-Q 2000 (Lugen, South Korea). The intensity of the bands was analyzed using ImageJ and normalized against β-actin [30].

Transient transfection and luciferase assay

The effects of HSHE on AP-1 activity were assayed in a Luciferase Reporter Assay System (Promega, Madison, WI, US) [8]. Upon reaching 60–70% confluency, the HaCaT cells were washed in PBS and the cells were then transfected with AP-1-Luc reporter vector (Affymetrix, Santa Clara, CA, US) using Fugene 6 (Promega, Madison, WI,US), according to the manufacturer’s protocol. After the 24 h transfection, cells were infected with P. acnes, medium was changed, and treated with various concentrations of hemp seed hexane extracts. The processed cells were cultured for an additional 24 h. Cells were washed with PBS, lysed with lysis reagent, and treated with the luciferase assay substrate. Luciferase activity was determined using the luminometer (Infinite F200 PRO, Tecan, Männedorf, Switzerland).

Confocal microscope analysis and gelatin zymography

Confocal microscopic analysis for NF-κB translocation and gelatin zymographic analysis of MMP activity on Hs68 cells was performed as described in our previous report [9].

Collagen synthesis-promoting assay

Collagen contents in the ECM of Hs68 cells were determined by Sircol collagen assay (Biocolor, UK) according to the manufacturer’s protocols. Collagen dye complexes formed in cell supernatants were added to the Sircol staining reagent and precipitated in soluble non-binding dyes. After centrifugation, the pellet was washed with ice-cold acid-salt wash reagent and reacted with alkali reagent. Samples were dispensed into a 96-well plate and the absorbance was read at 570 nm. The amount of collagen was calculated based on a standard curve obtained with the standard bovine type I collagen supplied with the kit [31].

Data analysis

Data was analyzed using the IBM Statistical Package for Social Sciences (SPSS, version 20). All the data were presented as mean ± standard deviation (SD) of triplicate experiments. One-way analysis of variance (ANOVA) with a Duncan multiple-comparison test was utilized to determine the statistical differences among groups. P-values <0.05 were considered statistically significant [32].

Results

Anti-microbial effect of hemp seed hexane extracts on P. acnes

At first, we investigated the anti-microbial effect of HSHE against P. acnes ( Fig 1 ). P. acnes was treated with 0, 15, 20 and 25% HSHE and the number of colonies was counted to examine anti-microbial activity against P. acnes of HSHE. 15 and 20% HSHE showed approximately 59% and 99% of anti-microbial activity compared to control. At 20% HSHE, complete inactivation of P.acnes was observed. Erythromycin (3 ppm), a conventional anti-microbial agent for acne, showed approximately 67% anti-microbial activity (Data not shown). These results suggest that HSHE is able to inactivate the growth of P. acnes.

All data are presented as mean±SD of three independent experiments. *p<0.05 indicates significant differences compared to control group.

Inhibitory effect of HSHE on P. acnes-induced inflammation in HaCaT cells

To investigate the effect of HSHE on the expression of inflammatory enzymes iNOS and COX-2, and pro-inflammatory cytokine IL-1β, western blotting was performed with P.acnes-infected HaCaT cells. As shown in Fig 2A , the expression of iNOS and COX-2 was enhanced upon the stimulation of P. acnes, however, HSHE down-regulated the induced expression of iNOS and COX-2. Moreover, the expression of P.acnes-induced IL-1β was significantly inhibited at 0.6% HSHE. In addition, HSHE significantly suppressed the production of NO caused by iNOS in P. acnes-infected HaCaT cells in a dose dependent manner ( Fig 2B ). At 0.6% HSHE, about 40% of NO production was inhibited, compared to the P. acnes-induced inflammation group. Prostaglandin E2 (PGE2), which was generated by COX-2, was also notably inhibited by HSHE ( Fig 2C ). The ELISA results showed that there was a reduction in pro-inflammatory cytokine IL-8 secretion caused by HSHE ( Fig 2D ). These results indicate that HSHE exert anti-inflammatory activity via regulating inflammation-related enzymes, their products, and pro-inflammatory cytokine secretion.

(A) Effects of hemp seed hexane extracts (HSHE) on iNOS, COX-2 and IL-1β expression in P. acnes-stimulated HaCaT cells. The expressions of iNOS, COX-2, and IL-1β were analyzed with ImageJ and normalized against β-actin. (B) Effects of HSHE on NO production in P. acnes-stimulated HaCaT cells. ELISA results demonstrate that HSHE reduced PGE2 (C) and IL-8 (D) in P. acnes-infected HaCaT cells. ELISA results demonstrate that HSHE reduced in P. acnes-infected HaCaT cells. All data are expressed as mean ± SD. *P<0.05 compared with P. acnes treated cells only.

Next, the anti-inflammatory effect of HSHE was investigated based on the NF-κB signaling pathway ( Fig 3 ). The expression levels of P.acnes-induced phosphorylated p-IKKα/β, p-IκB-α and p-NF-κB were investigated in P. acnes-stimulated HaCaT cells. The expression of P.acnes-induced phosphorylated IKKα/β, IκBα and NF-κB was significantly down-regulated by the action of HSHE. These results suggest that anti-inflammatory activity of HSHE was involved in suppression of NF-κB signaling pathway in P. acnes-stimulated HaCaT cells. In addition, suppression of nuclear translocation of p-NF-κB in P. acnes-infected HaCaT cells by HSHE was observed via confocal microscopy ( Fig 4 ). The nuclear translocation and accumulation of p-NF-κB in P. acnes-infected HaCaT cells were dramatically reduced upon treatment with HSHE. These results suggest that HSHE exerts anti-inflammatory effects by regulating the NF-κB signaling pathway and inhibiting p-NF-κB nuclear translocation.

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The expression of p-IKKα/β, p-IκB-α and p-NF-κB were analyzed with ImageJ and normalized against β-actin. Results are expressed as mean ± SD. *P<0.05 compared with P. acnes treated cells only.

Cannabidiol exerts sebostatic and antiinflammatory effects on human sebocytes

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Balázs I. Tóth

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

István Borbíró

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Koji Sugawara

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Attila G. Szöllõsi

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Gabriella Czifra

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Balázs Pál

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Lídia Ambrus

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Jennifer Kloepper

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Emanuela Camera

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Matteo Ludovici

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Mauro Picardo

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Thomas Voets

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Christos C. Zouboulis

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Ralf Paus

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Tamás Bíró

1 DE-MTA “Lendület” Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 2 Laboratory for Ion Channel Research and TRP Research Platform Leuven, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. 3 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan. 4 Department of Dermatology, University of Lübeck, Lübeck, Germany. 5 Neurobiology Research Group, Department of Physiology, University of Debrecen, Debrecen, Hungary. 6 Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy. 7 Departments of Dermatology, Venereology, and Allergology and Immunology, Dessau Medical Center, Dessau, Germany. 8 School of Translational Medicine, University of Manchester, Manchester, United Kingdom.

Associated Data

Abstract

The endocannabinoid system (ECS) regulates multiple physiological processes, including cutaneous cell growth and differentiation. Here, we explored the effects of the major nonpsychotropic phytocannabinoid of Cannabis sativa, (-)-cannabidiol (CBD), on human sebaceous gland function and determined that CBD behaves as a highly effective sebostatic agent. Administration of CBD to cultured human sebocytes and human skin organ culture inhibited the lipogenic actions of various compounds, including arachidonic acid and a combination of linoleic acid and testosterone, and suppressed sebocyte proliferation via the activation of transient receptor potential vanilloid-4 (TRPV4) ion channels. Activation of TRPV4 interfered with the prolipogenic ERK1/2 MAPK pathway and resulted in the downregulation of nuclear receptor interacting protein-1 (NRIP1), which influences glucose and lipid metabolism, thereby inhibiting sebocyte lipogenesis. CBD also exerted complex antiinflammatory actions that were coupled to A2a adenosine receptor-dependent upregulation of tribbles homolog 3 (TRIB3) and inhibition of the NF-κB signaling. Collectively, our findings suggest that, due to the combined lipostatic, antiproliferative, and antiinflammatory effects, CBD has potential as a promising therapeutic agent for the treatment of acne vulgaris.

Introduction

Acne vulgaris is the most common human skin disease, affecting quality of life of millions worldwide. In spite of heroic basic and applied research efforts, we still lack indisputably curative anti-acne agents, which target multiple pathogenetic steps of acne (sebum overproduction, unwanted sebocyte proliferation, inflammation) and, moreover, which possess favorable side effect profiles (1, 2). Investigations over the last two decades have confirmed unambiguously that the human body expresses such receptors, which are able to specifically bind and recognize characteristic terpene-phenol compounds of the infamous plant Cannabis sativa, collectively referred to as phytocannabinoids. These receptors, their endogenous ligands (the endocannabinoids [eCBs]), and the enzymes involved in the synthesis and degradation of the eCBs collectively constitute the eCB system (ECS), a complex intercellular signaling network markedly involved in the regulation of various physiological processes (3–6).

Investigation of the cutaneous cannabinoid system seems to be a promising choice when searching for novel therapeutic possibilities (7, 8). Indeed, we have shown previously that the skin ECS regulates cutaneous cell growth and differentiation (9, 10), and it reportedly exerts antiinflammatory effects (11). Of further importance, we have also demonstrated that the ECS plays a key role in the regulation of sebum production (12). According to our recent findings, prototypic eCBs, such as N-arachidonoyl ethanolamide (anandamide [AEA]) and 2-arachidonoylglycerol, are constitutively produced in human sebaceous glands. Moreover, using human immortalized SZ95 sebocytes, we have also demonstrated that these locally produced eCBs (acting through a CB2 cannabinoid receptor→ERK1/2 MAPK→PPAR pathway) induce terminal differentiation of these cells, which is characterized by increased neutral lipid (sebum) production of the sebocytes (12). These findings confirmed unambiguously that human sebocytes have a functionally active ECS; yet, we did not possess data on the potential effect(s) of plant-derived cannabinoids.

(-)-Cannabidiol (CBD) is the most studied nonpsychotropic phytocannabinoid (13–15). It has already been applied in clinical practice without any significant side effects (Sativex) (16), and numerous ongoing phase II and III trials intend to explore its further therapeutic potential (17). Hence, within the confines of the current study, we intended to reveal the biological actions of CBD on the human sebaceous gland. Since we lack adequate animal models (18), we used human immortalized SZ95 sebocytes, the best available cellular system (19), and the full-thickness human skin organ culture (hSOC) technique (20).

Results

CBD normalizes “pro-acne agent”–induced excessive lipid synthesis of human sebocytes.

We first assessed the biological effects of CBD (1–10 μM) on the lipogenesis of SZ95 sebocytes. Although eCBs are known to show intense lipogenic actions via the metabotropic CB2 receptors (12), neither semiquantitative Oil Red O nor quantitative Nile Red staining indicated changes in the basal neutral (sebaceous) lipid synthesis of SZ95 sebocytes following 24-hour CBD treatment (Figure ​ (Figure1, 1 , A–C) (or 48-hour CBD treatment; data not shown). Intriguingly, however, CBD markedly inhibited the lipogenic action of the prototypic eCB, AEA, in a dose-dependent manner (1–10 μM; Figure ​ Figure1, 1 , C–E).

(A, B, D, and E) Semiquantitative determination of lipid synthesis for (A) control, (B) 10 μM CBD, (D) 30 μM AEA, and (E) 30 μM AEA plus 10 μM CBD (sebum droplets: Oil Red O staining, red; nuclei: hematoxylin, blue). Scale bars: 10 μm. (C) Quantitative determination of lipid synthesis (Nile Red staining). **P < 0.01, ***P < 0.001 compared with the AEA-treated group. PL, polar lipids; NL, neutral lipids. (F) Neutral lipid synthesis (Nile Red staining). ***P < 0.001 compared with the respective AA- or LA-T–treated group. (C and F) Data are expressed as the percentage of the vehicle control (mean ± SEM of 4 independent determinations). The solid line indicates 100%. Two additional experiments yielded similar results. (G) Analysis of the sebaceous lipidome. Sebaceous lipid classes were analyzed by HPLC-ToF/MS in sebocytes. CH, free cholesterol; CE, cholesteryl esters; WE, wax esters; DG, diacylglycerols; TG, triacylglycerols; SQ, squalene; FFA, free fatty acids. Results are expressed as the percentage of the vehicle control (mean ± SD of 3 independent determinations). The solid line indicates 100%. Two additional experiments yielded similar results. *P < 0.05, **P < 0.01, ***P < 0.001.

We also tested its effect on actions of other lipogenic substances, which were shown previously to act through different, ECS-independent signal transduction mechanisms. Indeed, CBD effectively inhibited lipid synthesis induced by either arachidonic acid (AA) (21) or the combination of linoleic acid and testosterone (LA-T) (ref. 22 and Figure ​ Figure1F), 1 F), indicating that the effect of CBD is not “ECS specific” but a “universal” lipostatic action.

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Since cannabinoids have been very often shown to exert “biphasic” effects (i.e., opposing physiological actions at nM vs. μM concentrations) (23), we also tested the effects of lower (1–100 nM) CBD concentrations; notably, they did not influence either basal or AA-induced lipid synthesis of the sebocytes (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI64628DS1).

We also investigated the effects of CBD on the lipidome of SZ95 sebocytes under in vitro conditions that mimicked “acne-like” circumstances (the latter was achieved by using a key “pro-acne” inflammatory mediator, AA) (1, 2, 21, 24–26). Importantly, CBD almost completely normalized the AA-enhanced “pathological” lipogenesis of SZ95 sebocytes (Figure ​ (Figure1G). 1 G). This suggests that CBD may primarily normalize both quantitatively and qualitatively excessive and abnormal lipid production induced by acne-promoting stimuli.

CBD decreases proliferation, but not the viability, of human sebocytes both in vitro and ex vivo.

Besides the above lipostatic action, another desired effect of a proper anti-acne agent would be to inhibit the unwanted growth of sebocytes (2, 27, 28). Of great importance, proliferation of SZ95 sebocytes was significantly reduced in the presence of CBD (1–10 μM) (Figure ​ (Figure2A). 2 A). It should be noted, however, that CBD did not suppress the cell count below the “starting” number (measured at day 1), arguing for a “pure” antiproliferative effect. Indeed, the lack of its effects on the count of viable cells was further verified by showing that these concentrations of CBD did not decrease cellular viability or induce either apoptosis or necrosis of SZ95 sebocytes (Figure ​ (Figure2, 2 , B and C). Notably, administration of 50 μM CBD evoked apoptosis-driven cytotoxicity and, hence, led to decreased lipogenesis (Supplemental Figure 2, A–C). Likewise, elongated application of 10 μM CBD (6-day treatments) also decreased cell number and lipogenesis (Supplemental Figure 2, D and E).

(A) CyQUANT proliferation assay after 72-hour treatments. *P < 0.05, ***P < 0.001 compared with the 72-hour vehicle control. The solid line indicates the level of the 24-hour vehicle control. (B) MTT assay. Viability of sebocytes following 48-hour treatments. (C) Cell death [DilC1(5) and SYTOX Green double labeling] assays after 24-hour treatments. (AC) Results are expressed as the percentage of the vehicle control (mean ± SEM of 4 independent determinations). The solid line indicates 100%.Two additional experiments yielded similar results. (DG) hSOC of (D) control, (E) 10 μM CBD, (F) 30 μM AEA, and (G) 30 μM AEA plus CBD 10 μM (14 days; sebum: Oil Red O staining, red; nuclei: hematoxylin, blue). Scale bars: 50 μm. (H) Statistical analysis of the lipid production on 4 histological sections per group. Results are expressed as mean ± SEM. **P < 0.01. (I) Statistical analysis of the number of MKI67 + cells as compared with the number of DAPI + cells on 2 histological sections per group (hSOC; 48 hours). **P < 0.01 compared with the vehicle control. Results are expressed as mean ± SEM.

Clinically, the key question is whether the above in vitro observations could be translated into significant sebostatic (i.e., lipostatic and antiproliferative) effects of CBD on human sebaceous glands in situ. To explore this on the preclinical level, the full-thickness hSOC technique (20) was used. These hSOC assays, which mimic the human sebaceous gland function in vivo as closely as this is currently possible on the ex vivo level, clearly demonstrated that application of CBD completely prevented the lipogenic action of AEA in situ and, in line with our long-term in vitro observations (Supplemental Figure 2E), decreased basal lipogenesis as well (Figure ​ (Figure2, 2 , D–H). Likewise, CBD markedly suppressed the expression of the proliferation marker MKI67 (Figure ​ (Figure2I). 2 I). This suggests that CBD may also operate as a potent sebostatic agent in vivo when tested in appropriate clinical trials.

CBD exerts universal antiinflammatory actions.

We additionally found that CBD also prevented the “pro-acne” LA-T combination from elevating the expression of TNFA (Figure ​ (Figure3A), 3 A), a key cytokine in the pathogenesis of acne vulgaris (2, 24–30). These data suggested that CBD may exert antiinflammatory actions on human sebocytes (as had already been demonstrated for CBD in several other experimental models, such as diabetes, rheumatoid arthritis, etc.) (31). Therefore, in order to confirm the putative universal antiinflammatory action of the CBD on human sebocytes, we next assessed its effects by modeling both Gram-negative infections (applying the TLR4 activator LPS) and Gram-positive infections (using the TLR2 activator lipoteichoic acid [LTA]). We found that CBD completely prevented the above treatments from elevating TNFA expression (Figure ​ (Figure3). 3 ). Moreover, CBD also normalized LPS-induced IL1B and IL6 expression (Figure ​ (Figure3B) 3 B) (expression of these 2 cytokines was found not to be modulated by 24-hour LA-T or LTA treatment; data not shown). Taken together, these results strongly suggest that CBD’s universal sebostatic action is accompanied by substantial antiinflammatory effects, which would be very much desired in the clinical treatment of acne vulgaris (1, 2, 24–30).

(A) TNFA mRNA expression following 24-hour “pro-acne” lipogenic and TLR agonist treatments with or without CBD. *P < 0.05 compared with the corresponding CBD-free treatments. (B) IL1B, IL6, and TNFA mRNA expression following 24-hour LPS treatment with or without CBD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the corresponding CBD-free treatments. (A and B) Data are presented using the ΔΔCT method; GAPDH-normalized mRNA expression of the vehicle control was set as 1 (solid line). Data are expressed as mean ± SD of 3 independent determinations. Two additional experiments yielded similar results.

Sebostatic (i.e., lipostatic and antiproliferative), but not antiinflammatory, actions of CBD are mediated by the activation of transient receptor potential vanilloid-4 ion channels.

Next, we dissected the molecular mechanism(s) that underlie the remarkable lipostatic effects of CBD. As expected, neither CB1- nor CB2-specific antagonists (AM251 and AM630) were able to antagonize the lipid synthesis-inhibitory action of CBD (Supplemental Figure 3); hence, alternative options had to be considered.

First, we studied the effects of CBD on the ionic currents of SZ95 sebocytes. Using whole-cell patch-clamp configurations, membrane currents were elicited by voltage ramp protocols (Figure ​ (Figure4, 4 , A and B) and then normalized to cell membrane capacitance at two different potentials, i.e., at –90 and +90 mV (Figure ​ (Figure4C). 4 C). CBD (10 μM) induced a mostly outwardly rectifying current and a positive shift in the reversal potential, arguing for the activation of certain cation channels upon CBD application.

(A) Representative current-voltage traces of patch-clamp measurement of sebocytes using conventional whole-cell configuration with or without 10 μM CBD. (B) CBD-induced differential current (i.e., CBD minus control). (C) Averaged current densities measured at –90 mV and +90 mV of 7 cells. Results are expressed as mean ± SEM. **P < 0.01 compared with control.

It is well known that various cannabinoids can modulate the activity of certain transient receptor potential (TRP) channels, collectively referred to as “ionotropic cannabinoid receptors” (32–37). Moreover, we have shown recently that activation of TRP vanilloid-1 (TRPV1) on SZ95 sebocytes by capsaicin also exerts potent lipostatic actions (38). Therefore, we first systematically explored these candidate “CBD targets.”

We found that SZ95 sebocytes express TRPV1, TRPV2, and TRPV4 both at the mRNA and protein levels (Supplemental Figure 4, A–C). Among these TRP channels, TRPV4 showed the highest mRNA levels by far (expression of TRPA1 and TRPM8 was below the detection limit; data not shown).

Since the 3 identified TRPs are nonselective cation channels that are most permeable to Ca 2+ (39), we studied the effects of CBD on the calcium homeostasis of the sebocytes. Using a fluorescent Ca 2+ -imaging technique, we found that CBD significantly increased the intracellular calcium concentration ([Ca 2+ ]IC) of SZ95 sebocytes (Figure ​ (Figure5, 5 , A and B). This action was equally antagonized by (a) the decrease of the extracellular Ca 2+ concentration ([Ca 2+ ]EC); (b) the nonspecific TRP channel blocker ruthenium red; and, of great importance, (c) the TRPV4-specific antagonist HC067047 (HC) (Figure ​ (Figure5, 5 , A and B). We have also shown that the suppression of [Ca 2+ ]EC or the coapplication of HC also prevented the lipostatic action of CBD (Figure ​ (Figure5C); 5 C); notably, the TRPV4 antagonist alone did not affect basal lipid synthesis (Supplemental Figure 5).

(A) Fluorescent Ca 2+ imaging. Compounds were applied as indicated by the arrow. Fluorescence (measured in relative fluorescence units [RF]) was normalized to the baseline. “Low [Ca 2+ ]EC” indicates the use of nominally Ca 2+ -free Hank’s solution. Two additional experiments yielded similar results. (B) Statistical analysis of the fluorescent Ca 2+ -imaging data. Fluorescence (expressed in RF) was normalized to the baseline. Measured peak values were expressed as the percentage of the baseline (mean ± SEM of 3 independent determinations). The solid line indicates 100%. Two additional experiments yielded similar results. ***P < 0.001 compared with the CBD-treated group. (C) Neutral lipid synthesis (Nile Red staining). Data are expressed as the percentage of the vehicle control (mean ± SEM of 4 independent determinations). The solid line indicates 100%. Two additional experiments yielded similar results. “Low [Ca 2+ ]EC” indicates the use of low-Ca 2+ Sebomed medium. *P < 0.05, **P < 0.01, ***P < 0.001. (D and E) Neutral lipid synthesis (Nile Red staining) following selective gene silencing of TRPV4 channel (24-hour treatments, started at day 3 after the transfection). Data are expressed as the percentage of the untransfected vehicle control (mean ± SEM of 4 independent determinations). The solid line indicates 100%. Two additional experiments yielded similar results. *P < 0.05, ***P < 0.001 compared with the SCR cells. “siV4a” and “siV4b” mark 2 different siRNA constructs against TRPV4. SCR, scrambled control; UC, untransfected vehicle control.

To further confirm the functional expression of TRPV4 on human sebocytes, the TRPV4-specific ultrapotent agonist GSK1016790A (GSK) was applied. The agonist evoked membrane currents, which were prevented by the specific TRPV4 antagonist HC (Supplemental Figure 6, A and B), indicating that TRPV4 channels are indeed functionally expressed in human sebocytes. Moreover, GSK mimicked both the CBD-induced [Ca 2+ ]IC elevations (Supplemental Figure 6, C and D) and CBD’s lipostatic actions (Figure ​ (Figure5C). 5 C). Since the CBD-evoked lipostatic effects and the induced Ca 2+ signals were not influenced by the TRPV1-specific antagonists, capsazepine (Supplemental Figure 7, A–C) or AMG 9810 (data not shown), these electrophysiological, Ca 2+ -imaging and cellular physiology data collectively argued for the selective involvement of TRPV4 (but not of TRPV1) in mediating the effects of CBD.

To further validate this concept, knockdown of TRPV1, TRPV2, and TRPV4 by RNA interference (RNAi) was used (quantitative “real-time” PCR [Q-PCR] and Western blot analyses verified the successful silencing of the targeted TRPVs; Supplemental Figure 8, A–F). We showed that neither TRPV1 nor TRPV2 silencing significantly influenced the lipostatic action of CBD (Supplemental Figure 9, A and B). In contrast, TRPV4-specific “knockdown” was able to prevent this effect of CBD (Figure ​ (Figure5D) 5 D) as well as the increase of [Ca 2+ ]IC (Supplemental Figure 10) and the lipid-lowering action of the TRPV4-specific activator GSK (Figure ​ (Figure5E). 5 E). Collectively, these data unambiguously confirm that CBD activates TRPV4 and that this ion channel selectively mediates its lipostatic action.

Interestingly, we also showed that, similar to the lipostatic action, antagonism of TRPV4 was able to significantly prevent the antiproliferative effect of CBD (Figure ​ (Figure6A). 6 A). However, quite surprisingly, antiinflammatory actions of CBD were not affected by the antagonist (Figure ​ (Figure6B); 6 B); these latter findings suggested that these antiinflammatory actions might be a TRPV4-independent process.

(A) CyQUANT proliferation assay after 72-hour treatments. *P < 0.05 compared with the vehicle control. # P < 0.05. The solid line indicates the level of the 24-hour vehicle control. Dashed line indicates the level of the 72-hour vehicle control. Results are expressed as the percentage of the 24-hour vehicle control (mean ± SEM of 4 independent determinations). (B) TNFA mRNA expression following 24-hour LPS treatments with or without CBD and HC. *P < 0.05 compared with the vehicle control; # P < 0.05 compared with the CBD-free LPS-treated group. Data are presented using the ΔΔCT method; peptidyl-prolyl isomerase A–normalized (PPIA-normalized) TNFA mRNA expression of the vehicle control was set as 1. Data are expressed as mean ± SD of 3 independent determinations. Two additional experiments yielded similar results. (C) Validation of the key microarray results. mRNA expression of various target genes following 24-hour CBD treatments with or without HC. **P < 0.01, ***P < 0.001 compared with the vehicle control. ### P < 0.001. Data are presented using the ΔΔCT method; PPIA-normalized mRNA expression of the vehicle control was set as 1 (solid line). Data are expressed as mean ± SD of 3 to 6 independent determinations. Two additional experiments yielded similar results.

Sebostatic action of CBD is mediated by TRPV4-dependent interference with the ERK1/2 MAPK pathway and downregulation of nuclear receptor interacting protein-1.

To dissect the intracellular signaling pathways that underlie the above effects, we first investigated the putative participation of several kinases (i.e., PKC isoforms, PI3K, PKA) as well as calcineurin in mediating the lipostatic effects of CBD. Notably, inhibition of activities of these molecules had no effect on the lipostatic activity of CBD (Supplemental Figure 11, A and B).

Then, in order to identify target genes and pathways regulated (directly or indirectly) by CBD, genome-wide microarray analyses were performed on 3 independent sets of control and CBD-treated SZ95 sebocytes (10 μM CBD for 24 hours). Gene set enrichment analysis (GSEA) (40–42) of the microarray results revealed that numerous mitosis and cell cycle (e.g., “mitosis,” “G2/M transition,” “cell cycle,” etc.), inflammation (e.g., “cytokine production,” “cytokine biosynthetic process,” “TLR9 pathway,” “positive regulation of IκB kinase NF-κB cascade,” etc.), and lipid synthesis–related (“acyltransferase activity,” “lipid biosynthetic process,” “positive regulation of MAPK activity,” etc.) gene sets were identified among the downregulated ones, confirming our previous findings on the complex anti-acne effects of CBD. Moreover, downregulation of some “acne-related” gene sets (e.g., “IGF-1 pathway” and “mTOR pathway”) (2, 43) also argued for the putative in vivo anti-acne efficiency of CBD. Further, upregulation of the “calcium signaling pathway” gene set confirmed the involvement of (TRPV4-dependent) calcium signaling (detailed results of GSEA are available in Supplemental Excel files 1 and 2).

During further data processing, Biological Networks Gene Ontology (BiNGO) analysis (44, 45) was also performed (see Supplemental Excel files 3 and 4; the hierarchy of the different gene ontology terms enriched among the downregulated and upregulated genes is summarized in Supplemental Figures 12 and 13, respectively). In line with our previous results, this method also highlighted that CBD exerted “anti-differentiating” effects on sebocytes (terms like “negative regulation of fat cell differentiation” and “negative regulation of fatty acid biosynthetic process” were found to be enriched among the upregulated genes).

Although these analyses further confirmed our previous findings on the complex anti-acne effects of CBD, we still aimed to recognize target genes that might be involved in mediating the different anti-acne modalities and/or might further strengthen the putative in vivo efficiency of CBD. Therefore, using rigid exclusion criteria (at least 2-fold changes in the corresponding expression levels equidirectional changes in all cases, and global, corrected P < 0.05), we found that 80 genes were significantly downregulated, whereas 72 genes were significantly upregulated by CBD treatment (microarray results are accessible through GEO series accession number <"type":"entrez-geo","attrs":<"text":"GSE57571","term_id":"57571">> GSE57571; downregulated and upregulated genes, together with their averaged fold changes, are summarized in Supplemental Tables 1 and 2). By using Q-PCR, we have confirmed that, following CBD treatment, expression of Rho GTPase-activating protein 9 (ARHGAP9, an endogenous inhibitor of the prolipogenic ERK signaling) (46) was upregulated, whereas the proliferation marker MKI67 was downregulated (Figure ​ (Figure6C). 6 C). (This latter result perfectly confirmed our findings obtained in hSOC experiments [Figure ​ [Figure2I].) 2 I].) Moreover, also in line with our previous findings, we found that TRPV4 antagonism could successfully prevent both alterations (Figure ​ (Figure6 6 C).

It is well known that activation of the ERK1/2 MAPK pathway plays a crucial role in the regulation of cellular proliferation (47). Furthermore, we have demonstrated recently that this pathway is involved in mediating the “prolipogenic” action of AEA on human sebocytes (12). Considering that administration of CBD led to opposing cellular effects (i.e., decreased lipogenesis and proliferation) and upregulation the ERK inhibitor ARHGAP9, we hypothesized that CBD might inhibit MAPK activation. Indeed, AEA treatment was able to activate the ERK1/2 MAPK cascade (as monitored by assessing the level of phosphorylated ERK1/2 [P-ERK1/2]), an effect that was completely abrogated by the coadministration of CBD (Figure ​ (Figure7A). 7 A). In a perfect agreement with our previous data (Figure ​ (Figure5, 5 , C–E, and Figure ​ Figure6, 6 , A and C), this interference was found to be TRPV4 dependent, since the specific antagonist HC was able to fully prevent the effect of CBD (Figure ​ (Figure7A). 7 A). This, again, confirmed the crucial role of TRPV4 activation in initiating the lipostatic and antiproliferative signaling cascade(s) of CBD.

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(A) Western blot analysis of lysates of SZ95 sebocytes treated with 30 μM AEA, 10 μM CBD, and 1 μM HC for 5 minutes. Numbers on the OD row indicate the optical density of the P-ERK1/2 bands normalized to the corresponding ERK1/2 signals. (B) Quantitative determination of neutral lipid synthesis (Nile Red staining; 24-hour treatments started at day 3 after transfection). **P < 0.01, ***P < 0.001 compared with the scrambled (SCR) control group. “siNRIP1a” and “siNRIP1b” mark 2 different siRNA constructs against NRIP1. Data are expressed as the percentage of the SCR vehicle control (mean ± SEM of 4 independent determinations). The solid line indicates 100%.One additional experiment yielded similar results.

We have also demonstrated that expression of nuclear receptor interacting protein-1 (NRIP1, also known as RIP140; a corepressor essential for triglyceride storage in adipose tissue) (48) was downregulated in a TRPV4-dependent manner (Figure ​ (Figure6C). 6 C). We have shown that silencing of NRIP1 (validated by Q-PCR and Western blotting; Supplemental Figure 14, A and B) mimicked the lipostatic effect of CBD (Figure ​ (Figure7B), 7 B), suggesting that downregulation of NRIP1 is indeed an important final effector of the lipid synthesis-inhibitory activity of CBD.

Antiinflammatory action of CBD is mediated by upregulation of tribbles homolog 3 and inhibition of the NF-κB pathway.

Our microarray data have also highlighted the putative involvement of several innate immunity/inflammation-related genes in mediating the antiinflammatory action of CBD (Supplemental Tables 1 and 2). By using Q-PCR, we confirmed that expression of LL-37 cathelicidin (a key antimicrobial peptide expressed by and shown to be active in human sebocytes) (49) and tribbles homolog 3 (TRIB3, also known as SINK; a negative regulator of proinflammatory NF-κB signaling) (50) was upregulated by CBD. Importantly (again, in line with our previous results [Figure ​ [Figure6B]), 6 B]), these CBD-induced gene expression changes were not prevented by the coadministration of the TRPV4 antagonist HC (Figure ​ (Figure6C). 6 C). When assessing the functional role of TRIB3, we found that, after its selective silencing (Supplemental Figure 15, A and B), CBD was unable to exert its antiinflammatory action to prevent LPS-induced IL1B and IL6 upregulation (Figure ​ (Figure8A); 8 A); in contrast, its lipostatic activity was not altered (Supplemental Figure 15C).

(A) IL1B and IL6 mRNA expression following 5 μg/ml LPS treatment with or without 10 μM CBD (24-hour treatments started at the day 2 after the transfection). ***P < 0.001 compared with the corresponding CBD-free treatments. ### P < 0.001 compared with the SCR group receiving the same treatments. “siTRIB3a” and “siTRIB3b” mark 2 different siRNA constructs against TRIB3. (B) Western blot analysis of lysates of SZ95 sebocytes treated with 5 μg/ml LPS, 10 μM CBD, and 1 μM HC for 25 minutes. (C) Determination of the intracellular cAMP concentration following 1-hour CBD (10 μM) or vehicle treatment. Data are presented as mean ± SEM of 3 independent determinations. One additional experiment yielded similar results. (D and E) TRIB3 and TNFA mRNA expression following the indicated treatments (5 μg/ml LPS, 10 μM CBD, and 10 nM ZM). (A, D, and E) Data are presented using the ΔΔCT method; PPIA-normalized mRNA expression of the vehicle control was set as 1 (solid line). Data are expressed as mean ± SD of 3 independent determinations. One additional experiment yielded similar results. (CE) **P < 0.01, ***P < 0.001. (F) Western blot analysis of lysates of SZ95 sebocytes treated with 5 μg/ml LPS, 10 μM CBD, and 100 nM ZM for 25 minutes. (B and F) Numbers on the OD row indicate the optical density of the P-IκBα and P-P65 bands normalized to the corresponding β-actin signals.

TRIB3 is known to inhibit the NF-κB pathway (50), and, furthermore, CBD has already been reported to exert its antiinflammatory actions via inhibition of the NF-κB signaling (51). Importantly, we found that CBD cotreatment indeed prevented the LPS-induced phosphorylation (hence inactivation) of the inhibitory IκBα and phosphorylation (hence activation) of the p65 (RelA) NF-κB isoform (Figure ​ (Figure8B). 8 B). These data indicate that, irrespective of the investigated cell type, interference with the NF-κB pathway could be an important mechanism in the development of the antiinflammatory actions of CBD. It should also be noted that TRPV4 antagonism exerted only negligible effects on the action of CBD (Figure ​ (Figure8B), 8 B), again confirming that antiinflammatory activity of CBD is a TRPV4-independent process.

CBD induces a novel (A2a adenosine receptor→cAMP→TRIB3⊣NF-κB) antiinflammatory pathway.

Finally, we aimed at identifying the target molecule of CBD, which, via the upregulation of TRIB3, mediates the antiinflammatory action of the phytocannabinoid. Since previous data suggested that elevation of the intracellular cAMP level is one of the possible inducers of TRIB3 activation/upregulation (52), we have investigated the effects of CBD on the cAMP level. CBD treatment indeed elevated the intracellular cAMP concentration of the sebocytes (Figure ​ (Figure8C), 8 C), arguing that a Gs protein–coupled receptor might be the primary target of CBD. A previous finding that, in a murine model of acute lung injury, the Gs protein–coupled A2a adenosine receptor was found to mediate the antiinflammatory actions of CBD (53) made this receptor a very probable target in our system as well. Indeed, we found that the A2a receptor was expressed by human sebocytes both at the mRNA and protein levels (Supplemental Figure 16, A–C). In addition, we have also shown that application of a specific A2a receptor antagonist, ZM241385 (ZM), was able to significantly prevent the upregulation of TRIB3 by CBD (Figure ​ (Figure8D). 8 D). Moreover, ZM also suppressed the antiinflammatory effect of the phytocannabinoid as well as the CBD-evoked inhibition of LPS-induced NF-κB activation (Figure ​ (Figure8, 8 , E and F). These intriguing findings collectively argued that activation of the “A2a receptor→cAMP→TRIB3⊣NF-κB” axis indeed plays a crucial role in mediating the antiinflammatory actions of CBD.

Discussion

In this study, we provide the first evidence that the nonpsychotropic phytocannabinoid CBD, which is already applied in clinical practice (16), exerted a unique “trinity of cellular anti-acne actions.” Namely, CBD, without compromising viability (Figure ​ (Figure2, 2 , B and C), (a) normalized the pathologically elevated lipogenesis induced by “pro-acne” agents, both in a quantitative and qualitative manner (universal lipostatic effect; Figure ​ Figure1); 1 ); (b) suppressed cell proliferation (antiproliferative effect; Figure ​ Figure2A); 2 A); and (c) prevented the actions of TLR activation or “pro-acne” agents to elevate proinflammatory cytokine levels (universal antiinflammatory effect; Figure ​ Figure3). 3 ). Furthermore, we have shown that sebostatic actions of CBD also developed under “in vivo–like” conditions (hSOC; Figure ​ Figure2, 2 , D–I).

Besides the discussed “sebocyte-specific” steps of the pathogenesis of acne, promisingly targeted by the “cellular anti-acne trinity” of CBD, one should also keep in mind that there are additional factors, which contribute to the progression of the disease: the infundibular hyperproliferation/hyperkeratinization, leading to comedogenesis and subsequent overgrowth of “acnegenic” Propionibacterium acnes strains (2). It is very important to note that, based on the literature, administration of CBD holds out the promise to target these factors as well. Indeed, CBD was shown to inhibit proliferation of hyperproliferative keratinocytes (54), and it was demonstrated to possess remarkable antibacterial activity (55). Although its efficiency against “acnegenic” Propionibacterium acnes strains is not yet investigated, one can speculate that its putative indirect antibacterial activity (mediated by the upregulation of the expression of the antimicrobial peptide LL-37 cathelicidin [Supplemental Table 2 and Figure ​ Figure6C]) 6 C]) could be further supported by direct bactericide effects, arguing that CBD might be very likely to behave as a potent anti-acne agent in vivo.

Given that sebum production is the result of holocrine secretion, the amount of sebum produced is at least as dependent on the proliferative activity of basal layer sebocytes in the sebaceous gland as on the amount of lipogenesis that individual sebocytes engage in (27, 28). Therefore, the novel and significant antiproliferative activity of CBD on human sebocytes in vitro and ex vivo documented here (Figure ​ (Figure2, 2 , A and I) is expected to greatly reduce sebum production in vivo. Moreover, it is also important to emphasize that, clinically, it is highly desirable that basal sebogenesis and viability of sebocytes are unaffected (Figure ​ (Figure1, 1 , A–C, and Figure ​ Figure2, 2 , A–C) by CBD (at least in the noncytotoxic concentrations and after short-term treatments; Supplemental Figure 2, A–E), since a sufficient level of sebum production is a critical factor for maintaining proper function of the epidermal barrier, one of the central components of skin homeostasis (56).

CBD has already been shown to activate (e.g., certain TRP channels, α1 and 5-HT1a receptor, etc.), antagonize (e.g., TRPM8 and 5-HT3 receptor as well as “classical” [CB1 and CB2] and “novel” [GPR55] cannabinoid receptors, etc.), or allosterically modulate (e.g., μ- and δ-opioid receptors, etc.) the activity of a plethora of different receptors and, furthermore, to influence various other cellular targets (e.g., cyclooxygenase and lipoxygenase enzymes, fatty acid amide hydrolase, eCB membrane transporter, phospholipase A2, voltage-dependent anion channel 1, etc.) (15, 32–37, 57–60). Therefore, exploration of its exact mechanism of action appeared to be a great challenge. The fact that we have shown previously that activation of TRPV1 can evoke similar lipostatic effects (38) as those found for CBD (Figure ​ (Figure1 1 and Figure ​ Figure2, 2 , D–H), together with our present findings that CBD induced membrane currents on sebocytes (Figure ​ (Figure4), 4 ), prompted us to first investigate the role of TRP channels in mediating the above anti-acne modalities. We discovered that the lipostatic and antiproliferative effects of CBD were mediated by the activation of TRPV4 (and not TRPV1 or TRPV2) ion channels (Figures ​ (Figures5, 5 , C–E, and Figure ​ Figure6A) 6 A) and the concomitant increase in [Ca 2+ ]IC. Actually, the “negative regulation” of lipogenesis by the elevation of [Ca 2+ ]IC is not unprecedented, since it has already been described in sebocytes (38) as well as in adipocytes (61, 62). It is also important to note that, within the confines of another study, we have shown that extracellular Ca 2+ plays an important negative regulatory role in the sebaceous lipogenesis (C.C. Zouboulis et al., unpublished observations). Of further importance, we have also shown that the antiinflammatory activity of CBD is a TRPV4-independent process (Figure ​ (Figure6 6 B).

Importantly, our data are in perfect agreement with the recent findings of De Petrocellis et al. (37). Using heterologous expression systems, they demonstrated that CBD is a potent but less efficacious activator of rat TRPV4 (as compared with the “classical” agonists or certain other phytocannabinoids, such as cannabichromene [CBC] or cannabidivarin [CBDV]). Although the possibility that CBD might be a more efficacious activator of human TRPV4 than of rat TRPV4 should also be taken into consideration; preliminary data of our recently started assessment of the putative anti-acne effects of other phytocannabinoids also suggest that CBC and CBDV possess an even more pronounced lipostatic efficiency than CBD, which further argues for the central role of TRPV4 (A. Oláh et al., unpublished observations).

In order to identify additional downstream targets, genome-wide microarray experiments were performed on 3 independent sets of control and CBD-treated (10 μM for 24 hours) sebocytes. GSEA (40–42) and BiNGO analysis (44, 45) of the microarray results uniformly confirmed our results, arguing for complex anti-acne actions upon CBD administration, as indicated by downregulation of inflammation (e.g., “cytokine production”), lipid synthesis (e.g., “lipid biosynthetic process” and “positive regulation of MAPK activity”), proliferation-related (e.g., “mitosis” and “G2/M transition”), and “general pro-acne” (e.g., “mTOR pathway” and “IGF-1 pathway”) (2, 43) gene sets and BiNGO terms (Supplemental Excel files 1–4 and Supplemental Figures 12 and 13).

Besides the above results, microarray analyses also revealed that levels of 80 genes were downregulated upon CBD treatment, whereas expression of 72 genes was upregulated upon CBD treatment, among which multiple potential “anti-acne” effectors were identified (Supplemental Tables 1 and 2). Q-PCR validation of the most promising target genes revealed that (in agreement with our cell physiology data) expression of lipid synthesis–related (NRIP1 and ARHGAP9) and proliferation-related (MKI67) genes was altered in a TRPV4-dependent manner, whereas changes in the expression of “inflammation” genes were found to be TRPV4 independent (Figure ​ (Figure6C). 6 C). Moreover, alterations of ARHGAP9 expression (a known endogenous inhibitor of ERK signaling) (46) suggested that inhibition of the prolipogenic MAPK pathway (12) might play a role in mediating the lipostatic effects of CBD. Indeed, we found that CBD inhibited AEA-induced (prolipogenic) (12) ERK1/2 phosphorylation in a TRPV4-dependent manner (Figure ​ (Figure7A), 7 A), confirming again the crucial role of TRPV4 in mediating the action of CBD.

We also silenced another “lipid-regulating gene” (i.e. NRIP1) (Supplemental Figure 14, A and B). As expected (48), knockdown of NRIP1 was able to mimic the lipostatic effect of CBD (Figure ​ (Figure7 7 B).

Next, we aimed at revealing the signaling pathway of the antiinflammatory actions. Thorough assessment of the microarray data highlighted the putative role of TRIB3, a known inhibitor of proinflammatory NF-κB signaling (50). In addition, inhibition of NF-κB signaling plays a crucial role in the development of CBD-mediated antiinflammatory actions in other systems (51). RNAi-mediated selective gene silencing of TRIB3 in human sebocytes (Supplemental Figure 15, A and B) fully abrogated the ability of CBD to inhibit LPS-induced proinflammatory responses (Figure ​ (Figure8A). 8 A). Although a previous study would have suggested it (63), interestingly, TRIB3 was found not to participate in mediating the lipostatic effects of CBD in sebocytes (Supplemental Figure 15C).

It is also noteworthy that TRIB3 has been identified recently as a potent phytocannabinoid target gene (64–66). These results, together with our data presented here, strongly argue for the key participation of TRIB3 in mediating cellular effects of cannabinoids.

Although CBD-dependent upregulation of its several known target genes, such as activating transcription factor 4, asparagine synthetase, cation transport regulator-like 1, and DNA-damage-inducible transcript 3 (refs. 66, 67, and Supplemental Tables 1 and 2), also argued for the activation of a TRIB3-dependent signaling pathway, to further strengthen the “TRIB3-hypothesis,” we have also investigated the effects of CBD on one of the major cellular targets of TRIB3, i.e., NF-κB. As expected (51), CBD was able to inhibit LPS-induced NF-κB activation (again, in a TRPV4-independent manner; Figure ​ Figure8B), 8 B), which can fully explain its previously demonstrated antiinflammatory actions.

Finally, we aimed at identifying the upstream signaling of the TRIB3 activation/upregulation by CBD. We found that CBD elevated the level of cAMP (a known upstream regulator of TRIB3) (ref. 52 and Figure ​ Figure8C), 8 C), highlighting the putative role of a Gs-coupled receptor in initiating its antiinflammatory actions. We also demonstrated that sebocytes express Gs-coupled A2a receptors (which have already been shown to mediate antiinflammatory actions of CBD) (ref. 53 and Supplemental Figure 16, A–C). Further, the specific A2a antagonist (ZM) was able to prevent upregulation of TRIB3 upon CBD treatment (Figure ​ (Figure8D). 8 D). Then, we attempted to confirm the functional presence of the putative antiinflammatory A2a receptor→cAMP→TRIB3′ΔΤNF-κB axis. We found that coadministration of ZM abrogated the antiinflammatory action of CBD (Figure ​ (Figure8E). 8 E). Moreover, we were also able to show that it abolished the NF-κB–inhibitory action of CBD (Figure ​ (Figure8F). 8 F). Taken together, these data strongly argue that A2a receptor might be the primary orchestrator of the antiinflammatory actions of CBD. It should also be noted that, according to the data published by Carrier et al. (68), CBD-mediated activation of A2a receptor is very likely to be an indirect action, realized by the primary inhibition of the equilibrative nucleoside transporter(s) (e.g., ENT1, which mediates adenosine uptake of the cells) and the subsequently elevated “adenosine tone.”

Collectively, our data introduce the phytocannabinoid CBD as a potent “universal” anti-acne agent, possessing a unique “triple anti-acne” profile (Figure ​ (Figure9). 9 ). Multiple human studies have already investigated the safety of CBD (13, 14). Furthermore, it is already in use in many countries in clinical practice without any significant side effects (Sativex) (16). This is especially promising, because the currently available, most effective anti-acne agent, isotretinoin, is known to cause serious side effects (2, 69, 70). These data, together with our current findings, point to a promising, cost-effective, and, likely, well-tolerated new strategy for treating acne vulgaris, the most common human skin disease.