Cannabigerol (CBG) Modulates Neutrophil Activity and Ameliorates Rheumatoid Arthritis Pathogenesis

Source / Quelle: https://www.mdpi.com/1424-8247/19/4/560

Abstract

Background/Objectives: Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disease that primarily affects the joints. Current treatments aim to relieve pain and limit joint damage; however, many are associated with significant side effects or high costs. Neutrophils play a critical role in RA development and progression by driving synovial inflammation and tissue damage, yet no approved therapies directly target neutrophil-mediated pathogenic mechanisms. Cannabinoids have demonstrated anti-inflammatory potential. Although cannabinoids have been studied in RA, the direct modulation of neutrophil-driven mechanisms by purified CBG has not been systematically addressed. To harness the cannabinoid potential, we investigated the effects of the purified cannabinoid Cannabigerol (CBG) on neutrophil-mediated immune responses in RA. Methods: We assessed the effects of CBG on human blood isolated neutrophil cytokine secretion, signal transduction and migration as ex vivo models. In addition, collagen antibody-induced arthritis (CAIA) was applied in C57BL/6 wt mice, and immune-cell recruitment and cytokine secretion were examined after CBG treatment. Results: Ex vivo experiments demonstrated that CBG hampered the secretion of pro-inflammatory cytokines from human neutrophils in a dose-dependent manner (TNF-α and IL-6 by 68% and 72%, respectively). Furthermore, CBG downregulated inflammatory signal transduction, such as P38-MAPK, ERK1/2 and Akt phosphorylationpost neutrophil activation by 41%, 54% and 78%, respectively. Importantly, 60% of the CBG downregulation of IL-6 was consistent with the CB2 receptor axis in a selective way. In addition, CBG attenuated neutrophil migration toward IL-8 by 67%. To further evaluate CBG therapeutic capacity, we used CAIA as an in vivo model. CBG treatment resulted in improving mice arthritis clinical scores and body weight in comparison to RA-diseased mice. Moreover, CBG reduced leukocyte recruitment to the inflamed joints by 48%, primarily through the inhibition of neutrophil and monocyte cells to 27% and 49%, respectively. Additionally, CBG showed its anti-inflammatory effect by decreasing inflammatory cytokines like IL-6 and IL-1β by 98% and 60% in the blood. Also, CBG reduced MCP-1 and IL-1β cytokines in the joints by 22% and 38%, respectively. Conclusions: These results show that CBG has anti-inflammatory capacity and therapeutic potential in regulating neutrophil-mediated immunity in RA. These findings are preclinical and require further validation before therapeutic positioning.

Keywords:

 rheumatoid arthritis; neutrophils; cannabigerol (CBG); cannabis; cannabinoids

1. Introduction

Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disease that primarily affects the joints and is associated with autoantibodies that target various molecules including modified self-epitopes [1]. The innate immune system, including toll-like receptors (TLRs), triggers the initial inflammatory response in joint tissues. The synovium, normally a thin membrane lining the joint, becomes inflamed and thickened due to the influx of immune cells. Neutrophils, monocytes/macrophages, mast cells, T and B lymphocytes and other immune cells infiltrate the synovium. The inflamed synovium experiences new blood vessel growth (angiogenesis). Monocytes and macrophages differentiate into osteoclasts, which break down bones. These cells release cytokines and other inflammatory molecules, further amplifying the inflammatory response. The inflammation leads to cartilage and bone destruction [2].

Growing evidence indicates that synovial pathology and systemic inflammation in chronic immune-mediated diseases are sustained by convergent innate–immune and stress-response circuits, making them attractive pharmacological entry points. For example, synovial cell survival programs can be tuned through the ADORA2B–PI3K/Akt/mTOR axis, which has been linked to reduced apoptosis/autophagy in inflammatory joint settings [3]. Macrophage polarization and inflammasome control also emerge as central levers, as Substance P–driven M2 skewing can attenuate tissue inflammation via NF-κB/NLRP3 regulation [4], and NLRP3 activity can be restrained by ubiquitin–proteostasis mechanisms that limit pyroptosis and downstream inflammatory amplification [5]. In RA specifically, blocking the IL-23/IL-17/NF-κB axis has been shown to alleviate synovial inflammation, underscoring the importance of cytokine network interception in disease modification [6]. Beyond canonical cytokine signaling, mechanosensitive channels such as Piezo1 can shape fibroblast inflammatory phenotypes, supporting the concept that biophysical cues integrate with immune pathways in fibrotic/inflammatory microenvironments [7]. Systems-level approaches further reinforce multi-target immunoregulation, including network pharmacology coupled with in vivo validation for complex formulations that mitigate chronic inflammatory remodeling [8], as well as macrophage-targeted nanomedicine platforms designed to reprogram innate immunity for the durable control of inflammation [9]. Mechanistic parallels across immune diseases strengthen translational relevance: TLR4–NF-κB activation driven by upstream regulators has been implicated in inflammatory bowel disease, highlighting conserved innate–inflammatory wiring that may be therapeutically repurposed [10]. Likewise, mitochondrial stress and endothelial dysfunction are increasingly recognized as inflammatory amplifiers that can be ameliorated by metabolic resilience pathways such as SIRT3 signaling [11]. Finally, the growing appreciation that immune dysregulation intersects with neurodegenerative pathobiology emphasizes the broad value of immunomodulatory small molecules that can recalibrate inflammatory set points across tissues [12]. Collectively, these advances provide a strong conceptual basis for testing new candidates to modulate key innate effector functions—particularly neutrophil signaling, cytokine output and trafficking—to interrupt RA-relevant inflammatory cascades.

Of all cells implicated in the pathology of RA, neutrophils possess the greatest cytotoxic potential, owing to their ability to release degradative enzyme, reactive oxygen species and inflammatory cytokines [13]. Also, neutrophil extracellular traps (NETs) are a source of citrullinated autoantigens in RA [14]. NET components act as danger-associated molecular patterns (DAMPs) to activate NLRP3 inflammasomes and the complements in effector lymphocytes, amplifying inflammation. NETs promote the RA-related autoantibody production in B cells, such as anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF), fueling autoimmunity, while ACPAs further induce NETosis, creating a vicious feedback loop. NETs facilitate the release of pro-inflammatory cytokines (e.g., IL-6, IL-1β, TNF-α), exacerbating joint damage. Finally, NETs activate T cells, dendritic cells, and macrophages via boosting the RAGE/TLR9 pathway, thereby driving the proliferation and migration of fibroblast-like synoviocytes [15]. Furthermore, the inflammatory sites are characterized by low levels of oxygen and glucose and high levels of reductive metabolites. Hypoxia causes an inhibition of neutrophil apoptosis in human and murine neutrophils. Neutrophils possess the hypoxia-inducible factor (HIF)-1α and factor inhibiting HIF (FIH) hydroxylase oxygen-sensing pathway that contribute to synovial neutrophil survival in hypoxia despite systemic treatment [16]. Therefore, neutrophils play roles as both primary drivers and chronic amplifiers that make them an attractive target in rheumatoid arthritis, as they play a central role in driving joint inflammation.

Treatment aims to reduce pain and prevent or slow further joint damage. Early treatment with disease-modifying anti-rheumatic drugs (DMARDs) is crucial for achieving remission and preventing irreversible damage. Key treatment approaches include DMARDs, often combined with non-steroidal anti-inflammatory drugs (NSAIDs) and/or corticosteroids, and, in some cases, biological treatments like TNF-α, IL-1 and IL-6 blockers [17]. A new study shows Trimetazidine (TMZ) as an antirheumatic candidate, offering anti-inflammatory effects and a synergistic effect with Methotrexate (MTX) that together reduce the adverse effects of MTX while improving therapeutic efficacy [18].

To date, there is no treatment that specifically targets neutrophils, but the current standard therapeutic approaches show an indirect beneficial effect on their pathogenic profile. RA current treatments prevent neutrophil chemotaxis and migration and decrease degranulation and ROS production. In addition, treatments attenuate the production of inflammatory mediators and prevent the release of NETs [19].

Cannabinoids produce more than 100 naturally occurring chemicals, the most abundant of which are Δ-9-tetrahydrocannabinol (THC), cannabidiol (CBD), terpenes and flavonoids. THC and CBD bind with cannabinoid receptors (CB1 and CB2), which are present in the brain, immune system and many organs [20]. THC and CBD help to manage conditions ranging from chronic pain persistent inflammation, cancer, inflammatory bowel disease, and neurological disorders to even viral diseases such as Human Immunodeficiency virus (HIV) and SARS-CoV-2 [21]. Both THC and CBD exhibit promising therapeutic properties; however, impairments and the increased incidence of mental health diseases are associated with acute and chronic THC use, respectively, and significant side effects are associated with the chronic use of high-dose CBD [22]. Preclinical in vitro and in vivo studies show promising results regarding the anti-arthritic properties of cannabinoids, psychoactive and non-psychoactive alike [23]. Our previous studies demonstrated that a high-CBD extract (CBD-X) significantly reduced pro-inflammatory cytokine secretion in human-derived PBMCs, neutrophils and T cells [24]. More recent findings further show that CBD-X exerts anti-inflammatory effects in rheumatoid arthritis by inhibiting pro-inflammatory cytokine secretion, limiting immune-cell recruitment and attenuating inflammatory signal transduction [25]. However, CBD-X is an extract that has 37% CBD, 1.7% THC, 0.3% CBG and other unknown terpenes and flavonoids [25]. Therefore, we decided to focus our research on purified cannabinoids.

Cannabigerol (CBG) serves as the precursor molecule for the most abundant cannabinoids. While THC and CBD have been more widely studied than CBG, it has had increased attention due to the wide range of potential health benefits [26]. Studies indicate that CBG may have therapeutic potential in treating neurologic disorders (e.g., Huntington disease, Parkinson disease and multiple sclerosis) and inflammatory bowel disease, as well as having antibacterial activity [27]. Moreover, there are anti-inflammatory effects of CBG in rheumatoid arthritis synovial fibroblasts and peripheral blood mononuclear cell cultures [28]. Furthermore, research showed CBG has potential anti-inflammatory properties, including the diabetic kidney disease progression of rats subjected to a high-fat, high-sucrose diet [29].

CBG is a multi-target player that interacts with the endocannabinoid system and other key signaling pathways, such as CB1, CB2, transient receptor potential (TRP) channels and α2-adrenoceptor, potentially influencing inflammation, pain, neurodegeneration and other ailments. CB1 and CB2 are the two main types of cannabinoid receptors. Unlike THC and CBD, CBG does not bind directly to the CB1 receptors in the brain, which are responsible for the psychoactive effects of cannabis. CBG weakly agonizes the action of CB1 and partially acts as an agonist on CB2 receptor agonists [26]. Cannabinoid receptor 2 deficiency exacerbates inflammation and neutrophil recruitment [30]. The expression of the CB2 mRNA can be detected in neutrophils [31]. The CB2 receptor is primarily expressed only when there is active inflammation. Accordingly, CBG’s interaction with CB2 downregulates the inflammation [32]. Furthermore, CBG interacts with TRP channels, which play a crucial role in regulating the cytoplasmic calcium concentration from the extracellular sources as well as the calcium stored within the endoplasmic reticulum (ER). CBG serves as a strong agonist of TRPA1 and a weaker agonist of TRPV1, TRPV2 and TRPV4 while antagonizing TRPM8 (TRP melastatin type 8) [26]. CBG acts as a potent agonist of Peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor that suppresses NFκB or MAP kinases [33]. In addition, CBG is a potent serotonin 1A receptor (5-HT1A) antagonist, which is known to reduce the activity of NF-κB and the phosphorylation of its inhibitor α (IκB- α), resulting in a decrease in cytokine secretion [29].

Recent computational modeling indicates that CBG possesses a unique 3D conformational flexibility, characterized by ‘coiled’ and ‘stapled’ poses that are sterically distinct from the rigid bicyclic structure of CBD [34]. In addition, CBG exhibits affinity and activity characteristics between Δ9-THC and CBD at the cannabinoid receptors but appears to be unique in its interactions with the α-2 adrenoceptors, PPARγ and 5-HT1A [27].

However, CBG shows anti-inflammatory effects, but due to the lack of research, it is not widely used. Its physicochemical properties lead to considerable challenges, such as poor intestinal absorption, extensive first-pass metabolism, low bioavailability and variable brain penetration, all of which limit its effective application in clinical settings [35]. In addition, the regulatory status of cannabis, sources for cannabis and funding to support studies are difficult barriers to navigate [36].

There is growing interest in CBG and its promising therapeutic potential in treating immune-related diseases. Our aim in this study is to examine the regulatory effect of CBG in mitigating inflammatory RA disease severity through regulating neutrophil activity. We hypothesized that CBG through CB2 receptor mediation directly suppresses neutrophil activation and recruitment, thereby attenuating RA severity.

2. Results

2.1. CBG Downregulates TNF-α and IL-6 Secretion by Human-Neutrophils

Neutrophils are one of the first lines of host defense against pathogens, and they are involved in the early recognition and killing of infectious pathogens [37]. Synovial pathology reports reveal lymphocyte-predominant infiltration in most RA cases, with synovial neutrophils (SNs) observed in only 30% of patients. However, elevated neutrophil presence in RA synovium correlates with heightened clinical disease activity and an exacerbated inflammatory state [38]. Activated neutrophils function as a source of cytokines that initiate the inflammatory process, including TNF-α [39] and IL-6 [40]. Both cytokines play crucial roles in the activity and severity of RA [41].

Furthermore, Lipopolysaccharide (LPS) is an exogenous endotoxin; it is the canonical agonist for Toll-like Receptor 4 (TLR4) [42]. TLRs recognize Pathogen Associated Molecular Patterns (PAMPs) expressed on microbial pathogens or Danger Associated Molecular Patterns (DAMPs), which may be expressed by cells under stress [43]. In the context of RA pathology, TLR4 plays a pivotal role by recognizing endogenous DAMPs [44]. By utilizing LPS, we were able to induce a highly synchronized and reproducible activation of the TLR4, which effectively mimics the DAMP-driven sterile inflammation seen during RA.

CBG is a non-psychoactive cannabinoid that exhibits anti-inflammatory activities [26]. To determine whether CBG exerts inhibitory effects on neutrophil-driven inflammation, human peripheral blood neutrophils were isolated (purity ~95%, (Supplementary Figure S1)), treated with CBG and activated with LPS. The secretion of pro-inflammatory cytokine levels of TNF-α and IL-6 was determined. CBG treatment reduced the levels of both cytokines from neutrophils in a significant dose-dependent manner by 52% (48 ± 33.8, p < 0.001, n = 13, 95% CI (33.86, 69.99)) and 68% (32 ± 29.1, p < 0.001, n = 15, 95% CI (49.7, 85.8)) of TNF-α levels (Figure 1a) and by 50% (50 ± 32.1, p < 0.001, n = 13, 95% CI (34.06, 64.9)) and 72% (28 ± 25.3, p < 0.001, n = 15, 95% CI (56.25, 87.1))of IL-6 levels (Figure 1b). The shown results are relative to the normalized activated control treatment group (LPS, DMSO).

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