The CB₂ Receptor in Immune Regulation and Disease: Genetic Architecture, Epigenetic Control, and Emerging Therapeutic Strategies

1. Introduction

It is now accepted that the endocannabinoid system (ECS) is a sophisticated and evolutionarily preserved lipid signaling network that regulates various physiological and pathological processes, encompassing immune surveillance, inflammation, energy balance, emotional regulation, pain perception, and synaptic transmission[1]. Throughout history, cannabis has been utilized by ancient civilizations for its pain-relieving and therapeutic benefits[2]. However, it was only in the mid-20th century that the compounds derived from cannabis were identified structurally, leading to the discovery of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) as the primary active components of Cannabis sativa[3]. A pivotal advancement occurred with the uncovering of endogenous cannabinoids anandamide (N-arachidonoyl-ethanolamine; AEA) and 2-arachidonoyl-glycerol (2-AG), alongside their corresponding receptors, CB₁ receptor and CB₂ receptor, as well as the enzymes responsible for their synthesis and degradation, such as N-acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), diacylglycerol lipase (DAGLα/β), fatty acid amide hydrolase (FAAH), and monoacylglycerol lipase (MAGL)[4]. CB₁ receptors are highly represented in the central nervous system (CNS), primarily mediating the psychoactive effects of THC and regulating neurotransmitter release[5]. Conversely, CB₂ receptors were initially identified as peripheral receptors in immune tissues, such as the spleen, tonsils, and circulating leukocytes[6,7,8]. Nonetheless, growing evidence has shown that CB₂ receptors can also be inducibly expressed in the CNS during inflammatory or neuropathological conditions, particularly in microglia, astrocytes, and specific neuronal types[9]. This system functions as a precisely regulated homeostatic network activated as needed in response to stress, injury, or immune challenges[10].

Novel CB₂ receptor-selective drugs offer biased signaling and enhanced clinical specificity.

CB₂ receptor has attracted significant interest due to its non-psychoactive characteristics and diverse immunomodulatory roles[11,12]. In basic and translational studies, researchers are actively investigating its functions in inflammation, immune cell movement, mood regulation, neurodegeneration, and cancer immunity[13,14,15]. As the understanding of CB₂ receptor dynamics deepens, including its inducible expression, cell-type specificity, and distinct signaling capabilities, there is growing interest in leveraging its potential for therapeutic intervention. Herein, we provide an in-depth analysis of CB₂ receptor biology, covering aspects from molecular structure and epigenetic regulation to its involvement in inflammation, neurodegeneration, mental health disorders, and immune checkpoint regulation in cancer. The review synthesizes mechanistic studies, knockout models, and pharmacological interventions to create a framework relevant to understanding CB₂ receptor as a therapeutic target within the larger endocannabinoidome (eCBome).

2. Molecular Biology of the CB₂ Receptor

The CB₂ receptor is encoded by the CNR2 gene in humans, which is located on chromosome 1p36[16]. The typical isoform consists of a protein made up of 360 amino acids, featuring seven transmembrane α-helices that are typical of class A GPCRs, with an extracellular N-terminus that plays a role in ligand binding and an intracellular C-terminal domain essential for signaling and receptor transport[17]. The CB₂ receptor shares around 44% sequence homology with the CB₁ receptor but displays expression patterns and pharmacological characteristics that vary by tissue[18]. Notably, CB₂ receptor exhibits 82% sequence similarity between humans and rodents; however, there are species-specific differences in expression and function that must be considered when interpreting preclinical results[19]. CB₂ receptors act as context-sensitive signaling hubs, influenced by complex epigenetic, transcriptional, and post-translational regulatory mechanisms[20,21]. Their capability to variably interact with downstream effectors in a cell- and ligand-specific way makes them appealing targets for precision pharmacotherapy aimed at a range of immune, neurological, and psychiatric conditions[22]. Although present in low abundance in healthy CNS tissue, CB₂ receptor mRNA and protein expression increase significantly in microglia and specific neurons under inflammatory or neuropathological conditions. CB₂ receptor is highly prevalent in B lymphocytes within peripheral immune tissues, followed by NK cells, macrophages, and monocytes in these regions[23]. Importantly, Simard et al. (2022) provided a comprehensive transcriptomic analysis of human peripheral leukocytes, revealing that CB2 receptor mRNA is significantly upregulated in granulocytes (neutrophils and eosinophils) and PBMCs (monocytes, B and T lymphocytes)[8]. This receptor can also be found in stem cells derived from bone marrow and various epithelial and endothelial tissues[24,25]. In the brain, the CB₂ receptor has been identified in regions such as the hippocampus, substantia nigra, cortex, amygdala, and prefrontal cortex, particularly during pathological condition[26].

3. Epigenetic and Transcriptional Regulation of the CB₂ Receptor

The regulation of the CNR2 gene through epigenetic processes is crucial for modulating CB₂ receptor expression in response to various environmental and cellular stimuli[27]. Different layers of epigenetic control, including DNA methylation, histone modifications, and non-coding RNAs, regulate the accessibility and transcriptional activity of the CNR2 region[16,28]. These processes not only calibrate receptor expression but also enable CB₂ receptor to adjust its immune and neuroinflammatory roles in line with physiological demands.

3.1. DNA Methylation and Transcriptional Silencing

A key mechanism influencing CB₂ receptor expression is the methylation of DNA at CpG-rich regions within the CNR2 promoter[29]. In the absence of inflammation, DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) add methyl groups to cytosines in these CpG islands[29,30,31]. This methylation inhibits gene transcription primarily through two means: (1) it directly obstructs transcription factor binding, and (2) it recruits methyl-CpG-binding proteins like MeCP2[29,32,33,34]. MeCP2 then acts as a platform to attract histone deacetylases (HDACs) and chromatin remodeling complexes, which compact the chromatin and maintain a transcriptionally inactive state[29,35]. This repression, however, is dynamic and can be reversed. Environmental factors, such as inflammation, infection, psychological stress, or exposure to cannabinoids, can trigger TET enzymes (TET1, TET2, and TET3), which convert 5-methylcytosine to 5-hydroxymethylcytosine[36,37]. This modification promotes a shift toward a more open chromatin state, creating a permissive environment for gene transcription, which may include reactivation of the CNR2 gene under appropriate cellular conditions[36]. Furthermore, activation-induced cytidine deaminase (AID) and members of the APOBEC family aid demethylation by initiating base excision repair pathways that restore unmethylated cytosines[29]. These adaptive epigenetic alterations enable rapid increases in CB₂ receptor expression in both immune and neural cells during inflammatory or pathological conditions[4].

3.2. Histone Modifications and Chromatin Accessibility

Histone modifications work synergistically with DNA methylation to finely regulate the chromatin environment surrounding the CNR2 gene[16]. Enhancing histone marks, such as acetylation of histone H3 at lysine 9 (H3K9ac) and trimethylation at lysine 4 (H3K4me3), are closely linked to open chromatin and active gene transcription[16,38,39]. These activating changes are facilitated by enzymes such as CBP/p300, a group of transcriptional coactivators crucial for regulating gene expression, and MLL family methyltransferases[40,41,42,43,44]. Their activity is frequently stimulated by pro-inflammatory signals, immune activation, or engagement of the CB₂ receptor, which kick-starts downstream signaling pathways such as MAPK/ERK and PI3K/Akt[30]. These pathways further enhance the recruitment of co-activators and transcription factors to the CNR2 promoter, boosting gene expression[27].[36,37]

Conversely, repressive histone marks, such as H3K9me2 and H3K27me3, are introduced by histone methyltransferases G9a and EZH2, respectively, the primary components of the Polycomb Repressive Complex 2 (PRC2)[30,45,46,47]. These modifications lead to chromatin compaction and transcriptional repression. The dynamic interplay between activating and repressive histone modifications ultimately decides whether CB₂ receptor remains inactive or is actively transcribed, enabling the receptor to respond accurately to shifting physiological conditions[48].

3.3. Non-Coding RNAs: MicroRNAs and Long Non-Coding RNAs

Non-coding RNAs add an additional layer of regulatory complexity to the expression of CB₂ receptor[30,49]. MicroRNAs (miRNAs), such as miR-139 and miR-665, have been identified as binders to the 3′ untranslated region (3′ UTR) of CNR2 mRNA, inhibiting translation under resting conditions[50,51,52]. In cases of chronic heart failure, there is a notable increase in CB₂ receptor expression within human heart muscle tissue, indicating a possible adaptive mechanism involving the endocannabinoid system. Weis et al. (2010) found that levels of CB₂ receptor mRNA were significantly elevated in the myocardium of left ventricular failure, while CB₁ receptor expression showed a slight decline. This shift in receptor expression was correlated with a considerable increase in circulating endocannabinoids, indicating an overall activation of the endocannabinoid system[53]. Similarly, Möhnle et al. (2014) demonstrated that miR-665 functions as a post-transcriptional regulator of the CB₂ receptor. Their research indicated that miR-665 was notably downregulated in heart tissue samples from patients with heart failure and was predicted, through bioinformatic analysis, to target the 3′ untranslated region of CNR2 mRNA. The negative correlation observed between miR-665 and CB₂ receptor expression suggests a regulatory mechanism in which lower levels of miR-665 may alleviate the translational inhibition of CB₂ receptor, thereby promoting increased receptor expression. Collectively, these results highlight a coordinated mechanism in which modulation of microRNA and heightened endocannabinoid activity may facilitate CB₂ receptor-mediated responses as a compensatory adaptation in the failing heart[54].

3.4. Pharmacological Modulation of the CB₂ receptor: Biased Agonism, Allosteric Control, and Post-Translational Regulation

When activated by endogenous ligands (AEA, 2-AG) or synthetic compounds (such as JWH-133), CB₂ receptors associate with Gαi/o proteins, leading to the inhibition of adenylyl cyclase, decreased cAMP synthesis, and subsequent suppression of PKA[55,56,57]. Beyond traditional G protein signaling, CB₂ receptors also interact with β-arrestin pathways, which are responsible for receptor internalization, desensitization, and alternative signaling routes, including ERK1/2, p38 MAPK, and PI3K/Akt[58,59]. Evidence of ligand-biased signaling has shown that different ligands can preferentially engage either G protein or β-arrestin pathways, allowing for pharmacological fine-tuning[60,61]. Allosteric modulation is an innovative strategy to enhance or inhibit CB₂ receptor activity selectively[62]. Positive allosteric modulators (PAMs), such as EC21a and endogenous peptides like Pepcan-12, increase the sensitivity of CB₂ receptors to endogenous cannabinoids without acting as direct agonists, thus preserving physiological signaling dynamics[62,63,64]. Conversely, negative allosteric modulators (NAMs) can diminish receptor activity and may provide therapeutic advantages in conditions involving excessive CB₂ receptor activation[65,66,67]. CB₂ receptors also undergo post-translational modifications, including glycosylation and phosphorylation, which influence receptor transport, localization, and the accuracy of signaling[68,69,70]. G protein-coupled receptor kinases (GRKs) and β-arrestins regulate receptor desensitization and recycling, while receptor dimerization with CB₁ receptor or other GPCRs introduces another layer of functional complexity[71,72].

4. The Role of CB₂ Receptor in Immune Regulation

The CB₂ receptor serves as a crucial modulator of immune homeostasis, influencing both innate and adaptive immune responses through mechanisms that are specific to different cells and contexts[73]. It is highly expressed in the hematopoietic system and is essential for leukocyte activation, production of cytokines, cell migration, and infiltration into tissues[74]. In contrast to the CB₁ receptor, which has limited expression in immune cells, the CB₂ receptor has strong expression. It can be induced by inflammatory signals, making it a key therapeutic target for inflammatory and autoimmune disorders[75].

The activation of the CB₂ receptor results in the inhibition of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and interferon-gamma (IFN-γ), while it encourages the production of anti-inflammatory cytokines, including IL-10 and transforming growth factor-beta (TGF-β)[76,77,78]. This shift in cytokine profiles is achieved through the inhibition of NF-κB signaling, decreased cAMP levels, and reduced activity of MAPK pathways[79,80]. Additionally, CB₂ receptor influences inflammasome activation and the generation of reactive oxygen species (ROS), which contributes to its anti-inflammatory characteristics[81].

CB₂ receptor signaling promotes the M2 anti-inflammatory phenotype in monocytes and macrophages over the M1 pro-inflammatory state[11,82]. This polarization is driven by transcription factors, such as signal transducer and activator of transcription 6 (STAT6), and nuclear receptors, including peroxisome proliferator-activated receptor (PPAR-γ)[83,84]. M2 macrophages that are stimulated by CB₂ receptor agonists display increased phagocytic ability and diminished antigen presentation, both of which are vital for resolving inflammation and conducting tissue repair[85,86]. In dendritic cells (DCs), activation of CB₂ decreases the expression of MHC-II and co-stimulatory molecules (CD80, CD86), hinders maturation, and reduces their capacity to activate naïve T cells[87]. These consequences produce a tolerogenic DC phenotype that lessens T-cell activation and promotes immune resolution[88,89]. The ability of CB₂ receptors to inhibit the migration of dendritic cells to lymph nodes through the downregulation of CCR7 and changes in cytoskeletal structures further limits immune amplification[90,91]. T lymphocytes exhibit low baseline expression of the CB₂ receptor, but this expression increases significantly upon activation[8], [92,93,94,95]. CB₂ receptor signaling in CD4+ T cells restricts proliferation and differentiation into Th1 and Th17 subsets while encouraging the development of regulatory T cells (Tregs)[89,93,96,97,98,99,100,101,102,103,104,105]. This immunosuppressive action is partially facilitated by reducing the secretion of IL-2, IFN-γ, and IL-17, as well as modulating transcription factors like T-bet and RORγt. T cells that lack CB₂ receptor exhibit heightened inflammatory responses in colitis and encephalomyelitis models, reinforcing its role in immune regulation[97,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114]. B cells express high levels of the CB₂ receptor, and its activation influences antibody production, class switching, and migration[115,116]. CB₂ receptor influences germinal center dynamics and the formation of B cell memory, although the underlying mechanisms continue to be explored. CB₂ receptor supports tissue retention and chemotaxis in marginal zone B cells through gradients dependent on 2-AG[117,118,119].

CB₂ receptor also interacts with the wider eCBome through connections with prostaglandins, lysophospholipids, and nuclear receptors like PPARs[120,121,122,123]. For example, IL-10 production induced by CB₂ receptor works synergistically with PPARγ activation in macrophages to promote the resolution of inflammation[124,125,126]. Interactions with TRP channels and adenosine receptors also regulate leukocyte activity and migration[127,128].

In conclusion, CB₂ receptor orchestrates a complex immunoregulatory program that balances host defense and immune tolerance. Its ability to modulate cytokine networks, antigen presentation, leukocyte trafficking, and adaptive cell differentiation highlights its potential as a therapeutic target in chronic inflammation, autoimmunity, and tissue damage. Precisely targeting CB₂ receptor with full agonists, PAMs, or biased ligands presents a novel strategy for adjusting immune function without broadly suppressing host immunity.

5. CB₂ Receptor and Microglia in Neuroinflammation

Microglia, the brain's resident macrophages, are crucial for preserving CNS homeostasis and managing immune responses during injury and disease[129]. In a healthy brain, microglia exhibit a surveying state characterized by branching shapes and minimal expression of inflammatory proteins[130]. When faced with pathological triggers such as infection, trauma, or neurodegeneration, microglia quickly transform morphologically and functionally into reactive states, which are generally classified as M1 (pro-inflammatory) or M2 (anti-inflammatory/neuroprotective[131,132]. Although this M1/M2 classification is somewhat simplistic, it serves as a helpful conceptual framework for understanding immune polarization in the CNS. Notably, the CB₂ receptor appears to play a vital role in regulating this polarization. Under normal circumstances, CB₂ receptor expression in microglia is low or undetectable; however, it is rapidly increased in response to immune activation, oxidative stress, and neuroinflammatory stimuli[133]. This ability to be induced makes CB₂ receptor a context-dependent immunomodulator that can finely adjust the equilibrium between neurotoxic and neuroprotective states[134]. Activation of CB₂ receptor has been demonstrated to decrease the release of pro-inflammatory cytokines while enhancing anti-inflammatory mediators[135]. Mechanistically, activating the CB₂ receptor prevents the nuclear translocation of NF-κB, diminishes MAPK pathway activation, and modulates the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), thereby dampening the pro-inflammatory signaling cascade at several levels[136,137,138].

Recent studies conducted both in vivo and in vitro support the role of the CB₂ receptor in modulating the microglial phenotype. For instance, activation of the CB₂ receptor promotes a transition from M1 to M2 phenotypes, as evidenced by decreased expression of inducible nitric oxide synthase (iNOS) and increased levels of arginase-1 (Arg-1)[139,140]. In models of Alzheimer's disease, traumatic brain injuries, and stroke, pharmacological stimulation of CB₂ receptors has lessened neuroinflammation, reduced neuronal death, improved cognitive functioning, and minimized gliosis[141]. In conditions like neuropathic pain and multiple sclerosis, CB₂ receptor signaling has been found to reduce neurotoxicity caused by microglia and promote remyelination[142]. Epigenetic mechanisms also play a role in regulating the CB₂ receptor in microglia. Modifications to histones, such as increased H3K4me3 and decreased H3K27me3 at the CNR2 promoter, have been associated with the transcriptional activation of CB₂ receptor during inflammatory responses[143,144].

Furthermore, the expression of CB₂ receptor can be influenced by non-coding RNAs (like miR-139), which inhibit CNR2 translation under normal conditions but are downregulated during inflammation[145]. These regulatory processes highlight the dynamic adaptability of CB₂ receptor expression and its potential as a therapeutic target in neuroimmune disorders. Significantly, the activation of CB₂ receptors affects synaptic plasticity by modulating microglial behavior[146,147]. Activated microglia can affect neuronal circuits by pruning synapses and releasing neurotrophic factors. By influencing microglial activity, CB₂ receptor indirectly affects the stability and repair of neural networks. This aspect of neuromodulation has significant implications for disorders such as schizophrenia and depression, where abnormal synaptic pruning and ongoing neuroinflammation play a role[148]. In summary, the signaling of CB₂ receptors in microglia represents a significant intersection between immune modulation and neural repair. Targeting CB₂ receptor presents a comprehensive approach for reducing neuroinflammation, restoring immune balance, and safeguarding neural integrity, making it a foundational element in cannabinoid-based therapies for CNS disorders.

6. CB₂ Receptor in Depression and Psychiatric Disorders

Depression is increasingly acknowledged as a complex disorder involving the neuroimmune system, marked by a subtle interaction between chronic inflammation, improper stress response regulation, reduced neuroplasticity, and altered microglial cell activity[149]. This condition is not simply due to a lack of neurotransmitters; instead, its underlying pathophysiology consists of intricate relationships between the immune system and the CNS, shaped by both psychosocial influences and biological stress factors[150]. Significant factors contributing to this disorder include immune cell activation, increased levels of pro-inflammatory cytokines, and overactivity of the hypothalamic-pituitary-adrenal (HPA) axis[151,152]. These components result in synaptic malfunction, amplified glial reactivity, and long-lasting changes in neural circuits, creating a challenging environment for those affected by depression[153,154].

Within this complex framework, the cannabinoid receptor CB₂ has emerged as a crucial component in regulating the interaction between the immune system and neurological processes. Traditionally associated with immune responses in the periphery, recent studies have revealed the presence of CB₂ receptors in the brain, particularly in response to stress and inflammation[152,155,156,157]. The increased expression of the CB₂ receptor in glial cells and areas of the brain that respond to stress is now increasingly considered a compensatory strategy aimed at restoring equilibrium[145]. Through its signaling pathways, CB₂ can reduce neurotoxicity caused by cytokines, influence the release of glucocorticoids, and promote neurotrophic signaling[158]. This positions CB₂ as a central player in mechanisms that regulate mood and are influenced by the immune system. This section examines the various roles of CB₂ receptors in neuroimmune regulation during depressive episodes, with a focus on the dynamics of cytokines, modulation of the HPA axis, epigenetic changes, and behavioral traits observed in preclinical studies. The knowledge gained from these studies provides a strong justification for investigating the therapeutic potential of targeting CB₂ receptors in treating inflammation-related mood disorders, while avoiding the psychotropic side effects typically associated with CB₁ receptor activation.

6.1. Neuroimmune Pathology and CB₂ Receptor Expression

Numerous studies indicate a strong correlation between depressive symptoms and states of neuroinflammation. Elevated concentrations of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, are frequently found in the serum and cerebrospinal fluid of individuals with depression[159,160]. These cytokines interfere with neurotransmission by decreasing serotonin production (through the IDO-mediated metabolism of tryptophan), hindering neurogenesis in the hippocampus, and enhancing excitotoxicity. Furthermore, chronic stress triggers the activation of microglia and immune cells, leading to a systemic inflammatory response that worsens neurobiological impairments[161]. Research has shown increased levels of CB₂ receptor expression in brain tissues and immune cells of animal models and humans with major depressive disorder (MDD). This upregulation of CB₂ receptor appears to respond to inflammatory stimuli and stress hormones, functioning as a potential compensatory mechanism to restore balance[162,163]. Collectively, these findings suggest that the increase of CB₂ receptors in depressive states may reflect an adaptive neuroimmune strategy intended to counteract neuronal and behavioral deficits caused by inflammation.

6.2. Modulation of the HPA Axis and Inflammatory Feedback

The HPA axis regulates stress responses through the secretion of glucocorticoids. Chronic stress leads to persistent activation of this axis, which hampers the regulation of cortisol by the hippocampus and contributes to depressive disorders. Activation of CB₂ receptor has been found to alleviate excessive HPA activity, likely by reducing both central and peripheral inflammation. CB₂ receptor signaling diminishes the production of pro-inflammatory cytokines that typically contribute to HPA hyperactivity[164]. Notably, CB₂ receptors may play a dual regulatory function by modulating cytokine release from immune cells and by inhibiting neuronal hyperexcitability in stress-sensitive areas such as the hippocampus and prefrontal cortex[165,166]. Pharmacological stimulation of CB₂ receptors has been shown to normalize serum corticosterone levels, diminish glial reactivity, and restore expression of brain-derived neurotrophic factor (BDNF), all of which are crucial for mood regulation and synaptic plasticity[156,167,168].

6.3. Epigenetic Regulation and Transcriptional Modulation

Epigenetic processes closely regulate CB₂ receptor expression in the context of depression. Modifications to histones, such as increased levels of H3K4me3 and H3K9ac and diminished repressive marks (H3K9me2, H3K27me3) at the CNR2 promoter, promote CB₂ receptor transcription in response to stress and inflammation[52]. Additionally, the methylation status of the CpG islands located upstream of CNR2 can either repress or activate transcription, depending on exposure to environmental stressors[169]. Transcription factors such as nuclear erythroid 2-related factor 2 (NRF-2), NF-κB, and Activator Protein-1 (AP-1) have been associated with the regulation of the CB₂ receptor gene[170]. NRF-2 connects oxidative stress responses with the activation of the CB₂ receptor[171]. When exposed to stress, the translocation of these transcription factors to the nucleus can trigger the transcription of CNR2, suggesting a dynamic interaction among redox status, inflammation, and cannabinoid signaling[52]. MeCP2 plays a key role in maintaining the CNR2 gene in a transcriptionally repressed state under homeostatic conditions by binding to methylated CpG islands within its promoter region[172]. This repression is alleviated during stress or inflammatory responses through phosphorylation of MeCP2, which reduces its DNA-binding affinity and facilitates gene activation. The demethylation of the CNR2 promoter is mediated through two primary mechanisms: the deaminase pathway involving AID/APOBEC enzymes and the TET protein pathway, both of which initiate DNA base modifications that are resolved through the base excision repair (BER) system. These events ultimately replace methylated cytosines with unmethylated ones, enabling transcription of CNR2 and increased CB₂ receptor expression in microglia. This epigenetic switch illustrates how MeCP2 functions as a dynamic regulator of CB₂ receptor expression in response to environmental and inflammatory stimuli [172,173].

6.4. Behavioral Evidence from Animal Models

Research on rodents using CB₂ receptor agonists like JWH133 and β-caryophyllene has shown significant antidepressant-like effects in models of chronic stress and lipopolysaccharide (LPS)-induced depression[77,96,168]. These effects include reduced immobility in forced swim tests, enhanced sucrose preference, normalized HPA axis functioning, and restoration of neurogenesis in the hippocampus. In contrast, CB₂ receptor knockout mice display increased anxiety, social withdrawal, and heightened inflammatory responses that reflect critical features of depressive disorders. Notably, activation of CB₂ receptor reduces microglial activation and inhibits TLR4-mediated signaling, which is integral to the inflammatory mechanisms underlying depression. Moreover, microglia deficient in CB₂ receptor show increased production of nitric oxide and inflammatory cytokines, further underscoring the immunosuppressive role of this receptor[146].

6.5. Therapeutic Implications and Drug Development

Selective CB₂ receptor agonists are a new category of potential antidepressants that possess anti-inflammatory and neuroprotective characteristics without causing psychoactive effects associated with CB₁ receptor. Compounds such as HU-910, AM1241, and BCP exhibit anxiolytic and antidepressant effects in various preclinical studies[96,174]. Additionally, PAMs of CB₂ receptor could increase endocannabinoid levels without directly stimulating the receptor, providing more refined control with reduced side effects. Notably, therapies targeting CB₂ receptors could work in conjunction with selective serotonin reuptake inhibitors (SSRIs) or serotonin-norepinephrine reuptake inhibitors (SNRIs) by modulating the inflammatory environment, thereby diminishing the effectiveness of monoamines[149]. For instance, activating CB₂ receptors may restore glucocorticoid receptor sensitivity, boost BDNF levels, and enhance neuroplasticity, thus addressing the complex nature of depression. Overall, CB₂ receptors have a significant role in both immune regulation and neuroprotection in the context of depression. CB₂ receptor acts as a link between the immune system and CNS functions by modulating inflammation, releasing stress hormones, synaptic adaptability, and glial responsiveness. Future studies should focus on developing CB₂ receptor-selective compounds, examining their combined use with current antidepressants, and further investigating their epigenetic influences to maximize therapeutic outcomes.

7. CB₂ Receptor in Chronic Pain and Neuropathy

Chronic pain is a complex condition affecting millions around the world, often resulting from prolonged tissue damage, nerve injury, or disrupted neuroimmune signaling. It includes various types, such as inflammatory pain, pain related to cancer, and neuropathic pain[175,176,177,178]. Neuropathic pain occurs due to injuries or dysfunctions in the somatosensory system and often proves resistant to traditional pain relievers like nonsteroidal anti-inflammatory drugs (NSAIDs) or opioids[179,180,181]. There is growing evidence that the endocannabinoid system, particularly the CB₂ receptor, plays a significant role in managing chronic and neuropathic pain through immune regulation, glial signaling, and peripheral neuronal pathways.

7.1. Immune and Glial Contributions to Chronic Pain

Ongoing nociception is closely associated with the activation of the immune system. After a nerve injury, peripheral immune cells (such as macrophages and neutrophils) move into the site of injury, releasing pro-inflammatory substances like IL-6, TNF-α, and prostaglandins. These mediators make nociceptors more sensitive by altering ion channels and increasing receptor expression at the peripheral ends[182,183]. At the same time, microglia and astrocytes in the spinal cord and dorsal root ganglia (DRG) become active, furthering neuroinflammation by secreting cytokines, chemokines, and ROS. This interaction between glial and neuronal cells maintains central sensitization and allodynia. Significantly, following peripheral nerve injury, CB₂ receptor expression rises in DRG macrophages, spinal microglia, and astrocytes. This increase makes the CB₂ receptor a crucial molecular regulator that can help control excessive neuroimmune activation[184].

7.2. CB₂ Receptor Activation in Pain Modulation

CB₂ receptor agonists have demonstrated strong pain-relieving effects in various models of inflammatory and neuropathic pain[182]. In contrast to CB₁ receptor agonists, which might cause side effects in the CNS, such as sedation and dysphoria, CB₂ receptor-selective compounds provide pain relief without psychoactive effects due to their limited ability to penetrate the CNS or their preferential expression in activated immune cells[156]. Studies conducted in preclinical settings using CB₂ receptor agonists, such as JWH-133, AM1241, BCP, and HU-910, have exhibited significant reductions in mechanical and thermal hyperalgesia[140]. These outcomes are attributed to diminished glial activation, a reduction in pro-inflammatory cytokines, and the prevention of nociceptor sensitization. Activation of the CB₂ receptor leads to the downregulation of the NF-κB and MAPK signaling pathways, which in turn lowers iNOS, cyclooxygenase-2 (COX-2), and chemokine levels at the injury site. In both spinal cord slices and in vivo models, the administration of CB₂ receptor agonists has been shown to decrease excitatory neurotransmission by inhibiting glutamate release and restricting synaptic facilitation at dorsal horn neurons. Concurrently, CB₂ receptor activation boosts inhibitory GABAergic and glycinergic signaling, thereby restoring the excitatory-inhibitory balance disrupted in chronic pain conditions[163].

7.3. Endocannabinoid Tone and Peripheral Mechanisms

The two main endocannabinoids, 2-AG and AEA, are generated on demand by neurons and immune cells in reaction to injury. These substances activate CB₂ receptors in peripheral immune cells and spinal microglia, thereby modulating nociceptive signaling locally. The regulation of endocannabinoid tone through enzymes such as DAGL, MAGL, and FAAH is crucial for managing CB₂ receptor-mediated analgesia. Notably, inhibiting MAGL not only increases the levels of 2-AG but also reduces the production of prostaglandins, thereby further aiding in pain relief. Strategies that combine CB₂ receptor agonists with FAAH inhibitors have shown enhanced analgesic effects, highlighting the significance of endogenous ligand support for the control mediated by CB₂ receptor[4,139].

7.4. Cannabinoid Synergy and CB₁ Receptor/ CB₂ Receptor-Independent Pathways

Phytocannabinoids, such as cannabidiol (CBD) and cannabigerol (CBG), exhibit anti-inflammatory and pain-relieving effects, which are often partially mediated through CB₂ receptor signaling[4]. CBG functions as a weak antagonist of CB₁ receptor and a partial agonist of CB₂ receptor while also interacting with TRP channels and α2-adrenergic receptors[185]. In models of neuropathic pain, CBG alleviates hypersensitivity and mitigates the effects of microglial activation, which are reversed by CB₂ receptor antagonists, indicating its dependence on CB₂ receptors[186]. Importantly, the combination of CBD and CBG exhibits synergistic effects, likely resulting from their joint action on CB₂ receptors and TRP channels, as well as the suppression of cytokine release. This interaction between multiple receptors enhances the overall inhibition of neuroinflammatory pain pathways. Clinical investigations of balanced THC: CBD formulations such as nabiximols (Sativex®) have demonstrated effectiveness in treating neuropathic pain associated with multiple sclerosis, although the roles of CB₂ receptor compared to CB₁ receptor continue to be explored[187].

7.5. Immunological Insights and Translational Promise

From an immunological perspective, activating CB₂ receptors reduces the infiltration of peripheral macrophages, shifts macrophage phenotype from M1 to M2, and decreases spinal microglial activation, all of which are essential for resolving neuroinflammation[172]. In mice lacking the CB₂ receptor, observations have been made of heightened mechanical allodynia, prolonged cytokine levels, and increased glial reactivity following nerve injury, confirming the receptor's role in regulating pain through immune mechanisms[188]. Notably, CB₂ receptor signaling also influences mast cell degranulation and the movement of neutrophils in states of inflammatory pain, thereby broadening its relevance to visceral pain and autoimmune-related syndromes. In research models for rheumatoid arthritis and colitis, agonizing CB₂ receptor diminishes the trafficking of leukocytes and lessens nociceptive hypersensitivity, indicating the receptor's involvement in the broader immune-pain interaction[163]. There is a scarcity of clinical trials targeting the CB₂ receptor for pain management; however, selective ligands are beginning to undergo early-phase assessments. The limited expression of CB₂ receptor in disease-activated cells and the absence of CNS side effects underscore its potential as an appealing target for next-generation analgesics.

8. CB₂ Receptor in Schizophrenia and Dopaminergic Circuits

Schizophrenia is a multifaceted neuropsychiatric condition marked by positive symptoms (like hallucinations and delusions), negative symptoms (such as anhedonia and social withdrawal), and cognitive impairments. While earlier theories focused on dopaminergic dysregulation, particularly the heightened activity within the mesolimbic pathway, recent developments have broadened the etiological understanding to incorporate glutamatergic imbalance, neurodevelopmental irregularities, oxidative stress, and neuroinflammation[66,148]. Significantly, new evidence suggests that the endocannabinoid system, particularly the signaling of CB₂ receptors, plays a role in modulating various interconnected pathological areas.

8.1. CB₂ Receptor Expression in Schizophrenia-Relevant Brain Circuits

Historically, CB₂ receptor was viewed mainly as a peripheral receptor; however, it is now recognized to be inducibly expressed in numerous CNS regions relevant to schizophrenia, including the ventral tegmental area (VTA), nucleus accumbens (NAc), prefrontal cortex (PFC), and hippocampus. CB₂ receptor mRNA and protein have been identified in dopamine neurons, cortical pyramidal neurons, and glial cells under pathological circumstances through RNAscope, immunohistochemistry, and single-cell transcriptomics[66,189]. In the context of inflammation, excitotoxic stress, and dopaminergic overactivity, key features of the schizophrenic brain, CB₂ receptor expression is notably upregulated. Studies in rodents have demonstrated that CB₂ receptor is present in both the VTA's tyrosine hydroxylase-positive (dopaminergic) neurons and medium spiny neurons (MSNs) expressing D1 or D2 in the NAc[190]. The activation of CB₂ receptor in these areas leads to decreased neuronal excitability and reduced dopamine release, indicating a role in regulating dopaminergic tone.

8.2. CB₂ Receptor Modulation of Dopamine Signaling

The agonism of the CB₂ receptor results in neuronal hyperpolarization within dopaminergic circuits, which diminishes after depolarization and decreases dopamine release in key regions such as the striatum and PFC. Preclinical research utilizing selective CB₂ receptor agonists (JWH-133) has demonstrated that the activation of CB₂ receptors inhibits the hyperactivity of VTA dopamine neurons both in vitro and in vivo. Micro-dialysis investigations indicate that the administration of CB₂ receptor agonists into the NAc significantly decreases extracellular dopamine levels, while CB₂ receptor antagonists (AM630) enhance dopaminergic activity[140,190]. These effects do not occur in CB₂ receptor knockout mice, confirming the receptor specificity of the impact. This bidirectional modulation implies that CB₂ receptor signaling establishes a negative feedback mechanism within mesolimbic circuits. As hyperdopaminergic activity in the striatum is linked to psychotic symptoms, the attenuation by CB₂ receptor may serve to balance dopaminergic overactivity, presenting a potential mechanism for antipsychotic-like effects without the motor side effects associated with D2 antagonists.

8.3. Genetic and Epigenetic Links to Schizophrenia

Genetic research has further indicated the role of the CB₂ receptor in schizophrenia. Variations in the CNR2 gene, specifically single-nucleotide polymorphisms (SNPs), have been linked to an increased risk of psychosis, decreased CB₂ receptor expression, and altered inflammatory gene networks. For example, the Q63R polymorphism has been associated with reduced functionality of the CB₂ receptor and increased susceptibility to stress-related behavioral traits. In patients with schizophrenia, notable changes in methylation patterns and histone modifications are observed at the CNR2 gene locus due to epigenetic factors[191]. Environmental stressors, infections, and toxins recognized as risk factors for schizophrenia can trigger epigenetic alterations in CB₂ receptor expression within the CNS. These observations highlight the CB₂ receptor as a critical point of interaction between genes and environmental influences in this disorder.

8.4. CB₂ Receptor in Neuroinflammation and Glial Dysfunction

Neuroinflammation plays a significant role in the development of schizophrenia. Consistently, elevated levels of IL-6, TNF-α, and activated microglia have been observed in both postmortem studies and in vivo imaging of individuals with schizophrenia[192]. CB₂ receptors are potent regulators of immune responses, playing a crucial role in modulating the phenotype of microglia and the release of cytokines. In cases where the CB₂ receptor is absent, studies in mice have shown worsened inflammatory reactions and behavioral abnormalities similar to those seen in schizophrenia, such as reduced prepulse inhibition (PPI), altered social behavior, and heightened locomotor activity in response to psychostimulants. On the other hand, activating CB₂ receptor pharmacologically has been shown to reverse PPI deficits caused by MK-801 and restore abnormal dopamine signaling in models of schizophrenia[148]. Furthermore, the CB₂ receptor influences synaptic pruning by microglia, a process that is increasingly recognized as relevant to schizophrenia. Overactive pruning during the adolescent period may contribute to the cortical thinning and synapse loss observed in affected individuals. The activation of CB₂ receptor helps limit excessive microglial activity and may contribute to restoring synaptic balance, influencing neurodevelopmental pathways.

8.5. Endocannabinoid Tone and CB₂ Receptor Signaling Bias

The endocannabinoids 2-AG and AEA impact CB₂ receptor activity within the brain. In models of schizophrenia, levels of 2-AG are frequently found to be increased, and the dysregulation of enzymes that break down endocannabinoids, such as MAGL and FAAH, leads to circuit imbalances[193]. Importantly, the activation of CB₂ receptor can direct signaling toward either anti-inflammatory or neuroprotective pathways depending on the specific ligand and the cellular environment. Using biased CB₂ receptor ligands or PAMs could enhance the precision of therapeutic interventions. For instance, EC21a, a particular compound, selectively promotes CB₂ receptor-mediated anti-inflammatory and antinociceptive effects without fully activating the receptor, thereby minimizing potential side effects.

8.6. Translational Implications

Unlike conventional antipsychotic medications that primarily target dopamine D2 receptors and often lead to extrapyramidal symptoms, the modulation of CB₂ receptors presents a distinct mechanism of action with better safety profiles. CB₂ receptor agonists have the potential to address both hyperdopaminergic and neuroinflammatory conditions, thereby impacting various symptoms associated with schizophrenia. Although clinical trials focusing on the CB₂ receptor are currently limited, their number is increasing. Considering the multifaceted causes of schizophrenia, approaches that target multiple pathways, such as the combination of CB₂ receptor agonism with glutamatergic or neurotrophic strategies, could prove to be effective. The convergence of cannabinoid signaling, immune system regulation, and synaptic plasticity positions the CB₂ receptor as a promising target in the field of psychopharmacology.

9. CB₂ Receptor in Autoimmune and Inflammatory Diseases

Autoimmune and inflammatory disorders are intricate conditions characterized by dysfunctional immune responses, tissue damage, and prolonged inflammation, often instigated by a loss of immunological tolerance. Through the modulation of CB₂ receptors, the ECS has surfaced as an essential regulator in managing heightened immune responses and re-establishing tissue balance. It has demonstrated protective properties in various models of autoimmune diseases.

9.1. CB₂ Receptors in Asthma and Related Diseases: Immune Regulation, Innate Cell Crosstalk, and Therapeutic Paradox

Asthma is a long-lasting inflammatory condition of the airways characterized by fluctuating airflow obstruction, infiltration by eosinophils, excessive mucus production, and a prominent type 2 cytokine environment, often influenced by both innate and adaptive immune systems[194,195]. Growing research suggests that the CB₂ receptors act as a modulator of immune cell activation and tissue inflammation within this framework[114,196,197]. However, contrary to its anti-inflammatory role in various autoimmune contexts, CB₂R seems to exhibit a dual, and at times pro-inflammatory, function in allergic airway disease[73,114,197,198].

Ferrini et al. (2017) presented persuasive evidence that activating CB₂R fosters allergic airway inflammation in mice exposed to house dust mites (HDM). In their study, mice lacking CB₂ (CB₂⁻/⁻) displayed significantly lower levels of airway inflammation, with less eosinophilic infiltration, reduced recruitment of CD4⁺ T cells, and decreased mucus production, compared to wild-type (WT) controls[197]. Notably, this decline was not attributed to any shortcomings in adaptive immunity but instead connected to changes within the innate immune landscape of the lungs. CB₂⁻/⁻ mice exhibited a significant increase in the number of pulmonary NK cells, particularly those expressing DX5, NKp46, and granzyme A, which produced high levels of IFN-γ without generating Th2 cytokines. These NK cells negatively regulated group 2 innate lymphoid cells (ILC2s), which are critical producers of IL-5 and IL-13 during allergic reactions. The rise in IFN-γ-producing NK cells correlated with a suppression of ILC2 numbers and their function, implying a new immunoregulatory circuit through which CB₂R influences asthma development via the NK–ILC2 axis[197,199].

The causal significance of this pathway was further confirmed through both depletion and adoptive transfer experiments. Depleting NK cells in CB₂⁻/⁻ mice restored allergic airway inflammation and increased the presence of IL-13-expressing ILC2s, while transferring CB₂⁻/⁻ NK cells into WT mice inhibited HDM-induced inflammation. This evidence indicates that CB₂R signaling limits NK cell accumulation and IFN-γ production, thereby allowing for increased ILC2-driven type 2 responses. Mechanistically, endocannabinoids such as 2-AG inhibited IFN-γ secretion from activated NK cells in vitro, suggesting that CB₂ receptors act as a negative regulator of NK cell activity in the lungs[197,200]. Pharmacological investigations further supported this notion. Administering a CB₂ antagonist intranasally in WT mice replicated the phenotype observed in CB₂⁻/⁻ mice, reducing HDM-induced eosinophilia, CD4⁺ T cell influx, and goblet cell hyperplasia. Conversely, the CB₂-selective agonist HU-308 intensified allergic inflammation, aligning with the receptor’s pro-inflammatory impact in this scenario[197]. Additional research by Frei et al. (2016) also demonstrated that CB₂ receptor activation enhances eosinophil migration and responsiveness in allergen challenge models, suggesting that CB₂ receptor signaling may exacerbate eosinophil-dependent inflammation during asthma flare-up[198]. Moreover, Mimura et al. (2012) noted the role of endocannabinoids, such as 2-AG, in allergic inflammation, showing that endogenous ligands acting on the CB₂ receptor can affect immune cell migration and chemokine expression, including CCL2, a key player in Th2 polarization[201,202]. As such, 2-AG can be biosynthesized by eosinophils and is a potent inducer of their migration[203,204,205,206]. Ferrini et al. (2017) found that CB₂⁻/⁻ mice exhibited a significant reduction in CCL2 production and levels of type 2 cytokines (IL-4, IL-5, and IL-13) after allergen exposure[197].

In summary, these findings place CB₂R at a crucial crossroads between the regulation of innate immunity and allergic inflammation. Unlike its suppressive effects observed in autoimmunity, CB₂R signaling in asthma seems to restrain NK cells that would typically inhibit ILC2-driven eosinophilic responses. This paradox highlights the need for targeted strategies that are specifically tailored to the disease and cell type. Future treatment methods could benefit from selectively influencing CB₂ receptor signaling within specific immune subsets or utilizing biased agonism to refine their immune-regulatory effects in allergic conditions.

9.2. CB₂ Receptor in Multiple Sclerosis (MS)

Autoreactive CD4+ T cells invade the CNS and promote demyelination, neurodegeneration, and functional deficits in multiple sclerosis[207]. CB₂ receptor expression is elevated in activated microglia and immune cells penetrating the spinal cord during experimental autoimmune encephalomyelitis (EAE), a mouse model for MS[208]. The use of CB₂ receptor agonists like JWH-133 or BCP reduces the severity of the disease, diminishes CNS immune cell infiltration, and lowers levels of pro-inflammatory cytokines such as IL-17A, IFN-γ, and TNF-α[96,209]. Mechanistically, activating CB₂ receptor hinders NF-κB translocation and the formation of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, thus limiting the inflammatory response. Mice lacking CB₂ receptors exhibit aggravated EAE outcomes and more pronounced myelin damage, indicating that the endogenously increased CB₂ receptor offers a necessary regulatory mechanism against autoimmunity.

9.3. CB₂ Receptor in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is characterized by the infiltration of macrophages, T cells, and fibroblast-like synoviocytes (FLS) into the joints, resulting in cartilage degradation and bone damage. CB₂ receptor is predominantly expressed in synovial macrophages and FLS. The stimulation of CB₂ receptor leads to the inhibition of matrix metalloproteinase-3 (MMP-3) and MMP-9 secretion, decreases TNF-α and IL-6 levels, and diminishes oxidative stress by lowering iNOS and nitric oxide (NO) levels[210]. In models of collagen-induced arthritis, CB₂ receptor agonists reduce paw swelling and joint tissue damage[211]. Variants in the CNR2 gene, such as Q63R, have been associated with a higher risk of developing RA and more severe forms of the disease, underscoring the significance of the CB₂ receptor in the pathogenesis of RA[212]. Additionally, the CB₂ receptor facilitates M2 macrophage polarization and boosts regulatory T-cell (Treg) responses, which contribute to its immune-resolution capabilities.

9.4. CB₂ Receptor in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is characterized by hyperactive B cells, irregular dendritic cell signaling, and excessive production of type I interferons. The presence of CB₂ receptors in B cells can mitigate B-cell receptor-mediated growth and antibody generation. CB₂ receptor agonists have been shown to reduce the levels of anti-dsDNA autoantibodies and lessen the severity of glomerulonephritis in lupus-prone mice[213,214]. Notably, CB₂ receptor curtails TLR7/9-mediated IFN-α production from plasmacytoid dendritic cells (pDCs), which is a key pathway for cytokine activity in lupus[215]. It also promotes myeloid-derived suppressor cells (MDSCs) that secrete IL-10, aiding in immune regulation and reducing autoreactive responses. Changes in CNR2 expression and signaling among SLE patients highlight its potential as both a biomarker for the disease and a therapeutic target.

9.5. CB₂ Receptor in Inflammatory Bowel Disease

Inflammatory Bowel Disease (IBD), which includes Crohn's disease and ulcerative colitis, is characterized by chronic recurring inflammation of the gastrointestinal (GI) tract, resulting from an immune response to gut microbiota that is improperly regulated in individuals with a genetic susceptibility[216]. In IBD, the mucosal immune system becomes pathologically overactive, characterized by an exaggerated release of cytokines, dysfunction of the epithelial barrier, and persistent infiltration of immune cells, including T cells, neutrophils, and macrophages. Recently, CB₂ receptor has been recognized as a critical immunoregulatory modulator in the intestinal microenvironment.

CB₂ receptor is present at low levels in a healthy gut but shows a significant increase during periods of acute inflammation, particularly in lamina propria macrophages, CD4+ T cells, dendritic cells, and intestinal epithelial cells. In human biopsies from patients with IBD and preclinical mouse models, particularly those with dextran sulfate sodium (DSS)- and trinitrobenzene sulfonic acid (TNBS)-induced colitis, the expression of CB₂ receptor is linked to disease severity and immune cell activation[217]. Immunohistochemical studies highlight the presence of CB₂ receptor in the inflamed colonic mucosa, while flow cytometry confirms its elevated levels in infiltrating leukocytes. When activated by selective agonists such as JWH-133, HU-308, or AM1241, CB₂ receptor signaling has several protective effects within the gut. These effects include reducing the expression of pro-inflammatory cytokines (for example, TNF-α, IL-1β, IL-6, and IL-12), inhibiting neutrophil chemotaxis and oxidative bursts, and decreasing the expression of endothelial cell adhesion molecules, which helps to limit the migration of leukocytes into inflamed tissues. Additionally, CB₂ receptor activation enhances levels of anti-inflammatory mediators such as IL-10 and promotes tolerogenic dendritic cell phenotypes, leading to a more balanced mucosal immune response.

Importantly, CB₂ receptor also supports the integrity of the intestinal epithelial barrier, a key aspect of IBD pathology. Activation of CB₂ receptor restores tight junction proteins like occludin, claudin-1, and ZO-1, which are often downregulated in IBD, resulting in increased intestinal permeability, commonly referred to as leaky gut. Furthermore, CB₂ receptor stimulation reduces epithelial apoptosis and promotes wound healing, as demonstrated by organoid and epithelial scratch assays[140,218,219]. These benefits to the epithelium occur partly through interaction with the PI3K/Akt pathway, which is critical for cell survival and growth. CB₂ receptor signaling is also closely associated with other immuno-metabolic pathways in the gut, particularly PPAR-γ and TRPV1. The activation of CB₂ receptor enhances the nuclear translocation of PPAR-γ in macrophages and epithelial cells, bolstering anti-inflammatory transcriptional programs[220,221]. At the same time, CB₂ receptor reduces TRPV1-mediated neurogenic inflammation by modulating the signaling of capsaicin-sensitive afferent nerves and the output of cytokines[222,223,224]. This interconnected functional axis (CB₂ receptor–PPAR-γ–TRPV1) thus serves as a significant regulatory hub in intestinal inflammation.

Research using knockout models further emphasizes the contribution of CB₂ receptors to IBD pathogenesis. Mice lacking CB₂ receptor (Cnr2−/−) that were subjected to DSS or TNBS show more severe colitis, as evident by greater body weight loss, shortened colon lengths, increased histopathological scores, and elevated levels of inflammatory cytokines and ROS[225,226,227]. In contrast, pharmacological activation of CB₂ receptor alleviates these pathological features and reduces fibrosis, a common complication associated with chronic IBD, by inhibiting TGF-β signaling and preventing fibroblast activation. From a translational viewpoint, therapies targeting CB₂ receptor present promising adjunctive options for IBD management. CB₂ receptor activation has been associated with the upregulation of PPAR-γ signaling pathways, potentially contributing to anti-inflammatory responses in immune and epithelial cells through indirect crosstalk mechanisms[228]. Furthermore, because CB₂ receptor activation does not produce psychoactive effects, it represents a safer alternative compared to interventions targeting CB₁ receptor. Nonetheless, challenges persist in developing CB₂ receptor agonists that are specifically targeted to the gut and are peripherally restricted to minimize off-target effects while ensuring receptor selectivity. In conclusion, signaling through the CB₂ receptor plays a complex role in the development and resolution of IBD. It influences both immune cell functions and components of the intestinal epithelium to facilitate anti-inflammatory, barrier-protective, and antifibrotic responses. As a context-sensitive and inducible immunomodulator, CB₂ receptor represents a promising target for therapeutic strategies aimed at modifying chronic gut inflammation, restoring mucosal balance, and enhancing outcomes for patients with IBD.

9.6. Therapeutic Perspectives

Focusing on the CB₂ receptor provides an intriguing approach for treating autoimmune and chronic inflammatory conditions, as it can alter immune responses without causing psychoactive effects. The expression of CB₂ receptor can be induced in immune cells and inflamed tissues, making it a responsive and disease-related therapeutic target. In preclinical studies involving conditions such as MS, RA, IBD, and SLE, activation of the CB₂ receptor reduces the release of inflammatory cytokines, limits the recruitment of leukocytes, and aids in tissue repair.

10. CB₂ Receptor in Cancer and Immune Checkpoint Modulation

CB₂ receptor has been recognized as an unconventional but highly promising target for immunomodulation in cancer biology. Its presence in the tumor microenvironment (TME) and its role in immune regulation establish the CB₂ receptor as a modulator of tumor progression and a possible complement to immune checkpoint therapy[229]. This section examines the functions of the CB₂ receptor within the TME, its connection with immune checkpoint molecules, and its potential applications in clinical settings.

10.1. CB₂ Receptor Expression in the Tumor Microenvironment and Immune Checkpoints

CB₂ receptor is found at elevated levels in various tumors, including hematologic malignancies and solid tumors like breast, lung, colorectal, pancreatic, and glioblastoma[230,231,232]. Within the TME, CB₂ receptor is particularly concentrated in immunosuppressive niches, including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and exhausted CD8+ T cells[233,234,235,236,237]. CB₂ receptor influences leukocyte infiltration, angiogenesis, and the metabolic reprogramming of immune cells. In models of melanoma and glioma, activating CB₂ receptors has been shown to reduce pro-tumorigenic inflammation while restoring effector immune activities[238].

Emerging evidence suggests that CB₂ receptor expression within the tumor microenvironment contributes to the exhaustion of CD8+ T cells and NK cells, thereby impairing antitumor immunity. Its deletion enhances the response to immune checkpoint blockade, positioning the CB₂ receptor as a potential immunosuppressive checkpoint receptor in cancer[239]. CB₂ receptor signaling in TAMs and MDSCs contributes to immune suppression in the tumor microenvironment. Activation of CB₂ receptor promotes M2-like polarization and enhances IL-10 and Arg-1 expression, thereby facilitating the recruitment of Treg cells and inhibiting CD8+ T cell responses[240,241,242,243]. Conversely, CB₂ receptor blockade or deletion reprograms these myeloid cells toward a pro-inflammatory phenotype, boosting antitumor immunity[244].

Furthermore, CB₂ receptor activation enhances the presentation of antigens by DCs, leading to more effective T-cell priming and clonal expansion[245,246,247,248,249]. Notably, CB₂ receptor functionally interacts with traditional immune checkpoint pathways. Activation of the CB₂ receptor inhibits the STAT3 and NF-κB pathways, which in turn lead to the downregulation of PD-L1 expression on both tumor and antigen-presenting cells[250,251]. Additionally, it influences the IL-6/IL-10 axis, which regulates PD-1 expression in T cells[252]. In murine models of lung carcinoma and melanoma, CB₂ receptor agonists have been shown to decrease the expression of PD-1 and TIM-3 on CD8+ T cells, restoring their cytotoxic function and granzyme B production. CB₂ receptor activation encourages a shift from Treg to Th1-like immune responses, thereby enhancing anti-tumor immunity[253,254,255].

CB₂ plays a dynamic role in shaping the tumor microenvironment, particularly by influencing the behavior of immune cells and the tumor's vascular landscape[256]. Activation of CB₂ can shift TAMs away from an immunosuppressive, M2-like state toward a more inflammatory, M1-like profile, which better supports immune activation[257,258]. This shift helps reduce the presence of Treg and boosts the recruitment and effectiveness of CD8⁺ cytotoxic T cells, key players in anti-tumor immunity[257,259,260]. In tumors that typically exclude immune cells, often referred to as “cold” tumors, these changes can help break down barriers to effective immunotherapy. CB₂ also appears to regulate blood vessel growth by reducing levels of VEGF and related angiogenic signals, leading to more organized and less leaky vasculature[261,262]. This normalization of tumor blood vessels facilitates the entry and movement of immune cells through the tumor tissue.

Additionally, CB₂ influences the extracellular matrix by affecting enzymes such as MMPs and reducing TGF-β activity, thereby further improving immune cell access[263,264]. Some studies also suggest that CB₂ signaling can interfere with tumor cell proliferation by inhibiting growth factor pathways, such as EGFR and Ras[265,266]. Taken together, these effects position CB₂ not only as a target for controlling inflammation but also as a potentially valuable partner in cancer immunotherapy, especially in tumors that have been difficult to treat with checkpoint inhibitors alone. CB₂ receptor exists within a larger GPCR network involved in immune modulation. Importantly, CB₂ receptor counters immunosuppressive signals from adenosine A2A receptors (A2AR) and lysophosphatidic acid receptor 5 (LPAR5), both of which are associated with T-cell exhaustion. CB₂ receptor signaling mitigates A2AR-mediated suppression, thereby restoring mitochondrial function and glycolytic capabilities in T cells, which are key characteristics of sustained effector function[267,268,269].

Additionally, the CB₂ receptor influences intracellular calcium signaling and MAPK activation in DCs and CD8+ T cells, thereby bolstering immune surveillance[270,271,272]. In preclinical studies, selective CB₂ receptor ligands, such as JWH-133 and the phytocannabinoid BCP, have shown immunostimulatory and anti-tumor effects[140,272,273]. PAMs targeting CB₂ receptor further amplify these effects by making the receptor more responsive to endogenous ligands without disrupting normal signaling dynamics. Biased agonists that primarily activate MAPK or inhibit β-arrestin pathways present opportunities to fine-tune therapeutic responses and minimize side effects.

New delivery methods, such as tumor-targeted nanoparticles and liposomes that encapsulate CB₂ receptor agonists, are being developed to enhance bioavailability and reduce systemic exposure. These approaches are particularly relevant in solid tumors, where localized CB₂ receptor expression correlates with the disease severity. Although CB₂ receptor-specific drugs have not yet advanced to phase III clinical trials in oncology, early-phase studies and strong preclinical data suggest significant potential as a co-adjuvant with existing therapies. Stratification based on biomarkers will be crucial for patient selection and therapeutic optimization, which involves assessing CB₂ receptor expression levels, cytokine environments, and the types of immune cells present in the tumor microenvironment. CB₂ receptor appears to be a versatile, non-classical immune checkpoint receptor that reshapes the tumor-immune interface through various direct and indirect mechanisms.

11. Crosstalk Within the eCBome

The activity of CB₂ receptors within the growing concept of the eCBome does not function independently but collaborates with various signaling mechanisms, such as TRP ion channels, nuclear receptors, prostanoid pathways, and orphan GPCRs like GPR55 and GPR18[274]. This interconnected signaling framework captures the complexity of the roles of CB₂ receptors in immune modulation across both physiological and pathological conditions, including chronic inflammation, tumor immunity, and neuroimmune disorders.

One of the most significant interactions occurs between CB₂ receptor and TRPV1, a calcium-permeable ion channel that plays a vital role in pain signaling, oxidative stress, and immune response[275,276,277]. CB₂ receptor and TRPV1 are co-expressed in microglia, peripheral macrophages, and sensory neurons[278,279,280,281,282,283,284,285]. Evidence suggests that CB₂ receptor activation can mitigate TRPV1-driven neuroinflammation by reducing calcium influx, ROS generation, and the production of pro-inflammatory cytokines, such as IL-6 and TNF-α. Additionally, phytocannabinoids such as CBD and CBG, which exhibit weak binding to the CB₂ receptor, also act on TRP channels, displaying agonistic effects at TRPV1 and antagonistic effects at TRPM8, thereby enhancing the CB₂ receptor's contribution to an overarching anti-nociceptive and anti-inflammatory pathway[286,287,288]. In gastrointestinal disorders such as colitis and IBS, the combined modulation of CB₂ receptor and TRP channels improves epithelial barrier function, lowers neutrophil infiltration, and restores mucosal homeostasis.

The CB₂ receptor also exhibits a synergistic regulatory relationship with PPARγ, a transcription factor that regulates the expression of anti-inflammatory genes and mediates metabolic change[289]. Both endogenous and plant-derived cannabinoids, including AEA, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA), interact with CB₂ receptor and PPARγ to shift macrophages toward the M2 phenotype, increase IL-10 levels, and inhibit NF-κB signaling[290,291,292,293,294,295,296,297,298,299]. In DCs, this dual engagement promotes a tolerogenic phenotype, reducing antigen presentation and encouraging Treg development. Remarkably, stimulation of CB₂ receptors has been shown to enhance PPARγ transcription and function in monocytes and immune cells residing in adipose tissue, highlighting its role in mitigating adipose inflammation and preserving insulin sensitivity. In microglia, the interaction between the CB₂ receptor and PPARγ supports neuroprotection by limiting excitotoxicity and facilitating the clearance of phagocytic debris.

Another significant aspect of CB₂ receptor crosstalk relates to the prostaglandin system, particularly the COX-2-dependent conversion of AEA and 2-AG into prostaglandin-ethanolamides and prostaglandin-glycerol esters[300]. CB₂ receptor activation indirectly suppresses pro-inflammatory pathways that drive COX-2 expression and prostaglandin E2 (PGE2) production, potentially modulating immune suppression mediated by EP2 and EP4 receptors [301,302,303]. In the tumor microenvironment, this modulation can diminish the recruitment of MDSCs and Treg-mediated tolerance while facilitating the reactivation of CD8+ T cells[304,305,306,307].

CB₂ receptor signaling interacts with orphan GPCRs such as GPR55 and GPR18, which share endogenous ligands with the CB₂ receptor[308,309]. GPR55 generally stimulates pro-inflammatory responses in monocytes, microglia, and cancer-associated fibroblasts[310,311,312]. Conversely, activation of CB₂ receptor counteracts GPR55-induced cytokine production, indicating a receptor-level antagonism that may shape immune tone[313,314]. GPR18, a receptor involved in resolution-phase signaling and the chemotaxis of CD8+ T cells, shows some overlap with the CB₂ receptor in immune tissues[315,316,317,318]. The coordination of CB₂ receptor and GPR18 may play a role in reprogramming macrophages and responses during wound healing, although pharmacological separation remains challenging due to ligand promiscuity[319,320,321]. Collectively, CB₂ receptor stands out as a crucial signaling hub within the eCBome, integrating immune, metabolic, and neuronal signals through various receptor interactions. Its actions in regulating immunity are amplified and refined through interactions with TRP channels, PPARs, prostanoid pathways, and orphan GPCRs, creating a dynamic network that can finely adjust inflammation and immune resolution. These interactions carry significant implications for therapeutic approaches targeting polypharmacological modulation, particularly in conditions characterized by overlapping inflammatory and metabolic origins, such as cancer, neurodegenerative diseases, and autoimmune disorders. Thoughtful drug design that acknowledges this complexity, rather than isolating CB₂ receptor signaling, holds significant potential for developing the next generation of cannabinoid-based immunotherapies.