The Endocannabinoid System in Human Disease: Molecular Signaling, Receptor Pharmacology, and Therapeutic Innovation

The ECS is a fundamental regulatory system that maintains homeostasis across multiple organs, engaging functions that include neurological, immune, and metabolic endpoints. Despite the ECS's biological relevance, the system's complexity was not fully appreciated until the late 20th century. In the decades since, knowledge has evolved to position the ECS as an important system for neuroprotection, immune modulation, and gut–brain communication [1].

The ECS comprises three major elements: cannabinoid receptors, CB1 and CB2, endogenous ligands, anandamide and 2-arachidonoylglycerol, and metabolic enzymes, which catalyze the synthesis and degradation of endogenous cannabinoids and ECS activity [2]. CB1 receptors are expressed in the central nervous system (CNS) at areas of the brain including the hippocampus, the amygdala, and the cortex regions, where CB1 activity can alter neurotransmitter release, cognition, and emotions experienced. CB2 receptors are primarily expressed in immune cells, which help modulate inflammatory responses and maintain immune homeostasis [3,4].

There are also other receptors that involve GPR55 and TRPV channels, which may play a role in metabolic regulation and gut–brain signaling and further enhance the functional dimensions of ECS activity [5,6]. The primary endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), regulate ECS signaling through activation of CB1 and CB2 receptors. Anandamide, often referred to as the bliss molecule, typically displays affinity for activating CB1 receptors that regulate pain signaling, adaptation to stress, and mood [7]. Anandamide's functional relevance is circumstantially moderated by rapid treatment (functionally, degradation) via fatty acid amide hydrolase (FAAH). Contrastingly, 2-AG interacts with both CB1 and CB2 receptors, influencing synaptic plasticity, as well as immune activity with a specific inactivator, monoacylglycerol lipase (MAGL) [8,9].

In addition to playing a critical role in the nervous and immune systems, the ECS interacts with gut microbiota constituents to contribute to several functions, including gut epithelial barrier structure, immune function, and metabolic homeostasis. Gut microbiota generate short chain fatty acids (SCFAs) that can alter the activity of the ECS; thus, gut–brain communication and signaling via the ECS can modulate responses to inflammatory processes. Dysregulation of this system is associated with disorders including neurodegenerative disease, inflammatory bowel disease (IBD), metabolic disorders, and psychiatric illness, highlighting the ECS as a therapeutic target [10].

The ECS was first described in the 1960s, when an Israeli biochemist named Raphael Mechoulam isolated the active ingredient in Cannabis sativa, Δ9-tetrahydrocannabinol (THC). Sequencing efforts led to increased understanding of the physiological functions of the endogenous cannabinoid system, and, in the late 1980s, scientists identified CB1 and CB2 receptor subtypes as molecules that modulate the activity of the ECS. Subsequently, endogenous cannabinoids were discovered in the early 1990s. Since its identification, the ECS has been shown to be involved in many critical physiological functions, including pain perception, neuroprotection, metabolism, and immune regulation [11,12].

The ECS is central to maintaining a range of homeostatic physiological conditions because it integrates signals within complex interdependent systems (neurological, immune, metabolic).

To assess the role of the ECS as a functional bridge across neuroscience, immunology, and microbiota research, a table (Table 1) summarizing 25 important studies published in the last decade was created. The table summarizes the main aspects of research that integrated the ECS's influence within its interdependent physiological systems across a range of applications related to protective nervous functions (i.e., neuroprotection) or immunological functions (i.e., immune modulation), gut–brain communication, and therapeutic potential. The studies are presented to illustrate the progression of ECS research and the emergence of novel mechanistic and therapeutic insights. The table summarizes key aspects of each publication, including the model system, ECS targets, investigated mechanisms, and the therapeutic relevance of the findings. This summary will provide useful information describing the path the research trajectory has followed towards addressing an important and new area of clinical research, demonstrating the potential for cannabinoid-based therapies that may contribute to new approaches for treating complex multi-system diseases.

The ECS has recently been documented to play roles in metabolism, hormonal mediation, and circadian alignment. It is an energy balance reactor system that regulates appetite, glucose homeostasis, and deposition of lipids. It is the conductor of hormonal orchestras that regulates the hypothalamic–pituitary–adrenal (HPA) axis and reproductive health [37]. The ECS also interacts with circadian regulatory mechanisms, aligning biological processes with day–night cycles and maintaining temporal homeostasis across neural, immune, and metabolic systems. The ECS has demonstrated utility, even in the context of SARS-CoV-2 impacting the world, providing insight into how immunity can be modulated and even demonstrating antiviral behavior [38].

The ECS is a regulator of cross-talk between the nervous, immune, and gastrointestinal systems. This implies that the ECS is capable of integrating interactions among systems to regulate stable physiological homeostasis. The ECS represents a connection between the neurological, immune, and gastrointestinal systems as a functional and regulatory unity [39].

Neurospecific functions include CB1 receptors, which play roles in synaptic plasticity, the regulatory influence of neurotransmitter production, and neurogenesis. The ECS helps maintain cognitive functioning and the plasticity of neurons by modulating excitatory/inhibitory signaling to limit excessive excitation, which can relate to impairments like epilepsy [40]. Both ECS and the microbiota–gut–brain axis, which includes the HPA stress pathway, regulate stress and immune responses. While the HPA axis operates through systemic hormonal feedback loops, the ECS modulates stress and immune reactivity via localized, lipid-based signaling within neural, immune, and gut circuits [41,42,43].

The ECS is also a maintainer of immune homeostasis through interactions with immune cells' CB2 receptors, especially in macrophages, T cells, and microglia that modulate the intensity of immune and inflammatory responses. ECS-mediated signaling can also persist in limiting excessive inflammatory/immune responses that support tissue repair and immune tolerance [44]. CB2 receptor effects in the CNS can help manage neuroinflammation and excessive immune responses that can exacerbate neuroinflammatory processes [45,46].

Given its modulatory roles in the neuroregulatory, immune, and metabolic domains, the ECS will become an improved therapeutic target in complex disorders like Alzheimer's disease, IBD, and mood disorders. Dysregulation of ECS signaling has been reported in disease processes across organ systems, suggesting a critical space for intervention [47].

The ECS will be reported on as a key regulator of neuroscience, immunology, and microbiota interactions, which together integrate dynamics of signaling within these systems. This review provides an integrated analysis of its structural and functional character, paying particular attention to the cellular mechanisms that mediate cross-system interactions. In addition to reporting novel connections in signaling of the ECS, including the lesser-known receptors, systems approaches will be articulated focusing on emerging research opportunities in ECS's involvement in developing systems-mobilized immune responses, again relating that argument back to viral immune responses like those associated with SARS-CoV-2. Then, we will clearly identify conditions that could be improved through ECS interventions directly related to disorders where neuroinflammatory complaints, immune dysregulation, and metabolic imbalance are themes.

Integration of a multi-disciplinary perspective will provide opportunities for growth of knowledge but also further information beyond the boundaries of this review. The goal is to move towards an improved understanding of the ECS's potential for intervention in disease and develop viable innovative therapeutic pathways.

CB1R, a G-protein coupled receptor (GPCR), is one of the most abundant GPCRs in the mammalian brain and localized primarily to areas controlling higher-order functioning, such as the hippocampus (memory and learning), the basal ganglia (motor control), the cerebellum (co-ordination), and the prefrontal cortex (decision making) [49,50]. Activation of the CB1R is also dependent on depolarization, which is generated directly by activity and involves vintage signaling in which endocannabinoids (2-arachidonoylglycerol [2-AG] and anandamide [AEA]), generated postsynaptically, diffuse back to the presynaptic terminal to inhibit neurotransmitter release, particularly glutamate and GABA, which control excitatory and inhibitory synaptic transmission in these areas, thus modulating synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LDP) [51,52].

CB1R is also highly expressed in the enteric nervous system (ENS) outside of the CNS, where it modulates visceral nociception, motility, and permeability of the epithelial lining. Dysregulated CB1R signaling in the ENS has also been implicated in the pathophysiology of functional gastrointestinal disorders, such as irritable bowel syndrome (IBS), where dysfunctional gut–brain communication would be involved in the underlying mechanism of visceral pain and dysmotility [53].

Within the context of the immune system, the expression of CB1R is found in microglial and astroglial cells, although lower than CB2R, but it allows for anti-inflammatory modulation because it is capable of suppressing interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). In addition, CB1R is involved in the modulation of the hypothalamic–pituitary–adrenal (HPA) axis in order to attenuate stress-induced hyperactivation by maintaining systemic homeostasis [54].

CB2R shows low expression in the CNS from baseline but is abundant in tissues of immune and peripheral natures (macrophages, T cells, B cells, and dendric cells), where it regulates inflammatory balance [55]. Its activation results in a decrease in the concentration of pro-inflammatory cytokines (IL-6, TNF-α) and increases anti-inflammatory mediators, such as interleukin (IL)-10, which generate the stability of immune homeostasis and a decrease in autoimmunity [56].

CB2R is highly inducible during pathological inflammatory processes. In neurodegenerative diseases like Alzheimer's disease, upregulation of the receptors in microglial and astroglial cells is able to reduce oxidative stress and neuroinflammation, which results in neuronal survival [57]. This has rendered CB2R an important therapeutic target for autoimmune and neuroinflammatory disorders [45,58].

In the gastrointestine, CB2R may act in conjunction with immunological functions and the integrity of the epithelium of the intestine through potentiation of the proteins of tight junctions (occludin, claudin-1), which bind to enhance barrier function to prevent the translocation of antigens or microbes. This protective function is important in IBD, which includes Crohn's disease and ulcerative colitis. The signaling from CB2R may also effect a change in the composition of the physiological microbiota of the gastrointestinal tract and is able to mediate structurally and physiologically bidirectionally the interlocking interactions so that both immune tolerance and systemic inflammatory processes can be affected. Cannabinoid receptor type 2 (CB2R) is emerging as a therapeutic target for the treatment of infections like SARS-CoV-2, where activation of the receptor may lessen the release of cytokines from and promote reparative processes in damaged tissues (and thus potentially lessen morbidity and mortality) [59,60].

The cannabinoid receptors GPR55 and GPR18 and the TRP channels add to the complexity of ECS signaling. GPR55, formerly classified as an orphan receptor and showing a partial overlap in activity with CB1R/CB2R, binds to a number of endocannabinoids. Among them, lysophosphatidylinositol has been shown to mediate pro-inflammatory signaling in macrophages among other immune cells and recruitment of leukocytes and is involved in osteoarthritis, induction of cancer metastasis, and other inflammatory conditions [61,62].

The expression of GPR18 is found on several types of immune and endothelial cells; a key endogenous ligand for this receptor is N-arachidonylglycine (NAGly). The activation of GPR18 is involved in regulating the movement of immune cells into tissues, blood vessel constriction/dilation, and the level of inflammation. GPR18 agonists have been shown to prevent endothelial dysfunction and inhibit leukocyte infiltration in atherosclerosis, which demonstrates its potential use as a therapeutic target in reducing the inflammatory component of cardiovascular disease [63,64].

In addition to the above-mentioned effects of GPR18, it is involved in the modulation of mood through its participation in the gut–brain axis. Changes in the sensitivity of the GPR18 receptor may be caused by changes in levels of microbiota-derived metabolites, such as indoles, which are produced through alterations in the balance of the microbial composition of the gastrointestinal tract; these alterations may contribute to the development of stress-related mood disorders. GPR18 agonists administered to mice were shown to decrease stress-related behaviors, stimulate the generation of neurons in the hippocampus,, and normalize the microbial composition of the GI tract, which further supports its role in modulating the immune system and brain function [65,66].

The TRP channels, particularly the TRPV1 channel, historically identified with nociceptive and warmth sensing, can also be modified by the cannabinoid AEA, among others. TRPV1 abnormal functioning has been associated with pain responses to visceral stimulation and motility dysfunction, as well as IBD, which may be present in symptomatic subjects. TRPV1 functional dysregulation represents a possible target class of therapies [67,68].

Allosteric modulation of the CB1R and CB2R receptors appears to be a fertile area for therapeutic targeting. Allosteric modulators alter receptor activity through non-orthosteric modulation and titration of activity, avoiding side effects associated with respective full agonists or antagonists. This approach may have applications in chronic pain, metabolic syndrome, and diseases of neurodegeneration [69].

The ECS is an important part of maintaining gut barrier integrity and host interaction with the microbiota. The receptors CB1R/CB2R are associated with expression within the intestinal epithelial cells as well as regulation of junction proteins, such as zonula occludens-1 (ZO-1), which regulate intestinal epithelial permeability, as well as translocation of bacterial endotoxins (lipopolysaccharide, LPS, for instance), which reflect a protective strategy, in order to avoid systemic inflammatory response to obesity and metabolic syndrome and septic-related consequences [70,71,72].

The microbial metabolites, principally the short-chain fatty acids, also reflect a means through which there is modulation of the copy number and the synthesis of receptor component proteins and signaling, which engenders a feedback system and thus may integrate microbial messaging with respect to ECS-influenced regulatory-related pathways. Disruption of this microbiota–ECS interaction would appear responsible for disorders classed as psychiatric (depression, anxiety), metabolic (obesity), or inflammatory (IBD-related) [73].

Overall, it appears that cannabinoid receptors possess and act on gateways to complex intracellular signaling systems. These are at work with respect to physiological intranetworking network adaptation across systems. Sequentially, the next section will deal with endocannabinoid signaling systems and signaling pathways, demonstrating the respective interconnections that are thus able to act downstream from exogenous input stimuli to cellular functional processes and appropriate responses, illustrating the complexity of the system and correlating therapeutic benefits the ECS may provide [74].

ECS dysfunction appears to drive chronic disease states within the body; the lack of signaling drives synaptic downregulation, immune dysregulation, and breakdown of epithelial barriers. For example, neurodegenerative diseases, such as Alzheimer's or Parkinson's disease, demonstrate the links between ECS dysregulation and accelerating synaptic degeneration, neuroinflammation, and, possibly, oxidative stress [95]. Likewise, in the gastrointestinal tract, dysfunctional ECS signaling in IBD was shown to break down normal epithelial barrier function, thus sustaining gut inflammation. The ECS habitat is strongly implicated in metabolic diseases and insulin resistance, obesity, and systemic inflammation [96].

Therapeutic modulation of the ECS occurs through its integrated control over three major body systems, i.e., the nervous, immune, and gastrointestinal systems. The main ways to modulate the ECS include using CB2 receptor agonists to suppress neuroinflammation; using FAAH and MAGL inhibitors to elevate levels of endocannabinoids; and microbiota-directed treatments to enhance gut–brain–immune equilibrium [97].

In addition, emerging genetics and biotechnology strategies will also provide new options for the interventions described above. For example, CRISPR/Cas9-based genetic modification of the FAAH gene may be used to repair loss-of-function mutations of this gene and thus restore anandamide signaling in individuals with neurodegenerative disease. Similarly, genetically modified probiotic bacteria that produce endocannabinoid analogues are being investigated as a potential therapy to restore balance to the ECS in the gastrointestinal tract and treat systemic inflammation and disorders associated with dysbiosis [98].

The ECS operates via varied signaling mechanisms and thus operates as a homeostatic cornerstone and not just as part of disease pathology. In the next section, we will focus on how these signaling pathways contribute to both physiological and pathological states, including how they shape neurodevelopment, synaptic plasticity, and interactions between the gut, brain, and immune systems.

The ECS is functional in embryogenesis, where the presence of the CB1R in the neural progenitor cell (NPCs) enhances proliferation, migration, and differentiation. CB1R signaling represses adenylate cyclase and Gi/o protein predominating in pathways that modulate Wnt/β-catenin signaling and CREB signaling, which determines NPCs' fate. Also under the influence of CB1R is cytoskeletal remodeling, which through RhoA-ROCK signaling enhances neuronal migration and axonal elongation, which are important for creating the cortical and subcortical circuitry [99]. From the signal epigenetically is CB1R, which alters chromatin architecture through histone acetylation and methylation at loci like Sox2 and NeuroD1. The maladaptive changes occurring in this vital developmental period enhance the risk of development of disorders like autism spectrum disorder (ASD) and schizophrenia, and, in this context, the ECS represents an option for molecular scaffolding of neuroarchitecture [100,101].

With respect to what has been written about endocannabinoid signaling, the important issue is synaptic plasticity, which is the cellular basis of learning and memory. Endocannabinoids, such as 2-AG and anandamide, are produced in a localized area to allow for retrograde action to inhibit presynaptic release of glutamate and GABA and maintain the normal balance of excitation–inhibition [102]. The role of CB1R within the hippocampus is critical to long-term potentiation (LTP) and LTD—two processes essential for memory consolidation. ECS signaling through astrocytes modulates the expression of EAAT2, which is necessary for limiting glutamate-induced excitotoxicity. The CB1R also aids in supporting myelination in oligodendrocytes, which is important to support action potential conduction along axon segments in an effective and stable manner [103]. Overall, these findings indicate that the ECS is a modulator and stabilizer of neural network activity [104,105].

The immune cells of the CNS, microglia, are key effector cells of neuroinflammation and highly susceptible to modulation by ECS signaling. Under conditions of homeostasis, microglia constantly survey neural tissues for inflammation and tissue injury [106]. Upon activation, CB2R shifts microglia to an anti-inflammatory phenotype, upregulating IL-10 while downregulating IL-6 and TNF-α [107]. CB2R also rewires microglial metabolism from glycolysis to oxidative phosphorylation, resulting in reduced toxic reactive oxygen species and enhanced clearance of amyloid-β and cellular debris [108]. Another receptor in the ECS, GPR18, promotes directed microglial migration and phagocytosis in the presence of neuronal injury, further contributing to CNS repair [63].

Dysregulation of the ECS contributes to neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. CB1R signaling ameliorates glutamate-induced excitotoxicity and induces the expression of various anti-apoptotic proteins (e.g., Bcl-2), while CB2R induces enhanced autophagic clearance of aggregates of tau and α-synuclein [109]. CB1R also promotes mitochondrial stability through interaction with the mitochondrial calcium uniporter, resulting in a stable mitochondrial membrane potential and prevention of mitochondrial depolarization. CB2R enhances blood–brain barrier (BBB) integrity, leading to a decrease in immune cell infiltration to the CNS. Collectively, these findings describe the ECS as a target and regulator of neurodegeneration [96].

The ECS regulates mood, emotional resilience, and adaptation to stress, which are intimately linked to the gut–brain axis. The anandamide level is directly correlated with the activity of the HPA axis; activation of CB1R inhibits the release of corticotropin-releasing hormone and the release of downstream cortisol, decreasing chronic stress levels, but the synaptic levels of anandamide are low with decreased expression of CB1R and correlate with psychopathology, including anxiety, depression, and post-traumatic stress disorder (PTSD) [110]. Microbial metabolites stemming from the gut (e.g., short-chain fatty acids (SCFAs), butyrate) affect the ECS by leading to increased expression of CB1R and CB2R in the frontal cortex and hippocampus and provide a mechanistic rationale for microbiota-directed treatment of mood [111]. Inhibition of FAAH, which degrades anandamide, also increases the tone of endocannabinoids and thus normalizes gut microbiota towards a balance associated with stress, suggesting bidirectional therapeutic effects [112].

ECS effects also display circadian variations. Daily variations in the expression of CB1R in the suprachiasmatic nucleus synchronize the activities of neurons to light–dark cycles. Deviations in daily patterns (as with shift work or jet lag) contribute to dysregulation of the HPA axis and instability in mood. Therapies that modulate the ECS in belief of its pattern might enhance affective resilience [113].

Recent investigations have shed light on the mechanistic role of the ECS in the epigenetic regulation of neural plasticity. Stimulation of CB1R evokes histone acetylation in the promoters of the neurotrophic genes, such as BDNF, that enhance memory consolidation and functionality after injury [114]. The phenomenon of receptor heteromerization provides terrific diversity to ECS signaling. Thus, CB1R demonstrably heterodimerizes with TRPV1 in sensory components, leading to effects in pain and inflammation. GPR55–CB2R complexes are noted to be present in microglial elements, permitting dual points of therapeutic intervention in neuroprotection and neurorepair [99,112,115].

These modalities suggest that the ECS has an integrative capacity, referring to neurological, psychiatric, and immunological situations, and allows for the development of dual-agonist or microbiota effects companies in the larger area of personalized medicine. From a systems perspective, the ECS is responsible for processes of neurodevelopment, synaptogenesis, anti-inflammatory processes, and gut–brain communication such that it serves as a molecular substrate for reparative and adaptive processes. Its next roles can be seen in gut–brain–immune intercommunications and translational applications of its benefits to the health of the body.

The level of microbial metabolites is greatly influential on the ECS in terms of receptors and enzymes (and, in some cases, receptors) for all ligands. Also, fatty acid products, derivates of tryptophan, and secondary bile acids regulate CB1R and CB2R, forming and degrading the endocannabinoids, such that homeostatic intestinal repair is maintained [116,117].

The products of indole (tryptophan metabolism) produced by bacteria are responsible for the stimulation of anandamide production and stimulants to vagal afferents downstream such that there is a connection between microbial metabolism and regulation of emotions and stress. In germ-free species, it is observed that there is a decrease in cortical levels of anandamide and respiration related to increased stress reactivity and an inability to regulate emotional aspects of life [118].

The effects of some of the metabolites derived from the microbiota also communicate through CB2R in gut-associated lymphoidal tissue (GALT), where there is modulation of macrophagic and dentic cell functions towards modifications that alter the responsiveness of the immune system towards tolerance. Increased dysbiosis is seen in Crohn's disease, which gives rise to the imbalance that occurs, which alters mucosal inflammatory responses [119].

The ECS controls the balance of gut barrier integrity by means of its regulatory effects on tight junction proteins, such as the zonula occludens-1, and also claudins, where it prevents the translocation of antigens from the gut [120]. While the stimulation of epithelial healing and regeneration is related to CB1R, CB2R is closely linked with the inhibition of pro-inflammatory cytokines and stimulation of the synthesis of IL-10, which can be viewed as a means whereby recovery of the tissue is mediated [121,122,123,124].

The ECS is linked to the stimulation of mucous production from goblet cells, permitting colonization of microflora and protection of epithelial cells. Short-chain fatty acids (SCFAs) derived from the diet enhance this function, while high doses of saturated fats diminish the density of CB receptors and alter the integrity of the barrier [125,126,127,128].

These interactions indicate the importance for systemic and gut health of feeding, while attaching dietary–microflora–ECS together. This is seen in Figure 2, indicating the interaction of microflora, mediators of immune function, and the systems of the nervous system, including, in particular, the vagus nerve, with regard to the production of the ECS as well as that of the HPA axis, indicating the link between diet and resistance to stress.

Interactions of probiotics, prebiotics, and the ECS maintain gut barrier integrity and modulate inflammatory states. Particular strains of Lactobacillus and Bifidobacterium induce increases in levels of the endocannabinoid anandamide in intestinal tissues, resulting in enhanced epithelial resistance and reductions in visceral hypersensitivity via CB1R-dependent pathways [129]. Metabolites derived from probiotics also induce signaling via the CB2R on macrophages, leading to decreases in the release of pro-inflammatory cytokines, such as TNF-α and IL-6, but increases in the production of IL-10, which supports immune homeostasis. Prebiotic fiber sources, such as inulin and fructooligosaccharides, hydrate, ferment, and provide substrates for short-chain fatty acids, particularly butyrate, which acts a histone deacetylase inhibitor, inducing both CB1R and CB2R expression in conjunction with tight-junction expressed genes, maintaining epithelial integrity. Synbiotic supplementation of probiotics with prebiotics enhances both microbial diversity in the gut and CNS tone, decreasing colonic permeability and inflammatory activity [130]. Pharmacological augmentation of ECS tone can add further efficacy to these approaches. Inhibition of the enzymes FAAH and MAGL leads to elevations in levels of endogenous cannabinoids, restoration of tight junction functionality, and decreased levels of neutrophil infiltration in experimental models of acute colitis. Combination strategies targeted to the ECS and the gut microbiota would therefore appear to produce coordinated barrier-protective and anti-inflammatory effects, representing a possible experimental paradigm for future translational studies.

The microflora and the ECS can thus work together to present a unitary homeostatic apparatus of interaction, which can be modulated by circadian frequencies. It would in addition now appear that the microbial metabolites are capable of altering the diurnal release of CB1R in gut tissues, thus permitting ECS activity to be synchronized with appetitive periodicities. Loss of circadian synchronization caused by erratic sleep or diet induces changes to gut barriers and the release of systemic inflammatory mediators, thus showing the all-important, changing nature of chronobiology, indicating the necessity of taking this into consideration when devising remedial strategies in ECS dysregulation [131,132,133].

Still further, the microflora can influence the epigenetics of ECS elements, with butyrate-enhanced histone acetylation levels at the CB1R and CB2R gene promoter sequences being responsible for an increase in levels of expression of these receptors in epithelial and immune cells. This shows the way in which microbial diversity can dynamically change the level of expression of the host organism's genes and alter its responsivity of ECS [109].

In conclusion, the ECS mediates communication between the ECS, the gut, and the brain–immune axis, modulating the microbial and immunological signals into physiological homeostasis. Through appropriate maintenance of the purity of the barrier, regulation of the nature of the microbial ecology, and alteration of the levels of inflammation, the ECS presents us with a useful framework for understanding complex disease states and hence the development of rational medicines for the promotion of systemic health.

The ECS has many layers of influence on immune cells, balancing anti-inflammatory and pro-inflammatory immune system activity. CB2 receptors, mostly expressed on immune cells, are the master regulators of this balance, and CB2 activation prevents inflammatory processes through inhibition of inflammatory pathways, such as nuclear factor-kappa B (NF-κB), MAPK, and Janus kinase-signal transducer and activator of transcription (JAK-STAT), indirectly inducing cytokine storms while promoting immune tolerance and tissue repair by resolving the pro-inflammatory response [88,134,135].

Some of the most influential immune cells, macrophages, are affected by CB2 signaling and by promoting the transition from the pro-inflammatory phenotype (M1) to the reparative phenotype (M2), which is mediated by metabolic reprogramming. During this metabolic reprogramming, CB2 activation enhances fatty acid oxidation and mitochondrial biogenesis while reducing glycolysis, which promotes inflammation [136]. Viewing these changes from a regulatory perspective, a great benefit of this metabolic switch is that it promotes regeneration in the tissues and protects a macrophage's ability to survive long anti-inflammatory responses, which is necessary for chronic diseases that exhibit inflammation [137].

In T cells, the function of CB2 signaling helps to modulate the balance of pro-inflammatory T subsets, such as Th1 and Th17, with regulatory T cells (Tregs) through regulation of two transcription factors, T-bet and RORγt, suppressing the pro-inflammatory portion of Th1 and Th17. Additionally, if regulation of FoxP3 expression occurs, this will enhance Treg expansion. This is also a possible explanation for the ECS's involvement in inflammatory diseases, such as multiple sclerosis and systemic lupus erythematosus, that have the potential to push out Th1/Th17 acceleration [138,139,140].

More recently, we have started to isolate non-coding RNA, especially microRNAs (miRNA), that can mediate ECS function as modulators of the immune system. The ECS also has the ability to induce miR146a when activated by CB2, and it has a highly significant suppressor ability on the NF-κB signaling pathway by suppressing the inflammatory cytokine production ability of TNF-α and IL-6 [141]. The most notable of these miRNA mediators is miR-155, which inhibits T cell dysregulation and macrophage activation with some pro-inflammatory potential. The miRNA portion of this process is able to account for another dimension of the ECS's immune-modulatory influence and suggests that there is possibly an epigenetic influence associated with immune cells [142,143].

With regard to the CNS, the resident immune cells in microglia are in an energetic environment and respond to ECS signaling. Activation of CB2 may alter pro-inflammatory cytokine production from microglia and enhance the secretion of neurotrophic factors (BDNF) that could possibly translate to enhanced synaptic repair and provide protection against the inflammatory damage of the surrounding neuronal network [144]. Particular developmental influences of the ECS include modulating the microglial phagocytosis of cellular debris and toxic protein aggregates and, ultimately, downregulating inflammatory cascades through the degradation of cellular pattern recognition receptors, which stimulate the neurodevelopmental cascades that produce neurodegenerative effects, speeding up their influence on acceleration [57].

Table 2 intends to be an all-encompassing summary of original studies describing the ECS. We reviewed significant findings across various domains, including anxiety, pain insensitivity, drug discovery, and established ECS interactions with the gut microbiota. The table is divided into five columns: study reference, research focus, methodology, findings, and outcomes. Each study entry briefly describes how the research contributes to the current understanding of the role of the ECS in health and disease and, ultimately, the therapeutic possibilities.

Figure 3 provides a visual representation of the way cannabinoids bind to these receptors, illustrating that the ECS has a dual functionality in neurons and immune cells.

The ECS is essential in regulating autoimmune and inflammatory diseases by regulating interactions between different immune cell subsets and tissue-specific responses. In autoimmune diseases, such as multiple sclerosis (MS), the ECS reduces CNS inflammation by inhibiting the migration of autoreactive T cells across the blood–brain barrier. CB2 activation attenuates chemokine signaling, limits T cell invasion on patrolling inflammatory cells, and decreases astrocyte and microglia release of inflammatory mediators, such as IL-1β, TNF-α, etc. In preclinical models of MS, CB2 agonists display a decrease in demyelination and neuronal loss, demonstrating that the ECS has therapeutic possibilities in neuroinflammation [162,163,164].

In the gastrointestinal tract, the ECS maintains intestinal homeostasis by regulating the immune responses that underlie IBD. CB1 and CB2 signaling promote epithelial barrier function through tight junction proteins (occludin and ZO-1) that limit microbial translocation and prevention of systemic inflammation that leads to colitis. Evidence has shown that CB2 activation can reduce inflammatory cytokines released from macrophages and T cells, which would lead to tissue repair and reduced severity in IBD. Clinical trials testing cannabinoid-based therapies in patients with Crohn's disease and ulcerative colitis have shown efficacy when testing treatments to impact outcomes of disease and markers of disease activity, which support therapeutic targeting of the ECS in gut inflammation [53,165,166,167].

The role of ECS modulation is important in systemic autoimmune diseases like systemic lupus erythematosus (SLE). One example of this is the use of CB2 agonists, which reduced hyperactivation of B cells and inhibited pathogenic autoantibodies that promote the tissue damage that drives SLE. Furthermore, the ECS could promote Tregs' ability to inhibit autoreactive T cells and restore immune tolerance to reduce the number of flares in the disease.

The ECS is capable of facilitating tumor-associated immunity in the highly dynamic and hostile tumor microenvironment. By activating CB2 on tumor-associated macrophages (TAMs), the phenotype can flip from a tumor-supportive M2-like state to an M1-like anti-tumor state while simultaneously promoting recruitment and activation of cytotoxic T cells and inhibiting the release of pro-tumor cytokines like IL-10 and transforming growth factor-beta (TGF-β). The ECS can effectively remodel the tumor microenvironment and destroy immunosuppressive barriers/modulations/reservations that tumors depend on for immune evasion [168,169,170].

Cannabinoid-based therapies can also target/impose immune checkpoint pathways tumors use to inhibit T cell activity. Recent studies have suggested that ECS signaling may alter PD-L1 expression in tumor cells, thus altering PD-L1 activity as a method to inhibit T cell activation. CB1 signaling can alter angiogenesis via endothelial cells, thus removing the vascular supply tumors utilize for growth and metastases [171,172,173].

ECS-targeted strategies have also been looked at from an agricultural perspective, as it has been reported that some cannabinoids can affect plant stress responses and pest resistance behaviors. A series of recent studies have demonstrated that cannabinoid analogs can be applied to crops to improve drought response capabilities via lipid signaling pathways similar to those underlying ECS functioning in humans. As an aside, from an agricultural point of view, further studies specific to ECS biology are also forthcoming, and these results present exciting opportunities to harness ECS-based research for sustainable agriculture [174,175].

The implications of the ECS for tumor immunology continue to expand to include potential synergy with already existing treatments. By modulating the ECS early and reducing inflammation, it may be theoretically possible to enhance immune cell infiltration into tumors, particularly while employing immune checkpoint inhibitors and adoptive T cell therapies. The ECS is also an attractive target for integrative cancer immunotherapy due to its potentially multi-faceted effects, as well as its already established effectiveness in mitigating common side effects in cancer patients [176].

The ECS, as an active and developing area of interest in biomedical research, is also at the verge of innovative studies with tools like artificial intelligence (AI), machine learning, and multi-omics approaches. The emergence of AI/machine-learning-based approaches has the potential to revolutionize how we study ECS functioning in health and disease and enables a deeper, more collaborative approach when studying ECS functions. There is exciting potential for these new technologies to transform ECS-focused therapeutic discovery, as they can utilize precision medicine, where medications, including ECS-targeted drugs, can be matched to patients' unique phenomena based on individual patients' distinct molecular make-up as it pertains to ECS function. New and exciting applications utilizing AI and machine learning in ECS fields have begun.