The immune system consists of both adaptive and innate immunity. Innate immunity is the rapid and non-specific response to pathogens mediated by myeloid cells and natural killer (NK) cells. On the other hand, adaptive immunity is a slower but specific response that generates immunological memory, involving the activation of B and T lymphocytes (Netea et al., 2019). During normal inflammation, innate immunity is activated within minutes to hours as a first line of defense against pathogen infection, followed by the elimination of the threats carried out by both the innate and the adaptive immune responses (Netea et al., 2019). Ending inflammation and returning to homeostasis is a process known as resolution. However, failure to remove the inciting stimulus efficiently can lead to the development of chronic inflammation and progression of tissue damage (Feehan and Gilroy, 2019). This kind of chronic, unresolved inflammation contributes significantly to various pathogeneses, including that of asthma (Pothen et al., 2015), COVID-19 (Effenberger et al., 2021), atherosclerosis (Galkina and Ley, 2009), chronic obstructive pulmonary disease (Sevenoaks and Stockley, 2006), inflammatory bowel disease (Actis et al., 2019), neurodegenerative disease (Perry, 2004), multiple sclerosis (Sá, 2012) and rheumatoid arthritis (Masoumi et al., 2021).

Cannabis (Cannabis sativa) has been used as medicine for the treatment of inflammation for millennia, but its use in modern medicine has been hampered by a lack of scientific knowledge (Ryz et al., 2017). Previous studies reported that cannabis extracts and inflorescence inhibited inflammatory responses in vitro and in pre-clinical and clinical studies. For example, a high-CBD cannabis ethanolic extract reduced the release of skin inflammation mediators in keratinocytes (Sangiovanni et al., 2019). Similarly, a study on a mouse model of colitis showed that oral or intraperitoneally treatment with high-CBD cannabis extract led to a reduction in intestinal inflammation and hypermotility, in contrast to pure CBD treatment at matched doses (Pagano et al., 2016). Moreover, two clinical trials on patients with Crohn’s disease reported that daily treatment with THC-rich cannabis inflorescence had beneficial effects against the disease symptoms with no significant side effects and reduced the need for other medications (Naftali et al., 2011; Naftali et al., 2013). In another clinical trial, daily cannabis treatment was associated with lower levels of pro-inflammatory biomarkers in cerebral fluid (CSF) of HIV patients (Watson et al., 2021).

Great efforts have been made to suppress chronic inflammation. Cannabis and its compounds were shown to have anti-inflammatory activity (see Appendix A for methodology), but to exploit the full potential of cannabis it is important to define the active molecules and understand the cellular and molecular mechanisms that underlie its anti-inflammatory activity.

Monocytes are the major starting entities of inflammation. Once released from bone marrow, monocytes migrate through the blood into various tissues and undergo the tissue-specific maturation required to become inflammatory macrophages that respond to infection, injury, or damage. The various sub-populations of activated macrophages may differ in morphology, release of inflammatory mediators and functional properties, but in inflammation they have three major functions: phagocytosis, antigen presentation and immunomodulation (Fujiwara and Kobayashi, 2005). The process of inflammation is orchestrated via inflammatory mediators. Pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β are released from activated macrophages in response to infection (Figure 1; Abdulkhaleq et al., 2018). TNF-α and IL-1β act through specific cell membrane-bound receptors and participate in the recruitment of polymorphonuclear neutrophils (PMNs) into the site of infection and their activation (Hackel et al., 2021).

TNF-α facilitates the release of other pro-inflammatory cytokines from immune effector cells, including interferon alpha (IFN-α), interferon gamma (IFN-γ), IL-1β, IL-6, IL-8, Transforming growth factor beta (TGF-β) and chemokines (Silva et al., 2019). Further, in cases of enhanced inflammation, when the cell is stimulated, typically by bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines, there is induction of inducible nitric oxide synthase (iNOS). An increase in iNOS levels generates significant amounts of nitric oxide (NO) radicals or cyclooxygenase 2 (COX2); COX2 catalyzes the conversion of arachidonic acid to prostaglandins (PGs), prostacyclin and thromboxane A₂ (Salvemini et al., 2013; Cinelli et al., 2020).

The signal transduction of inflammatory responses involves several signaling pathways including mitogen-activated protein kinase (MAPK), toll-like receptor (TLR), Janus kinase/signal transducers and activators of transcription (JAK-STAT), and nuclear factor kappa B (NFκB) pathways (Figure 1; Zhao et al., 2021). The activation of these pathways involves a series of phosphorylation events leading to the induction of various anti-apoptotic target genes and the expression of cytokines, chemokines, and adhesion molecules (Taniguchi and Karin, 2018; Fitzgerald and Kagan, 2020). Moreover, during inflammatory processes, reactive oxygen species (ROS) are commonly multiplied and can contribute to host cell and organ damage. Further, intracellular redox changes induced by ROS augment NF-κB activation through the phosphorylation and degradation of IκB by increasing IkB kinase ß (IKK) or Akt kinase activity (Haddad, 2002).

Resolution of inflammation may involve increased production of IL-10, among others. IL-10 is an anti-inflammatory cytokine, which inhibits the release of lipid mediators and pro-inflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α; Figure 1; Panigrahy et al., 2021).

The endocannabinoid system (ECS) is a modulator of multiple physiological activities, including in the nervous, endocrine, immune, blood circulation, gastrointestinal tract and reproductive systems (Di Marzo et al., 1998). Accordingly, dysregulation of the ECS is involved with various pathological conditions, including inflammation among others (Di Marzo and Piscitelli, 2015; Hillard, 2018), whereas therapeutic modulation of ECS activity has beneficial effects on various medical conditions, including those associated with inflammation (Ambrose and Simmons, 2019; Giacobbe et al., 2021). ECS is involved in both innate and adaptive immunity and in several chronic inflammatory diseases (Chiurchiù et al., 2015). ECS includes cannabinoid receptors types 1 and 2 (CB1 and CB2, respectively) and multiple other receptors such as the peroxisome proliferator-activated receptors (PPARs) and ion channels (e.g., the transient receptor potential ankyrin [TRPA] family and the transient receptor potential vanilloid [TRPV] family) (Biringer, 2021). Also included in the ECS are the receptors’ ligand, arachidonic acid derived endocannabinoids, and enzymes for endocannabinoid metabolism (Di Marzo et al., 1998).

Most immune cells express endocannabinoids, the enzymes regulating their biosynthesis and degradation, and endocannabinoid receptors (Chiurchiù et al., 2015). Both CB1 and CB2 are expressed in immune cells, with CB2 being expressed 10–100 times higher than CB1 in these cells (Jean-Gilles et al., 2010; Rahaman and Ganguly, 2021). Moreover, CB receptor activation regulates anti-inflammatory responses. For example, activation of CB2 receptors by its agonist inhibited the release of the pro-inflammatory cytokine IL-12 and IL-23 and enhanced the release of the anti-inflammatory cytokine IL-10 from cultured activated macrophages. This study suggested that the inhibitory effect of CB2 on IL-12 production was mediated by ERK1/2-MAPK (Correa et al., 2009).

In another example, a CB2 receptor agonist reduced in human peripheral blood mononuclear cells LPS-induced ERK1/2 and NF-kB-p65 phosphorylation and release of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 (Capozzi et al., 2021). A selective/inverse agonist of CB2 induced the differentiation of Th0 cells into regulatory T cells (Treg) cell phenotypes in a naïve CD4⁺ T lymphocyte population isolated from a mouse spleen. The Treg phenotype is important for suppressing immune response by inhibiting T cell proliferation and cytokine production. The Treg phenotype was induced via P38 phosphorylation and STAT5A activation and was characterized by the expression of FoxP3, TGF-β and IL-10. Accordingly, treatment with this CB2 selective/inverse agonist reduced colitis severity in vivo (Gentili et al., 2019).

Cannabis produces a large number of phytocannabinoids (Hanuš et al., 2016). Phytocannabinoids are aromatic oxygenated hydrocarbons, derived from meroterpenoids with a resorcinyl core structure with isoprenyl, alkyl or aralkyl substitutions. The characteristic alkyl side chain typically contains an odd number of carbon atoms (Hanuš et al., 2016; Gülck and Møller, 2020). They are produced in the plant in their acid form and are decarboxylated to the active form (Gülck and Møller, 2020). Among the phytocannabinoids, Δ⁹-trans-tetrahydrocannabinols (Δ⁹-THCs) and cannabidiols (CBDs) are the most abundant (Table 1). Cannabigerol (CBG) in its acid form (CBGA) serves as a core intermediate that diverges to provide the phytocannabinolic acids (Table 1; Hanuš et al., 2016; Tahir et al., 2021).

In addition to phytocannabinoids, cannabis produces a plethora of non-cannabinoid constituents including a vast array of terpenes as the second-largest class of cannabis constituents (El Sohly et al., 2017). Cannabis biosynthesizes flavonoids as well, among them cannflavins, which are prenylated (C5) and geranylated (C10) flavones (Bautista et al., 2021).

The synergy between phytocannabinoids (Mazuz et al., 2020; Anis et al., 2021) as well as between phytocannabinoids and terpenes (Namdar et al., 2019) has been demonstrated. Pre-clinical evidence suggests an ‘entourage effect’ might be inferred from the superior medical activities of full-spectrum cannabis extracts versus single molecules (Koltai et al., 2019). Furthermore, in some cases a “parasitage effect” might be detected, as there might also be negative molecular interactions in vitro (Namdar et al., 2020).

Indeed, as detailed above, phytocannabinoids are potent anti-inflammatory and immunomodulatory agents and in some cases they act via different signaling pathways. For example, although both THC and CBD decreased inflammation in LPS-activated microglial cells of a BV-2 mouse, they acted through different, although partially overlapping, mechanisms. CBD but not THC inhibited the NF-κB-dependent pathway, yet both CBD and THC regulated the IFNβ pathway activity (Kozela et al., 2010).

In order to take advantage of the synergy and to diminish negative interactions between phytomolecules, activity might be improved by selecting the most active phytomolecules while eliminating parts of the whole extract. This approach was demonstrated in the reduction of inflammation in colon cells and tissues. A THCA-rich fraction from the cannabis strain was shown to have superior activity against inflammation over the crude extract (Nallathambi et al., 2017) suggesting that the selection of active compounds may reduce the presence of inactive compounds or even those that have pro-inflammatory effects.

Moreover, in some cases, the activity of a combination of phytomolecules was found to be superior over that of a single molecule. This was demonstrated in an in vivo study on inflammation, where treatment with CBD combined with cannabis extract overcame the bell-shaped dose-response of purified CBD, suggesting that components found in the extract synergize with CBD to achieve the desired anti-inflammatory action (Gallily et al., 2015). In addition, a phytocannabinoid formulation showed superior activity reducing lung inflammation over the cannabis-derived fraction in vitro. Moreover, this particular phytocannabinoid and CBD formulation had superior activity over CBD alone (Anil et al., 2021).

Cannabis compounds, in some cases via the endocannabinoids system, were shown to affect some of the cornerstones of chronic inflammation. However, in light of the large number of active molecules produced by cannabis and their sometimes-synergistic interactions, there is a need to better specify cannabis-based treatments and the active compounds, while utilizing the synergy identified between cannabis phytomolecules. Thus, even if CBD or THC are considered potentially leading molecules, additional cannabis-derived compounds may be selected for improved activity.

Future approaches for improved usage of cannabis demand the development, transformation and formulation of full-spectrum cannabis extracts into active plant ingredients (APIs) to achieve higher effectivity. This might be done via careful selection of phytomolecules composition (Koltai et al., 2019; Koltai and Namdar, 2020). Notably, selecting only a few compounds for drug formulation may be compatible with modern medicine due to the potential for standardization, and careful dosing of API-based products. Importantly, once the mode of action of phytocannabinoids and that of their combination is known, APIs might be targeted towards specific mechanisms involved with inflammation.

Moreover, it might be that cannabis components can be combined with other pharmaceutical drugs to reduce inflammation. On the one hand, complementary effects might be identified due to different and perhaps complementary modes of action of cannabis compounds and pharmaceutical drugs. For example, THC was shown to reduce gastric inflammation caused by diclofenac, which may facilitate diclofenac’s effective usage against inflammation (Kinsey and Cole, 2013). On the other, CBD and THC were shown to have metabolism-dependent inhibition for Cytochrome P450 (CYP) enzymes. CYPs are responsible for drug metabolism, including detoxication and metabolic activation of xenobiotics (Yamaori et al., 2011). Hence, combined treatment with cannabis and anti-inflammatory drugs should be carefully considered.