**4. Endocannabinoids**

The endocannabinoid system (ECS) modulates many physiological processes, including the cardiovascular, gastrointestinal and immune systems, pain, learning, memory, perception, mood, appetite, metabolism, emotions, and sleep [22,112,113,277–285]. The bioactive endocannabinoid lipid mediators have potent anti-inflammatory activities [286–291]. In addition, they promote neural progenitor cell proliferation and differentiation, and have neuroprotective effects [20,292–294]. The effect on neural cell proliferation is mediated by both the CB1 and CB2 receptors [293,295,296].

#### *4.1. The Endocannabinoid System*

The endocannabinoid system is composed of: (1) the lipid active endogenous ligands *N*-Arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG); (2) their biosynthetic enzymes (e.g., diacylglycerol lipases (DAGL), *<sup>N</sup>*-acyl-phosphatidyletha nolamine phospholipase D-like esterase (NAPE-PLD), and Ca2+-dependent and Ca2+- independent N-acetyltransferases); (3) their degradative enzymes (e.g., fatty acyl amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)); and (4) the CB1 and CB2 cannabinoid receptors [15,297,298]. The precursors of endocannabinoids (e.g., *<sup>N</sup>*-acylphosphatidylethanolamine (NAPE) and phosphatidylinositol-4,5-bisphosphate (PIP2)) are present in the lipid membranes, and the endocannabinoids are produced upon demand, usually after activation of certain G-protein-coupled receptors (GPCRs) and in response to an increase in the intracellular calcium levels [299–302].

#### *4.2. The Production of AEA and 2-AG*

The production of endocannabinoids requires one or two enzymatic steps, followed by their release into the extracellular space. AEA is usually produced from N-arachidonoylphosphatidylethanolamine phospholipid, and 2-AG is produced primarily from membrane phospholipid 1-stearoyl-2-arachidonoyl-sn-glycerol [297]. The synthesis of 2-AG involves two steps: first, the hydrolysis of its precursor phospholipid by a phospholipase (PLCβ, PLC γ2, or PLCε), followed by further cleavage by diacylglycerol lipase (DAGL) [303–305].

The biosynthesis of these endocannabinoids occurs in areas of the brain functionally related to cognitive processes, motivation, and movement control [306,307]. 2-AG was found to be present at 170 times higher concentrations than AEA in brain lysate [308]. While AEA was initially detected in the brain [135] and 2-AG in the canine gu<sup>t</sup> [309], today it is known that these host-derived endocannabinoid lipid hormones are found in various peripheral tissues (e.g., the intestine) and in the serum, and produced by certain immune cells [23,290,309–317]. For instance, lipopolysaccharides induced the production of AEA in adipose tissue macrophages [318]. T and B cells produce elevated levels of 2-AG upon activation [290]. Astrocytes were found to produce AEA, as well as homo-γlinolenoylethanolamine (HEA), docosatetraenoylethanolamine (DEA), oleoylethanolamine (OAE), and palmitoylethanolamine (PEA) [319].

#### *4.3. The Circulating Levels of AEA and 2-AG*

The circulating endocannabinoid levels are affected by various factors, and under physiological conditions, the AEA serum level was found to be between 1 to 5 nM, and the 2-AG serum level between 10–500 nM [316,320]. Physical exercise mobilizes endocannabinoids, which could contribute to the analgesic and mood-elevating effects of exercise [316]. The circulating levels of 2-AG show a circadian rhythm that gets altered when sleep is disrupted [316,320]. CBD inhibits the degradation of AEA and 2-AG, which is associated with the anti-inflammatory and anti-oxidative activities [321].

#### *4.4. Endogenous Receptors for AEA and 2-AG*

AEA and 2-AG act as agonists of the CB1 and CB2 receptors [135,322–325]. While 2-AG binds with high affinity to CB1 and CB2 cannabinoid receptors, AEA binds with low affinity to these receptors [323,324]. Although phytocannabinoids and endocannabinoids bind to the same CB1 and CB2 receptors, their chemical structure is quite different [297] (Figures 1 and 3). Both AEA and 2-AG have an alkyl-amide (alkamide) chemical structure, while cannabinoids are terpenophenolic compounds.

In addition to acting on CB1 and CB2, AEA activates the ionotropic TRPV1 channel, resulting in the opening of the ion channel and Ca2+ influx [312,326–331], the Gprotein-coupled receptor GPR55 [332,333], and the cation channel TRPA1 [334], while it inhibits the TRPM8 channel [334]. In addition, AEA activates PPARγ, and 2-AG activates PPARα [335]. The vasodilation action of AEA was found to be mediated via activation of TRPV1 [336]. Endocannabinoids activating TRPV1 have been included in the endovanilloid system [337–339]. Recent studies sugges<sup>t</sup> that potassium channels are also the targets of endocannabinoids [340].

In the brain, endocannabinoids serve as retrograde synaptic messengers [299,341]. They are released from postsynaptic neurons and inhibit the release of presynaptic neurotransmitters, such as glutamate and gamma-aminobutyric acid (GABA) by binding to the CB1 receptor and TRPV1 expressed in the presynaptic terminals [299,342,343]. This has led to the hypothesis that endocannabinoids regulate over-excitability and promote synaptic homeostasis [344]. Endocannabinoids differ from the classical neurotransmitters in that they are not stored in vesicles but are released immediately after their production.

The solubility of endocannabinoids is low in water, raising the question of how AEA diffuses through the synaptic cleft [345]. There is evidence that AEA can interact with cholesterol and ceramide, which are required for their insertion into and transport through the membrane [345–347]. In the brain, the lipid-binding protein α-synuclein is involved in the transport of arachidonic acid [348]. Fatty acid binding proteins have been shown to be intracellular carriers of AEA [349].

**Figure 3.** The chemical structures of some endocannabinoids.

Another communication system that exists between neurons is the release of lipidbased transport systems such as exosomes from neurons following a synaptic response, that are taken up by neighboring cells [350,351]. Gabrielli et al. [352] observed that endocannabinoids are secreted on extracellular membrane vesicles. In this study, extracellular vesicles secreted by microglial cells were found to carry AEA on their surface that was

able to stimulate the CB1 receptor expressed on neurons and inhibit presynaptic transmission [352]. Microglial cells release endocannabinoids at much higher levels than neurons and astrocytes [319,353,354] and are thought to play a role in regulating the synaptic activity by a process termed gliotransmission, which functions to bridge the non-synaptic inter-neuronal communication [355].

#### *4.5. Other Endocannabinoids and Endocannabinoid-like Compounds*

Other endocannabinoids include the oleoyl- and palmitoyl-ethanolamines (OEA and PEA) that affect intestinal permeability by acting on TRPV-1 and PPAR α [356,357], and 2-AG-ether and O-arachidonoylethanolamine (virodhamine) [22,358] (Figure 3). PEA is produced by neurons, microglia, and astrocytes in the central nervous system [359,360] where it plays an important role in neuroprotection [361,362]. Moreover, it was shown to have both anti-nociceptive and anti-inflammatory activities [363–366]. Immune cells release PEA that activates the CB2 receptor, resulting in downregulation of the inflammatory processes [367,368]. PEA, which is synthesized along with AEA, potentiates the action of AEA by increasing receptor affinity or reducing the degradation of AEA by FAAH [357,369–371]. The study of Lo Verme et al. [372] showed that PPAR α was required for the anti-inflammatory effect of PEA. Borrelli et al. [365] observed that PEA alleviates the inflammation in a murine colitis model through acting on CB2, GPR55, and PPAR α. OEA acts on PPAR α and is secreted in the proximal intestine where it controls appetite, exhibits anti-inflammatory properties, and stimulates lipolysis and fatty acid oxidation [373–376].

The endocannabinoid noladin ether acts on CB2 and inhibits the intracellular effector adenylyl cyclase [377]. The endocannabinoid virodhamine, which is composed of arachidonic acid and ethanolamine joined by an ester linkage, is a partial agonist with an antagonist activity on CB1, while being a full agonist on CB2 [378]. At low concentrations, virodhamine activates GPR55, while at high concentrations it acts as an antagonist [379]. The endocannabinoid N-arachidonoyl-dopamine (NADA), which is highly expressed in the striatum, hippocampus, and cerebellum, activates TRPV1, induces the release of substance P and calcitonin gene-related peptide from dorsal spinal cord slices, and enhances hippocampal paired-pulse depression [380]. NADA and its epoxide metabolites also act as an agonist for the CB1 and CB2 receptors and show anti-inflammatory activities [337,381–383]. Other dopamine-related endocannabinoids include N-oleoyldopamine (OLDA), N-palmitoyldopamine (PALDA), and N-stearoyldopamine (STEARDA) [384]. OLDA is only a weak ligand of CB1, but it induced calcium influx, reduced the latency of paw withdrawal from a radiant heat source, and produced nocifensive behavior [384].

*N*-Arachidonoyl-L-serine (AraS) is an endogenous bioactive lipid found both in the central nervous system (CNS) and in the periphery, with a similar structure and physiological functions as AEA [385,386] (Figure 3). It possesses vasoactive, pro-angiogenic, pro-neurogenic, and neuroprotective properties [386–388]. Since AraS binds weakly to CB1 and CB2, it is not classified as an endocannabinoid, but rather has been coined as an "endocannabinoid-like" substance [386]. The pro-angiogenic activity of AraS is achieved by activation of GPR55 [387]. Moreover, AraS stimulates phosphorylation of MAPK and Akt protein kinases [385].

#### *4.6. Anti-Microbial Activities of Endocannabinoids and Endocannabinoid-like Compounds*

The anti-microbial effect of endocannabinoids depends on the strain studied and the endocannabinoid used [16–18,389,390] (Table 2). Among the tested organisms, *Streptococcus salivarius*, *Bacteroides fragilis*, and *Enterococcus faecalis* were the most susceptible bacteria to AEA and *N*-Linoleoylethanolamine (LEA) [390]. MSSA and MDRSA become immediately growth arrested by AEA, an effect that was transient and relieved upon time [16]. On the other hand, the growth of *Lactobacillus gasseri* species becomes enhanced by LEA and OEA [390].


**Table 2.** Anti-microbial activities of endocannabinoids and endocannabinoid-like compounds.


4.6.1. AEA and AraS Exert Bacteriostatic Activity on Both Drug-Sensitive and Drug-Resistant *Staphylococcus aureus*

Feldman et al. [18] observed that the MIC of AEA toward three MRSA species (MRSA ATCC 33592, MRSA ATCC 43300, and a MRSA clinical isolate) was above 256 μg/mL. AraS had a MIC of 16 and 128 μg/mL on MRSA ATCC 33592 and MRSA ATCC 43300, respectively, and a MIC above 256 μg/mL for the clinical MRSA isolate [18]. A kinetic study of AEA on a multidrug-resistant *Staphylococcus aureus* (MDRSA) clinical isolate and the MSSA ATCC 25923 strain showed that AEA caused a transient bacteriostatic effect that was overcome with time [16]. The bacteriostatic effect of AEA was independent of the drug-resistant phenotype [16]. Further analysis showed that AEA inhibited cell division just prior to daughter cell separation [16]. Gene expression studies showed that AEA reduced the expression of some autolysin genes, which might in part contribute to the growth arrest [16]. AEA altered the membrane structure of the MDRSA and caused an immediate membrane depolarization that recovered with time [16]. Both AEA and AraS reduced the hydrophobicity index of MRSA at a concentration of 16 μg/mL [18].

4.6.2. AEA and AraS Sensitize Drug-Resistant *Staphylococcus aureus* to Antibiotics

Importantly, it was observed that AEA and AraS sensitize MRSA and MDRSA strains to various antibiotics, including β-lactam antibiotics (ampicillin and methicillin), gentamicin, tetracycline, and norfloxacin [16,17]. For instance, the MIC of ampicillin against MRSA ATCC 33592 and ATCC 43300 was 128 and 256 μg/mL, respectively, but in the presence of 8–16 μg/mL AEA, it was reduced to 8 μg/mL [17]. The MIC of gentamicin against MRSA ATCC 33592 was 128 μg/mL, but in the presence of 8 μg/mL AEA, it was reduced to 4 μg/mL [17]. Treating a MDRSA clinical isolate with 50 μg/mL AEA reduced the MIC of methicillin from above 500 μg/mL to 50 μg/mL [16]. AEA was found to prevent drug efflux, resulting in intracellular drug accumulation, which might explain, at least in part, the sensitization of the bacteria to antibiotics [16]. Gene expression analysis shows that AEA reduces the expression of some efflux pump genes, including *norB*, *norC*, *mepA*, *kdpA*, and *opp1C* in MDRSA [16], but it is likely that the alterations in the membrane structure caused by AEA also contribute to intracellular drug retention.

It is notable that the sensitization of MRSA to methicillin takes place even when bacterial growth is inhibited by AEA [16], suggesting that the anti-bacterial effect of methicillin and other β-lactams does not require cell division as previously documented when used

as a single agen<sup>t</sup> [391,392]. Indeed, FtsZ inhibitors that arrest bacterial cell growth, also sensitize drug-resistant *Staphylococcus aureus* to β-lactam antibiotics, which was related to membrane relocalization of penicillin-binding proteins (PBPs) [393]. Further studies are required to fully understand the antibiotic-sensitization mechanisms of AEA and AraS.

4.6.3. AEA and AraS Exhibit Anti-Biofilm Activity against Drug-Sensitive and Drug-Resistant *Staphylococcus aureus*

AEA and AraS prevent biofilm formation of MRSA and MDRSA with a maximum effect at 12.5–35 μg/mL [16,18]. AEA and AraS had a rather weak effect on preformed biofilm of MRSA and MDRSA, where concentrations as high as 64 μg/mL were required to eradicate 50% of the biofilms after a 24 h incubation [16,18]. The simultaneous treatment of the MRSA and MDRSA strains with endocannabinoids and antibiotics significantly lowered the effective dose of the two compounds [16,17]. For instance, the MBICs of AEA and ampicillin on MRSA ATCC 33592 were, respectively, 33.8 and 128 μg/mL, while in combination, 8 μg/mL of each compound was required for inhibiting biofilm formation [17]. When combining the sub-MBIC concentration 3.125 μg/mL of AEA with 50 μg/mL norfloxacin, which, as a single agent, had no anti-biofilm effect, an 80% reduction in biofilm formation by MDRSA was observed [16]. A 90% reduction in preformed MDRSA biofilm was observed when 50 μg/mL of AEA was combined with 50 μg/mL methicillin, which is the synergistic condition required for killing the bacteria [16]. The latter observation shows that the combination of AEA with antibiotics is also effective against biofilm-embedded bacteria.

Gene expression studies showed that AEA reduced the expression of the regulatory *RNAIII* and the virulence gene α-helical phenol-soluble modulin (*psmα*) in MDRSA [16]. Additionally, the genes *fnbB*, *hla*, and *hld* encoding for the virulence factors fibronectin binding protein, α-hemolysin and δ-hemolysin (δ-toxin), respectively, were downregulated by AEA [16]. As Psmα plays a central role in *Staphylococcus aureus* biofilm formation by stabilizing the biofilms through amyloid formation [394–396], the inhibition of its expression might be one mechanism for the anti-biofilm effect of AEA (Figure 4).

**Figure 4.** The anti-bacterial actions of anandamide on *Staphylococcus aureus*.

4.6.4. AEA and AraS Inhibit Yeast-Hypha Transition of *Candida albicans* and Prevent Adhesion of *Candida albicans* Hyphae to Epithelial Cells

AEA and AraS were found to inhibit yeast-hypha transition of *Candida albicans* at 125 and 250 μg/mL [389]. At 50 μg/mL, there was only a partial inhibition on the yeasthypha transition, but this concentration was sufficient to prevent hyphal extension [389]. Importantly, *Candida albicans* hyphae that have been exposed to AEA at 50 μg/mL and higher concentrations showed strong reduction in their ability to adhere to the HeLa cervical epithelial carcinoma cells [389]. AraS-treated *Candida albicans* hyphae showed deficient adherence to HeLa cervical carcinoma cells similar to AEA-treated fungi, while 2-AG treatment had only a minor effect at the concentrations analyzed (up to 250 μg/mL) [389]. None of the endocannabinoids affected the adherence of the *Candida albicans* hyphae to polystyrene tissue culture plates within the first hour of incubation, while AraS and 2-AG, but not AEA, reduced the biofilm mass formed on the polystyrene tissue culture plates after a 24 h incubation [389].

Gene expression studies showed that AEA increased the expression of *NRG1*, which is a transcriptional repressor of filamentous growth, but reduced the expression of the hyphal cell wall protein 1 (*HWP1*), the Agglutinin-like protein 3 (*ALS3*), the Hypha-specific G1 cyclin-related protein 1 (*HGC1*), the Ras-like protein 1 (*RAS1*), the enhanced filamentous growth protein 1 (*EFG1*), the cell surface hydrophobicity-associated protein *CSH1*, and the extent of cell elongation protein 1 (*ECE1*). The combined effect of AEA on the expression of these genes might cumulate in the observed effects of AEA on *Candida albicans* adherence and hyphal growth (Figure 5).

**Figure 5.** Effect of AEA on *Candida albicans* gene expression [381]. Genes in red are downregulated by AEA aftera2h incubation. Genes in green are upregulated by AEA. Genes in light blue are unaffected by AEA. Open circles are genes that have not ye<sup>t</sup> been analyzed. Ultimately, AEA prevents the adherence and hyphal extension of *Candida albicans*.

#### *4.7. Dialog between the Gut Microbiota and the Endocannabinoid System*

An intercommunication system has been found to exist between the gu<sup>t</sup> microbiota and the endocannabinoid system [285,298,397–402]. The gu<sup>t</sup> microbiota, representing more than 100 trillion microorganisms, including at least 1000 distinct species, lives in symbiosis with the host and assists in controlling the metabolic health of the host by degrading nutrients that the host is unable to digest and by providing a whole battery of small signaling molecules, metabolites, and nutrients beneficial for the host physiology [298,403]. The gu<sup>t</sup> microbiome differs from individual to individual, and the composition of the microbiota is believed to affect various metabolic disorders, such as obesity, hyperglycemia, and dyslipidemia, which are risk factors for type 2 diabetes, hepatosteatosis, and arteriosclerosis [404]. Reduced diversity of gu<sup>t</sup> microbiota has been linked with various pathophysiological conditions, such as depression, schizophrenia, neurological disorders, and chronic fatigue [405–409]. Microbiota can affect the endocannabinoid system and the nervous system, and vice versa; the nervous system and the endocannabinoids can influence the enteric microbiota composition [112,285,390,397,410,411].

4.7.1. The Relationship between Gut Microbiota, the Endocannabinoid System, and Depression

The effect of the gu<sup>t</sup> microbiota on depressive-like behaviors in mice was found to be mediated by the endocannabinoid system [411]. These authors showed that the transfer of microbiota from stress-induced depressive mice to naïve unstressed hosts induced a depressive-like state in the recipients. This was accompanied by a reduced adult hippocampal neurogenesis that was related to decreased hippocampal 2-AG levels and deficient CB1-mediated activation of the mTOR signaling pathways [411]. The detrimental effects on hippocampal neurogenesis could be restored by a MAGL inhibitor that prevents 2-AG degradation, addition of the 2-AG precursor arachidonic acid to the diet, or by complementation with *Lactobacillus plantarum*WJL [411]. The microbiota from the stressinduced depressive mice showed an increase in *Ruminococcaceae* and *Porphyromonodaceae* species, with a decrease in *Lactobacillaceae* [411]. The complementation with *Lactobacillus plantarum*WJL restored hippocampal 2-AG to normal levels, as well as increased the levels of AEA, n-3, and n-6 polyunsaturated fatty acids (PUFAs) [411]. *Lactobacillus* species can regulate fatty acid metabolism, absorption, and fatty acid composition of the host [412,413], which in turn affects the endocannabinoid system [411]. Rousseaux et al. [414] observed that oral administration of *Lactobacillus acidophilus* induced the expression of both the μ1 opioid receptor and CB2 receptor on colon epithelial cells, resulting in reduced abdominal pain in a rat colorectal distension model.

#### 4.7.2. Association between Gut Microbiota, PEA, and Anhedonia/Amotivation

PEA was shown to mediate the association between gu<sup>t</sup> microbial diversity and anhedonia/amotivation [410]. Increased serum levels of PEA were associated with antidepressive effects [357,415], while increased stool levels of PEA, indicative of increased excretion of PEA, were associated with alterations in synaptic plasticity, learning, and emotional responses [410,416,417]. The stool PEA levels were associated with gu<sup>t</sup> microbial diversity, with implications on host mental health [285,357,410,418]. The relative abundance of microbes of the *Blautia* and *Dorea* taxa was particularly associated with fecal PEA and anhedonia/amotivation [410]. Reduced microbial diversity corresponded with increased excretion of PEA and more severe anhedonia/amotivation [410]. PEA was also found to counteract autistic-like behaviors in BTBR T+ *tf*/J mice by dampening inflammation, reducing oxidative stress, reducing gu<sup>t</sup> permeability, and altering the gu<sup>t</sup> microbiota, besides its neuroprotection through induction of PPARα [419]. These authors found that PEA treatment increased the ratio of *Firmicutes*/*Bacteroidetes*, which was due to an increase in *Firmicutes* (e.g., *Clostridials*) and a decrease in *Bacteroidetes* [419].

#### 4.7.3. The Relationship between Gut Microbiota, AEA, and Acute Respiratory Distress Syndrome

AEA was found to attenuate acute respiratory distress syndrome through modulating the gu<sup>t</sup> microbiota [420]. In this study, the researchers investigated the effect of AEA on staphylococcal enterotoxin B (SEB)-mediated acute respiratory distress syndrome. SEB caused an increase in pathogenic bacteria in both the lungs and the gu<sup>t</sup> [420]. AEAtreated mice showed increased level of anti-microbial peptides in the lung epithelial cells and prevented the increase in pathogenic bacteria induced by SEB [420]. AEA increased the level of several bacterial species (e.g., *Lachnospiraceae* and *Clostridia*) that produce elevated levels of SCFAs, such as butyrate and valerate, important for stabilizing the gut–lung microbial axis and suppressing inflammation [420]. In addition, AEA treatment increased the abundance of *Muribaculaceae* and reduced the abundance of *Pseudomonas* and *Enterobacteriaceae* [420].

#### 4.7.4. The Relationship between Gut Microbiota, the Endocannabinoid System, and Obesity

Obesity is often characterized by low-grade inflammation, with increased levels of endocannabinoids in the plasma and adipose tissues and altered expression of CB1 [421]. Activation of CB1 and CB2 receptors reduces motility, limits secretion, and decreases hypersensitivity in the gu<sup>t</sup> [422–424]. Impaired CB1 signaling protected against the development of obesity and steatosis [425–427]. Lipopolysaccharides from Gram-negative bacteria induce the production of endocannabinoids under inflammatory conditions that dampen the inflammatory response [318,428–430]. Vice versa, the activation of CB1 in mice increases circulating levels of lipopolysaccharides due to reduced expression of the tight junction proteins occludin and zonula occludens-1 (ZO-1), resulting in increased gu<sup>t</sup> epithelial permeability [421,431].

The gu<sup>t</sup> microbiota was found to modulate colon CB1 receptor expression in both normal and obese mice [421]. Obese mice fed with the prebiotic oligofructose showed reduced CB1 expression, lower AEA content, and increased expression of FAAH [421]. Obese mice treated with the CB1 antagonist SR141716A (Rimonabant) improved the gu<sup>t</sup> barrier function and reduced body weight gain [421]. Mehrpouya-Bahrami et al. [432] observed that SR141716A attenuated diet-induced obesity and inflammation that was correlated with increased relative abundance of *Akkermansia muciniphila* and decreased abundance of *Lanchnospiraceae* and *Erysipelotrichaceae* in the gut. Interestingly, SR141716A prevented the intracellular replication of macrophage-phagocytosed *Brucella suis* by activating the macrophages, which was related to the inhibition of CB1 [433]. It would be interesting to study whether SR141716A also has a direct anti-microbial effect. In this context, it is worth mentioning that SR141716A could potentiate the anti-fungal activity of amphotericin B against *Candida albicans* and *Cryptococcus neoformans* by increasing cellular oxidative stress and cell membrane permeability [434].

Mice fed on high-fat, high-glucose diet showed altered microbiome with concomitant increase in AEA and 2-AG in the plasma [398]. The relative abundances of *Adlercreutzia*, *Barnesiella*, *Coprobacillus*, *Eubacterium*, and *Parasutterella* in the ileum were negatively associated with AEA levels [398]. The level of the AEA congener N-docosahexaenoylethanolamine (synaptamide, DHEA), which is required for normal brain development [435], was negatively associated with *Barnesiella*, *Enterococcus*, *Eubacterium*, *Flavonifractor*, and *Intestinimonas* in the ileum [398]. These authors also found a negative correlation between the *Delftia* genus and N-linoleoylethanolamine (LEA), while the *Lactobacillus* genus was associated with increased 2-docosahexaenoyl-glycerol (2-DHG) levels [398].

Repeated administration of OAE to mice fed on normal chow pellet diet for 11 days led to alteration in fecal microbial composition with an increase in *Bacteroidetes* (e.g., *Bacteroides* genus) and a decrease in *Firmicutes* (*Lactobacillus*), which is considered a "lean-like" phenotype [436]. OAE also reduced intestinal cytokines expression by immune cells isolated from Peyer's patches [436].

4.7.5. The Relationship between Gut Microbiota, the Endocannabinoid System, and Inflammatory Bowel Diseases

Both AEA and PEA have been observed to reduce inflammation in murine models of colitis and inflammatory bowel disease [437–439]. Elevating the levels of the endocannabinoids by inhibiting FAAH could relieve colitis and inflammatory bowel disease [440,441]. Butyrate that is produced by gu<sup>t</sup> microbiota (e.g., bacteria of the *Ruminococcaceae* and *Lachnospiraceae* family) [442] reduces inflammation and pain in colitis animal models, which can in part be mediated through the endocannabinoid system [443]. Vijay et al. [443] studied the association of the endocannabinoids AEA, 2-AG, OEA, and PEA with gu<sup>t</sup> microbiome composition upon exercise. Under resting condition, AEA and OEA were positively associated with alpha diversity and with SCFA producing bacteria such as *Bifidobacterium*, *Coprococcus 3*, and *Faecalibacterium*, while being negatively associated with *Collinsella* [443]. AEA, OEA, and PEA increased with exercise, and changes in AEA correlated with bacterial butyrate production [443]. The increases in AEA and PEA correlated with decreased expression of the inflammatory mediators TNF α and IL-6 and increased expression of the anti-inflammatory cytokine IL-10 [443].

PEA was found to increase the phagocytosis and intracellular killing of encapsulated *Escherichia coli* K1 by activated microglial cells and macrophages [444,445]. Pre-treatment with PEA significantly increased the survival of mice challenged with *Escherichia coli* K1 [445]. Similarly, Heide et al. [446] observed that prophylactic PEA attenuated inflammation and increased the survival of mice challenged with intracerebral *Escherichia coli* K1 infection. Lower bacterial loads were observed in the spleen, liver, and blood of the PEA pretreated animals [446]. This was related to the anti-inflammatory effect, since PEA at 1 μg/mL had no effect on *Escherichia coli* growth in vitro [446].

The gu<sup>t</sup> microbiota of IBD patients differs from healthy individuals, with a decrease in butyrate- and indole-producing bacteria, decrease in bile salt-sensitive bacteria, while an enrichment in bile acid-metabolizing bacteria [390,447–449]. Among others, *Escherichia coli*, *Lactobacillus gasseri*, *Ruminococcus gnavus*, and *Blautia producta*, were more abundant in IBD, while *Bacteroides cellulosilyticus*, *Bacteroides fragilis*, and *Streptococcus salivarius* were depleted [390]. Fornelos et al. [390] observed that certain *N*-Acylethanolamines (NAEs), such as LEA, PEA, OEA and AEA, are elevated in the stool of IBD, Crohn's disease, and/or ulcerative colitis patients, and stimulate the growth of bacterial species overrepresented in IBD while inhibiting bacterial species lacking in IBD. *<sup>N</sup>*-acylethanolamine levels were highest in samples with most differences in the microbiome, suggesting a connection between *N*-Acylethanolamines and altered microbiota in IBD [390]. These researchers observed that LEA inhibited the growth of *Bacteroides fragilis*, *Bacteroides cellulosilyticus*, and *Enterococcus faecalis*, while slightly enhanced the growth of *Escherichia coli*, *Ruminococcus gnavus*, and *Blautia producta* [390]. AEA also transiently inhibited the growth of *Bacteroides fragilis* and *Enterococcus faecalis* that recovered with time [390]. The growth of *Lactobacillus gasseri*, which is enriched in IBD, was enhanced by OEA and LEA, and to a lesser extent by AEA [390]. LEA and AEA partly inhibited the growth of *Alistipes shahii* and *Ruminococcus lactaris* that are underrepresented in IBD subjects [390]. The growth of *Streptococcus salivarius* was completely prevented at a concentration of 50 μM LEA, AEA, or OEA, but only slightly inhibited by PEA [390].

#### 4.7.6. Effect of *N*-Acylethanolamines on the Microbial Composition of Stool Chemostats

Fornelos et al. [390] also studied the effect of *<sup>N</sup>*-acylethanolamines, including endocannabinoids, on the composition of two different stool chemostats. In the control chemostat A, the *Enterobacteriaceae*, *Clostridiaceae*, and *Veillonellaceae* taxa dominated, while in the presence of AEA, the bacterial community was almost entirely overtaken by *Enterobacteriaceae* [390]. LEA-treated chemostat A was dominated by *Enterococcaceae*, *Veillonellaceae* and *Enterobacteriaceae* at the expense of *Streptococcaceae*, *Erysipelotrichaceae*, *Porphyromonadacea*, *Bacteroidaceae*, and *Rikenellaceae*, while OEA treatment did not impact *Enterobacteriaceae* abundance but increased the relative abundances of *Enterococcaceae* and *Streptococcaceae*

and decreased those of *Bacteroidaceae* and *Rikenellaceae* [390]. AEA and LEA also reduced the abundance of *Barnesiella intestinihominis*, *Alistipes*, and *Bacteroides* species, while they increased the abundance of *Escherichia* species [390]. In Chemostat B, the abundance of *Blautia producta*, *Clostridium clostridioforme*, *Klebsiella pneumoniae*, and *Proteus mirabilis* was increased in the presence of AEA or LEA [390]. These data indicate that LEA, AEA, and other *N*-Acylethanolamines can shift the microbiome of a healthy individual into an IBD profile [390].

Transcriptional analysis showed that AEA upregulates both the anaerobic, reductive, and oxidative branches of the citrate cycle concomitant with increased energy metabolism and increased respiratory electron transport chain activity, especially in *Enterobacteriaceae* [390]. Metabolic changes occurring upon exposure to AEA, LEA, and other NAEs include the activation of bacterial processes involved in NAE metabolism [390].

Searching for an action mechanism of LEA and AEA on *Bacteroides fragilis*, the researchers found that the most upregulated genes are those encoding for membraneassociated efflux transport proteins, and the most downregulated gene was the long chain fatty acid (LCFA) importer *fadL* [390]. They further showed that two other genes involved in fatty acid metabolic processing were repressed: a *fadD* homolog that catalyzes esterification of incoming fatty acids into CoA thioesters and a *fadE* homolog involved in downstream fatty acid breakdown [390]. These data indicate that bacteria have developed mechanisms that can respond to endocannabinoids.
