Mast cells (MCs) contribute to the control of local inflammatory reactions and become hyporesponsive after prolonged TLR4 activation by bacterial LPS. The molecular mechanisms involved in endotoxin tolerance (ET) induction in MCs are not fully understood. In this study, we demonstrate that the endocannabinoid 2-arachidonoylglycerol (2-AG) and its receptor, cannabinoid receptor 2 (CB2), play a role in the establishment of ET in bone marrow–derived MCs from C57BL/6J mice. We found that CB2 antagonism prevented the development of ET and that bone marrow–derived MCs produce 2-AG in a TLR4-dependent fashion. Exogenous 2-AG induced ET similarly to LPS, blocking the phosphorylation of IKK and the p65 subunit of NF-κB and inducing the synthesis of molecular markers of ET. LPS caused CB2 receptor trafficking in Rab11-, Rab7-, and Lamp2-positive vesicles, indicating recycling and degradation of the receptor. 2-AG also prevented LPS-induced TNF secretion in vivo, in a MC-dependent model of endotoxemia, demonstrating that TLR4 engagement leads to 2-AG secretion, which contributes to the negative control of MCs activation. Our study uncovers a functional role for the endocannabinoid system in the inhibition of MC-dependent innate immune responses in vivo.

Inflammation is a mechanism developed by the immune system to respond to tissue damage in which distinct cells migrate from the blood vessels to the damaged tissues to destroy the harmful agent (1). Prolonged or chronic inflammation has been related to many diseases, such as diabetes, fibrosis, arthritis, multiple sclerosis, and neurodegenerative disorders; moreover, repeated exposure to proinflammatory stimuli has been associated to the generation of an unresponsive state (tolerance) that modifies systemic immune responses (2) and affects human populations exposed to high polluted environments (3). Therefore, knowing the mechanisms controlling chronic inflammation is of special interest to identify possible targets for the development of treatments for inflammation-related diseases.

The endocannabinoid system has emerged as a potent natural mechanism to control the intensity of inflammatory reactions, preventing the onset of pathological proinflammatory responses (4, 5). This system is composed of the CB1 and CB2 cannabinoid receptors, the endocannabinoids (i.e., the endogenous ligands for these receptors), of which N-arachidonoylethanolamine (anandamide [AEA]) and 2-arachidonoylglycerol (2-AG) are the best studied, including the enzymatic systems involved in their biosynthesis and degradation (6). In line with the notion of an inhibitory action of endocannabinoids on inflammatory responses, mice lacking fatty acid amide hydrolase, the endocannabinoid-degrading enzyme, show elevated endocannabinoid levels and reduced allergic responses (7). Conversely, CB1 and CB2 receptor double knockout mice exhibit stronger allergic inflammation than wild-type (WT) mice (8).

Mast cells (MCs) are important players in the regulation of local and systemic inflammatory reactions (9, 10). As other cell lineages in the immune system, MCs initially derive from precursors generated at the yolk sac in the embryo and, in the adult, are progressively substituted (with a tissue-specific kinetics) by bone marrow–derived precursors that mature in each one of the vascularized tissues where they reside (11, 12). Mature MCs generate a microenvironment-determined transcriptome associated to the production of proteins that allow them to perform specialized functions (13). The role of MCs as regulators of local inflammatory responses is exerted by the production of a number of different mediators (9) that are secreted using distinct pathways (14, 15). Despite their well-known participation in type I hypersensitivity reactions, multiple lines of evidence support their protective role in the defense against microbes such as Mycoplasma pulmonis, Salmonella typhimurium, Listeria monocytogenes, and Klebsiella pneumonie (1619). Also, a positive participation of MCs in the survival to peritonitis provoked by cecal ligation and puncture has been reported (20); a function that relies on the activation of the TLR4 (21). The signaling cascade activated by TLR4 receptor triggering requires the activation of the MyD88-dependent pathway (22), which leads to the activation of MAPK and IKK, the nuclear translocation of NF-κB transcription factor and the synthesis of TNF, IL-6, and other proinflammatory cytokines (23).

Chronic exposure to LPS, a component of the wall of Gram-negative bacteria, leads to a phenomenon known as endotoxin tolerance (ET), in which cells or organisms enter into an immune unresponsive state and are unable to respond to further challenges with endotoxin to avoid exaggerated inflammatory reactions (24). Development of ET in innate immune cells can occur via a direct TLR4 desensitization pathway that requires the synthesis of negative regulators of main transduction steps, such as the inactive kinase IL-1R–associated kinase M (IRAK-M), the ubiquitin ligase A-20, the phosphatidylinositol phosphatase SHIP1, and the p50 subunit of NF-κB (2427). Recent evidence has shown that ET can also occur by an indirect mechanism, in which the activation of TLR4 receptor leads to the synthesis of mediators, such as TGF-β and IL-10, which (acting in an autocrine manner) contribute to heterologous desensitization of TLR4 signaling cascade (24, 25, 28, 29).

Development of ET in MCs has only started to be studied, and it is considered an important field of research, because the intense production of MCs-derived cytokines has been shown to exert deleterious effects in high intensity infections. For example, in the high-severity infection caused by extensive cecal ligation and puncture, a detrimental role of MC-produced TNF has been reported (30). Also, MC-derived IL-4 blocks the clearance of bacteria by macrophages in sepsis (31), and MCs (through the production of TNF) enhance the proliferation of i.p.-injected S. typhimurium (30). Remarkably, both positive and negative effects of ET on FcεRI-induced responses in MCs have been reported, suggesting that the desensitization of the TLR4 signaling pathway alters the outcome of MC-dependent allergic reactions (32, 33).

Endocannabinoids are gaining importance in the regulation of inflammatory conditions, and it has been reported that they exert inhibitory actions on MCs. For example, in MCs of human hair follicles, inhibition of the CB2 receptor increased MCs degranulation without altering their proliferation (34). Likewise, MC-dependent edema, triggered by compound 48/80 in the mouse ear pinnae, was reduced by a selective CB2 agonist (35). In the current study, we tested the hypothesis that TLR4 receptor stimulation on MCs leads to the production of 2-AG that, by an autocrine action, binds to CB receptors, which activation contributes to the development of ET to control MC-mediated inflammation. We also investigated if the effects of 2-AG on MCs responsiveness to LPS could be observable in vivo using a murine model of endotoxemia.

Mice C57BL/6J (stock no. 000664), MC-deficient c-Kit Wsh/Wsh mice (stock no. 005051), and TLR4 knock out mice (TLR4−/−, stock no. 007227) from The Jackson Laboratory (Bar Harbor, ME) were used. Animals were kept under controlled conditions of temperature (22–24°C) and humidity, with free access to food and water. All experimental procedures were performed in accordance with our Institutional Committee for the Care and Use of Laboratory Animals, which follows the rules of the Mexican Official Norm for the use and care of laboratory animals (NOM-062-ZOO-1999) under authorized protocols (numbers 137-15, 23-12, and 75-13).

A DNP-specific mouse IgE (SPE7 clone from Sigma-Aldrich) was used for MC sensitization as previously described (36). Abs for CB1 receptor (sc-10066), CB2 receptor (sc-25494), p-AKTSer473 (sc-7985-R), p-ERK 1/2 (sc-7976), p-p38 (sc-11852), ERK 2 (sc-154), β-actin (sc81178), SHIP (sc-8425), IRAK-M (sc-100389), β-arrestin 2 (β-Arr2) (sc-13140), Rab11A (sc-166523), Rab7 (sc-271608), and Lamp-2 (sc-8100) were from Santa Cruz Biotechnology. Abs against p-IKK (catalog no. 2697) and p-p65 (catalog no. 3033) were from Cell Signaling Technology, and the Ab for early endosome Ag 1 (EEA-1) was from eBioscience. Secondary Abs or reagents used for immunoblotting were the anti-mouse IgG-HRP and anti-rabbit IgG-HRP (Jackson ImmunoResearch Laboratories). For immunofluorescence, secondary Abs were anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 488 or Alexa Fluor 647, and anti-goat Alexa Fluor 647 from Life Technologies.

Reagents for cell culture and Western blot were obtained from Sigma-Aldrich and Invitrogen. 2-AG dissolved in acetonitrile, LPS (serotype 026:B6), and the antagonist for the CB1 receptor, AM251, were obtained from Sigma-Aldrich. The antagonist for the CB2 receptor, AM630, was from Tocris Bioscience. Both antagonists were dissolved in ethanol before use. ELISA kits for TNF and 2-AG detection were from Novex (Life Technologies) and Cloud-Clone, respectively. The inhibitor of monoacylglycerol lipase, MNJ110, was from Calbiochem. The inhibitor of SHIP (3-α-aminocholestane [3-AC]) was from Echelon Biosciences and kindly provided by Dr. M. E. Cruz Muñoz from the Autonomous University of Morelos State, Mexico.

Bone marrow was isolated from 5–6-wk-old mice as described (36, 37). Briefly, bone marrow was placed on culture media supplemented with FBS and IL-3, changing the medium every week. For some experiments, stem cell factor was also added to the medium. After 5–6 wk, generation of bone marrow–derived MCs (BMMCs) was monitored by flow cytometry using specific Abs against FcεRI α-chain (clone MAR-1; eBioscience), and only cultures showing more than 95% of positive cells were used. When stated, BMMCs were sensitized with 100 ng/ml of a monoclonal anti-DNP IgE (clone SPE-7) for 24 h at 37°C. For FcεRI stimulation, the specific Ag dinitrophenol coupled to human serum albumin was used. To confirm cell functionality, degranulation assays were performed, as described elsewhere (38).

Pellets from sensitized cells (2 × 106 cells per sample) were solubilized in 1 ml of TRI Reagent (Sigma-Aldrich) at room temperature. First-strand cDNA synthesis was generated with the Fermentas Life Sciences First-Strand Synthesis System. Primers for amplification of CB1, forward, 5′-CGTGGGCAGCCTGTTCCTCA-3′; reverse, 5′-CATGCGGGCTTGGTCTGG-3′ (39) and CB2 (forward 5′-CCGGAAAAGAGGATGGCAATGAAT-3′; reverse 5′-CTGCTGAGCGCCCTGGAGAAC-3′ (39), were obtained from Sigma-Aldrich. PCR conditions were the following: 95°C for 10 min, 95°C for 45 s, 58.4°C for 1 min (CB1) or 60.9°C for 1 min (CB2), 72°C for 45 s, and a final step of 72°C for 7 min. Thirty-five amplification cycles were performed for the detection of both receptors. PCR products were separated on 2% TBE/agarose gels and dyed with ethidium bromide (40). Pictures of representative gels were taken using the MiniBIS Pro from Bio-Imaging Systems.

IgE-sensitized BMMCs (2 × 106 cells per sample) were treated with different stimuli at 37°C to analyze TNF secretion. After the stimulation indicated in each figure, cells were centrifuged at 9500 × g for 10 min at 4°C for collection of the supernatant. TNF concentration was determined by ELISA (Novex Life Technologies) following the manufacturer’s instructions.

IgE-sensitized BMMCs (3 × 106 cells per sample) from WT and TLR4−/− mice were preincubated with 1 μM MNJ110 for 15 min at 37°C. After this treatment, LPS was added for different times, indicated in the corresponding figure. After LPS addition, cells were centrifuged at 3750 × g for 10 min at 4°C. Supernatants were collected, and 2-AG production was analyzed by ELISA following the manufacturer’s instructions.

IgE-sensitized BMMCs were stimulated as indicated in each figure legend. After treatments, the reaction was stopped by collection of the cells and the addition of 150 μl of 1× Laemmli buffer (Sigma-Aldrich) supplemented with orthovanadate (4 mM) and 2-ME (0.28 mM) to the cell pellets. Samples were boiled for 15 min. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat milk (Svelty; Nestlé) and incubated overnight with primary Abs at the following dilutions: p-AKTSer473 1:1000, SHIP 1:5000, IRAK-M 1:3000, p-IKK 1:5000, p-p65 1:5000, p-ERK 1:10000, and p-p38 1:5000. After incubation with primary Abs, membranes were washed two times with TBS/0.1% Tween 20 before being incubated with the respective secondary Abs: anti-mouse (1:10,000) or anti-rabbit (1:20,000). Membranes were washed two times to remove the secondary Ab, and chemiluminescent HRP substrate (MilliporeSigma) was used for the detection of protein bands. Relative quantitation of immunoblots was performed by densitometry using Image Studio Software (v. 5.2.5).

BMMCs were incubated in fibronectin (10 μg/ml)–coated slides for 45 min. After that time, cells were fixed with 4% paraformaldehyde for 15 min and treated with 50 mM of ammonium chloride for 10 min. Blocking was performed using a solution composed by 1% BSA, 5% donkey serum, and 0.1% Tween 20. After 1 h of blocking at room temperature, cells were incubated with the specific primary Ab overnight. Dilutions of primary Abs were as follows: β-Arr2 1:50, EEA-1 1:100, Rab11 1:100, Rab7 1:200, and Lamp2 1:100. PBS washes were performed, followed by incubation with secondary Abs (anti-mouse, anti-rabbit, or anti-goat 1:200) for 1 h. After washing the secondary Ab, DAPI at 300 nM was added to the slides for 5 min. ProLong Gold Antifade Reagent (Thermo Fisher Scientific) was applied, and slides were sealed with nail polish. Cells were analyzed by confocal microscopy using a Zeiss LSM 780 and the numerical aperture of the objective was 0.55 (WD 26 mm). Images were analyzed with the ImageJ software (V. 1.51m9).

MC-deficient mice (c-KitWsh/Wsh; Wsh) with genetic background on C57BL/6J were kept under sterile conditions as recommended by the provider. For MCs reconstitution, reported protocols were used (36, 41, 42). Briefly, 2 × 106 mature BMMCs derived from WT or TLR4−/− animals were i.p. injected to 8-wk-old Wsh mice. The reconstituted Wsh (Wsh rec) mice were used 4 wk later.

Age-matched C57BL/6J, Wsh, and Wsh rec mice were i.p. injected with LPS (1 mg/kg) or different dosages of 2-AG (0.03, 0.1, 0.3, or 1 mg/kg) plus LPS 1 mg/kg. Animals were euthanized 1 h after LPS injection in a CO2 chamber. Thereafter, 2 ml of saline solution was injected into the peritoneal cavity and, after a gentle massage of the abdominal surface, peritoneal content was recovered with a sterile syringe. Recovered material was centrifuged at 9500 × g for 5 min at 4°C. Supernatants were collected, and TNF concentration was determined by ELISA following the manufacturer instructions.

Data are expressed as the mean ± SEM of at least three independent experiments using different cell cultures. For in vivo experiments, at least four animals were used for each treatment. For paired comparisons, the Student t test was used. For multiple group comparisons, one or two-way ANOVA followed by specific post hoc tests (indicated in each figure) were used. Statistical analyses were performed with GraphPad Prism Software (v.7.0).

Because cannabinoids exhibit anti-inflammatory effects and inhibit cytokine release from different cell types, we investigated whether the direct addition of 2-AG, like LPS (43), could induce tolerance to endotoxin in BMMCs by measuring TNF secretion in response to LPS. Cells were pretreated or not (0 h) with either LPS (500 ng/ml) or 2-AG (10 nM, equivalent to 3.8 ng/ml) for 1 or 2 h before stimulating them in fresh media with LPS for 3 h. After this, the amount of TNF released was determined. Fig. 1A shows that pretreatment with LPS inhibited up to 50% the TNF secretion induced by the second LPS challenge (from 551 ± 52.68 pg/ml TNF in the LPS-treated cells to 274.7 ± 52.03 pg/ml TNF in tolerant cells). Interestingly, preincubation with 2-AG induced a similar tolerant phenotype in BMMCs because a similar inhibition of TNF release was observed in cells preincubated with the cannabinoid (from 541.4 ± 38.2 to 357.3 ± 34.76 pg/ml). Inhibition of TNF secretion after LPS challenge in tolerant BMMCs was not related to the depletion of intracellular TNF stores because tolerant cells were still able to secrete important amounts of TNF when stimulated through the high affinity IgE (FcεRI) receptor with the specific Ag dinitrophenol coupled to human serum albumin (Supplemental Fig. 1). To test if the inhibitory effect of 2-AG was dependent on the presence of cannabinoid receptors, we analyzed the expression of CB1 and CB2 receptors in BMMCs. Both RT-PCR analysis using reported primers and Western blot experiments using specific Abs detected CB1 and CB2 mRNAs and proteins, respectively. As can be observed on Fig. 1B, the expected sizes of RT-PCR fragments were obtained, and the proteins detected were in the range of 55–64 kDa (corresponding to glycosylated CB1 receptor) and 36 kDa (corresponding to the CB2 receptor). To test the functionality of these receptors, MCs were stimulated with 2-AG, and the phosphorylation of ERK1/2 MAPK and AKT on Ser 473 were examined. Fig. 1C shows that 2-AG induced ERK1/2 and AKT phosphorylation in a dosage-dependent manner in BMMCs, validating the functional expression of both CB receptors in these cells.

To further characterize the mechanism by which 2-AG induces tolerance to LPS in MCs, we sought to identify the relevant endocannabinoid receptor in this phenomenon. To this end, we tested the effect of the CB1 receptor antagonist, AM251, and the CB2 receptor antagonist, AM630, on the inhibition of LPS-induced TNF secretion provoked by 2-AG in dosage/response experiments. Cells were preincubated during 15 min with specific antagonists of CB receptors, and then, 2-AG was added for 2 h. At the end of this time, cells were washed and stimulated in fresh media with LPS for 3 h, and TNF content in the supernatant was measured by ELISA. Cells treated with LPS alone, or LPS plus antagonists (without 2-AG) were used. Fig. 2A shows that the CB1 antagonist, AM251, did not prevent the inhibitory actions of 2-AG at any of the concentrations tested. In contrast, the CB2 receptor antagonist, AM630, inhibited the effects of 2-AG on TNF secretion in response to LPS when used at low nanomolar concentrations. As RBL2H3 MCs are able to produce the precursors of endocannabinoids AEA and palmitoylethanolamine in response to ionomycin or IgE/Ag stimulation (44), we evaluated whether LPS, acting through TLR4 receptors, could induce 2-AG secretion in BMMCs that could activate CB2 receptors in an autocrine manner. BMMCs derived from WT or TLR4-deficient mice were preincubated in the presence of the inhibitor of the monoacylglycerol lipase MNJ110, a highly relevant enzyme for 2-AG degradation. Cells were then stimulated for different times with LPS, and the content of 2-AG in cellular supernatants was determined by ELISA. As can be observed in Fig. 2C, LPS induced 2-AG secretion of up to 13 ± 2 ng/ml (corresponding to values close to 34 nM of 2-AG) at 10 and 15 min after LPS addition. This effect required the expression of the TLR4 receptor because it was not observed in BMMCs derived from TLR4-deficient mice.

To analyze possible TLR4 signaling pathways that were blocked by CB2 receptor stimulation, we studied the effect of 2-AG on the LPS-dependent activation of IKK and NF-κB. BMMCs were preincubated with LPS for 2 h or 2-AG for different times (15, 60, or 120 min) before a challenge with LPS for 15 min was performed. Fig. 3A and 3B shows that both preincubation with LPS and with 2-AG inhibited the IKK and p65 phosphorylation observed after LPS challenge (Fig. 3A). Maximal inhibition of IKK and p65 phosphorylation was observed when 2-AG was added 2 h before LPS (Fig. 3A, 3B). Of note, a nonsignificant increase on IKK phosphorylation was observed when 2-AG was added 15 min before LPS without any associated increase in p65 phosphorylation (Fig. 3A, 3B). When LPS-dependent phosphorylation of MAPKs was analyzed, only preincubation with LPS was able to prevent ERK1/2 and p38 phosphorylation induced by a second addition of LPS (Fig. 3C, 3D). These data suggest that the CB2 receptor engagement by 2-AG inhibits the TLR4-dependent NF-κB activation but not the TLR4-dependent MAPK activation.

It has been reported that during the development of ET, cells express different negative regulators that inhibit the TLR4 signaling pathway, such as SHIP1 and IRAK-M (45). To analyze whether 2-AG induces the synthesis of these negative regulators, we analyzed their cellular amounts. Fig. 4A shows that LPS-induced BMMC stimulation promoted the synthesis of SHIP and IRAK-M in a time-dependent manner, being significant 8 h after LPS challenge (Fig. 4B). Likewise, addition of 2-AG to BMMCs produced an important induction of SHIP and IRAK-M at 4 h (Fig. 4C, 4D). As expected, the effects of 2-AG on SHIP expression were also observed in BMMCs derived from TLR4−/− mice (Fig. 4E), reaching maximal levels at about 4 h (Fig. 4F). Because tolerance to LPS was evident at 2 h (Fig. 1A) and no detectable amounts of SHIP were observed by Western blot at that time, the possibility that SHIP could not been involved in early events leading to ET was explored. In this experiment, the SHIP inhibitor 3-AC was used to pharmacologically diminish SHIP activity before the induction of ET. Cells were preincubated with 10 μM 3-AC [a concentration that has been proven to effectively inhibit SHIP in distinct innate immune cells (46, 47)] for 15 min and, then, LPS or 2-AG were added during 2 h to induce tolerance. After this, supernatants were discarded, and cells were washed and resuspended in fresh media, where stimulation with LPS alone was performed for 3 h. Finally, supernatants were analyzed by ELISA to quantify secreted TNF. As can be observed in Supplemental Fig. 2, LPS- and 2-AG–induced tolerance was still observed in 3-AC–pretreated cells, suggesting that early events leading to LPS- and 2-AG–induced ET are independent from SHIP activity.

Because the collected evidence showed effects of TLR4 stimulation on CB2 receptor activation in an autocrine manner, we asked whether LPS stimulation could impact the cellular distribution and vesicular trafficking of the CB2 receptor. We therefore analyzed the colocalization of CB2 receptors with molecular markers, described as important for trafficking of different G protein–coupled receptors (GPCRs), in vehicle and LPS-treated cells. Short-term treatments with LPS were performed to detect possible rapid changes on CB2 intracellular trafficking potentially triggered by the early 2-AG production, whereas long-term treatments with LPS were performed to analyze CB2 intracellular trafficking when tolerance was already established. We first analyzed the colocalization of the CB2 receptor with β-Arr2 and the EEA-1 (Supplemental Fig. 3). The CB2 receptor was localized in the cytoplasm of BMMCs in vehicle and in LPS-treated cells, where it partly colocalized with β-Arr2 (∼20%) and EEA-1 (∼5%). No evidence of a change in colocalization of CB2 with β-Arr2 (Supplemental Fig. 3A) or EEA-1 (Supplemental Fig. 3B) was noticed after either short- or long-time exposure to LPS.

To analyze if TLR4 stimulation could induce the recycling of CB2 receptors, we determined CB2 receptor colocalization with Rab11, as this receptor has been described to enter slow recycling endosomes during trafficking (48). Results show that, in BMMCs, only a small percentage (8.86%) of CB2 receptors colocalize with Rab11 under basal conditions and that a 4 h treatment with LPS induces a 40% increase of CB2 receptor location in this recycling compartment (Fig. 5A, 5B), indicating that only a small amount of this cannabinoid receptor recycles back to the plasma membrane after LPS stimulation.

To determine whether CB2 receptors could enter the degradation pathway in tolerant cells, we analyzed its location in Rab7-positive late endosomes as well as in Lamp2-positive lysosomes. Assuming that localization of CB2 receptors in these compartments could require longer times after LPS treatment, we decided to perform the experiments 4 and 7 h after endotoxin stimulation. Fig. 6A and 6B show that, under basal conditions,∼19% of the CB2 receptor is located in Rab7-positive vesicles and that stimulation with LPS for 4 and 7 h induces a sizable increase.

Late endosomes can fuse with lysosomes, where cargo degradation takes place (49). We therefore analyzed the colocalization of the CB2 receptor with the lysosomal marker Lamp2. As seen in Fig. 6C and 6D, under basal conditions,∼23% of the receptor is located in lysosomes, and this percentage did not change after 4 h of incubation with LPS. However, after 7 h of endotoxin treatment, the detection of CB2 receptor in lysosomes markedly increased to around 34%. These results suggest that, in tolerant cells, a significant percentage of CB2 receptors go to late endosomes and then enter lysosomes for degradation.

To analyze whether 2-AG participates in the control of MC-dependent inflammatory reactions in vivo, we examined its effect on the secretion of MC-derived TNF in the peritoneal cavity of C57BL/6J mice. It has been reported that 1 h after i.p. administration, LPS induces MC-derived TNF without the participation of other cell types (21, 36). As seen in Fig. 7A, i.p. injection of 2-AG (1 mg/kg) prevented the LPS-induced TNF release by 50%, whereas smaller doses of 2-AG did not show any significant effect. To demonstrate that this effect was indeed dependent on MCs in the peritoneal cavity, we performed the same experiment in MC-deficient mice Kitwsh/wsh and in Kitwsh/wsh mice reconstituted with MCs (Kitwsh/wsh rec). As can be observed on Fig. 7B, no TNF secretion was observed in KitWsh/Wsh mice after LPS stimulation, and in consequence, 2-AG administration did not have any effect. By contrast, when mice were reconstituted with WT BMMCs, both TNF secretion and the inhibitory effect of 2-AG in response to LPS stimulation were recovered, supporting that they were both MC dependent.

Endocannabinoids are considered homeostatic guardians of the immune system, preventing the onset of pathological proinflammatory responses (4, 5). Bacterial LPS-induced activation of the TLR4 receptor triggers innate inflammatory responses. One mechanism by which endocannabinoids may inhibit innate immune responses could involve the negative control of TLR inflammatory signaling (50). Based on previous data indicating a role of endocannabinoids in dampening MC-mediated inflammatory mediators (5154), we tested the hypothesis that the endocannabinoid 2-AG could be produced by MCs after TLR4 stimulation and participate in the development of tolerance to endotoxin.

The main findings of our present study are the following: 1) addition of 2-AG to BMMCs produces tolerance to LPS as the stimulation of TLR4 receptor does; 2) both CB1 and CB2 cannabinoid receptors are expressed and functional in BMMCs, but only the CB2 receptor is involved in the 2-AG–dependent induction of ET; 3) activation of TLR4 receptor leads to the production of 2-AG; 4) 2-AG prevents some of the main signaling events induced by TLR4, such as IKK and p65 phosphorylation, and promotes the synthesis of negative regulators of the TLR4 signaling pathway (SHIP1 and IRAK-M); 5) TLR4 triggering in MCs produces trafficking of CB2 receptors, which are found in Rab11-, Rab7-, and Lamp2-positive vesicles; and 6) 2-AG prevents LPS-induced TNF secretion in vivo in a MC-dependent model of endotoxemia.

In the present work, 2-AG was capable, like LPS itself, to induce tolerance to LPS in BMMCs. This is in line with previous reports that have shown that 2-AG and AEA exhibit anti-inflammatory effects when used acutely and that the activation of cannabinoid receptors inhibits TLR receptor–mediated responses (5560). Inhibitory effects of 2-AG on TLR4-induced immune responses, including the reduced expression of the proinflammatory cytokines IL-6, TNF, and COX2, have also been reported in macrophages (56, 61). Therefore, 2-AG involvement in the reduced inflammatory response that characterizes tolerance to LPS constitutes further evidence of its anti-inflammatory properties.

Although our results showed that both the mRNA and protein for CB1 and CB2 receptors are expressed and functional in BMMCs, 2-AG–induced ET was mediated by the specific activation of CB2 receptors without the participation of CB1 receptors. Indeed, only the CB2 receptor antagonist, AM630, but not the CB1 receptor antagonist, AM251, was able to prevent the development of ET produced by 2-AG. Cannabinoid receptors are expressed in different immune cells that include MCs, monocytes/macrophages, B lymphocytes, and NK cells (62, 63). Coexpression of CB1 and CB2 receptors was reported earlier in two MC lines (RBL2H3 and P815) (51); however, to our knowledge, our data are the first evidence for its coexpression in BMMCs, albeit only CB2 proved to be functionally involved in establishing tolerance. 2-AG is recognized with high affinity by CB1 and CB2 receptors, and it has been proposed to be the endogenous ligand for both of them (64). Notwithstanding, the physiological roles of 2-AG in inflammatory reactions and/or immune responses are thought to be mediated by its interaction with the CB2 receptor (65, 66), likely because 2-AG represents a full agonist toward this receptor subtype (67). Our results are in line with this assumption (6872).

To our knowledge, our data showing that LPS triggers 2-AG production and release in BMMCs via the activation of TLR4 is a novel finding. Although 2-AG is an endocannabinoid produced by several immune cells (4, 73), its association with TLR4 activation is limited. Previously, increases in 2-AG levels were reported to occur after in vitro exposure to LPS (200 μg/ml) in rat platelets (74) and rat-circulating macrophages (75). However, to our knowledge, our work provides the first evidence that TLR4 activation induces the synthesis of 2-AG in MCs because this phenomenon is not observed in those derived from TLR4−/− mice. The mechanism by which TLR4 could activate 2-AG synthesis is still an open question. It has been demonstrated that TLR4 triggering can lead to phospholipase C activation (76), increasing the levels of diacylglycerol (DAG) and thereby providing a substrate for DAG lipase (DAGL), the enzyme responsible for 2-AG synthesis. DAGL is widely expressed in immune cells (4), and although its regulation has not been described yet, its structural analysis allows to surmise several signaling pathways that could lead to its activation. For example, DAGL contains several potential sites of phosphorylation by distinct kinases, such as PKC, PKA, Src, and CAMKII (77). In particular, an increase in DAGL activity is observed upon PKA phosphorylation (78, 79). Because TLR4 has been shown to increase cAMP production and to activate PKA (80), it is possible to speculate that TLR4-dependent activation of that kinase could be involved in the 2-AG synthesis observed in BMMCs. Likewise, the production of reactive oxygen species leads to an increase in the activity of DAGL, provoking the accumulation of high 2-AG levels in macrophages in a NADPH oxidase (NOX)–dependent manner (81). Because TLR4 triggering leads to NOX activation (82), an increase in reactive oxygen species due to NOX activity after LPS addition could lead to the activation of DAGL and the production of 2-AG.

In MCs, activation of TLR4 receptors leads to the synthesis of proinflammatory mediators through the MyD88-dependent pathway (83) with the participation of early phosphorylation events mediated by Src family kinases such as Lyn (84) and Fyn (85). The canonical MyD88 cascade in MCs also leads to the ERK-dependent phosphorylation of the TNF-α–converting enzyme, which processes TNF precursor to facilitate the secretion of mature TNF (40). Long-term incubation with LPS has been shown to induce ET disabling of LPS signaling. This involves a variety of well-characterized signaling events, such as the synthesis of negative-feedback regulators of TLR4 signaling (IRAK-M, MyD88s, and A20), and epigenetic changes that prevent the transcription of the genes for proinflammatory cytokines (86, 87). Our observation shows that long-term incubation with 2-AG also induces ET with a similar, but not identical, signaling profile because phosphorylation of the MAPKs ERK1/2 and p38 was not affected by 2-AG. However, we show that 2-AG, like prolonged LPS exposure, inhibits IKK and p65 NF-κB phosphorylation caused by TLR4 receptor stimulation. However, MAPK phosphorylation was not inhibited by 2-AG. The blockage of IKK phosphorylation and downstream events leading to NF-κB activation by cannabinoids has been observed in innate immune cells, such as macrophage cell lines (88) and microglia (89), although the mechanism is still under investigation. CB2 receptors activate Gi/o heterotrimeric G proteins, that, upon stimulation, dissociate, and the Gi/o α subunit inhibits adenylyl cyclase. At the same time, βγ dimers lead to the activation of MAPK and the PI3K (9092). Because activation of PI3K leads to the inhibition of IKK, but not ERK, phosphorylation triggered by LPS in monocytes and macrophages (93), a similar mechanism can be proposed for the observed 2-AG actions on TLR4 signaling pathway in BMMCs.

Tolerance to LPS is mediated by the synthesis of a number of negative regulators of the TLR4 signaling pathway (24). Among them, the phosphatase SHIP and the inactive kinase IRAK-M are increased after LPS addition in monocytes and MCs (24, 25). We detected in BMMCs the synthesis of SHIP and IRAK-M 4 h after stimulation with 2-AG, suggesting that their production requires de novo synthesis. Because the inhibition of LPS-induced TNF release is observed as early as 2 h after 2-AG addition, it is likely that early 2-AG–induced ET events are not related to the activity of those enzymes. In agreement, we observed that pharmacological inhibition of SHIP did not show any effects on ET induction by LPS or 2-AG at 2 h. Early ET events induced by 2-AG included the inhibition of IKK and NF-κB phosphorylation, which was observed 2 h after endocannabinoid treatment. Late induction of SHIP and IRAK-M could, however, contribute to modulate long-term effects associated to ET. Our results, to our knowledge, are the first showing an early blockage of TLR4 signaling system by 2-AG and the expression of markers of ET by a prolonged exposure to endocannabinoids in immune cells, similar to LPS actions.

One of the main findings of the current study is the fact that LPS, in addition to inducing endocannabinoids’ production in BMMCs, also induces intracellular trafficking of the CB2 receptor, further confirming an important role of inflammation triggering in the function of the endocannabinoid system. GPCRs can traffic between different cell compartments to interact with different signaling pathways (94). Vesicle trafficking of GPCRs is an important process that leads receptors into two main destinies; in some cases, receptors are recycled back to the plasma membrane or to vesicles from which they can continue signaling, but in other cases, they are targeted to lysosomes to be degraded. Different molecules participate in vesicle trafficking of GPCRs, among them, the small GTPases, belonging to the Rab family, control intracellular movement of specific vesicles from distinct intracellular compartments (95). Rab5, Rab4, Rab7, and Rab11 have been involved in GPCR trafficking (49). Our data show that CB2 receptors are located in BMMCs’ intracellular compartments, and this condition was not altered by the addition of LPS or the dynamin inhibitor dynasore (data not shown). In line with these findings, it has been reported that, in immune cells, CB receptors have primarily an intracellular location. For example, in primary human B cells, CB2 receptors are present at both the plasma membrane and intracellular positions, but the majority of the protein is located within the cell. Also, in T cells and monocytes, CB2 receptor expression is restricted to intracellular locations, and in peripheral blood leukocytes, CB2 receptors are mainly found inside the cells (96). After LPS stimulation, a slight increase on CB2 immunoreactivity was detected in the nucleus, suggesting that novel signaling pathways triggered by GPCRs could be active in BMMCs (97). Further research will contribute to the definition of the intracellular signaling mechanisms of CB receptors in MCs. An important mechanism of internalization of GPCR is associated with their interaction with β-Arr2 (98). However, we were not able to detect any important interaction between CB2 receptor and β-Arr2 neither under basal conditions, nor after its activation. It is known that CB2 receptors belong to class A, rhodopsin-like GPCR family, which does not show a stable interaction with β-Arr2 (99), a fact that could have contributed to the lack of CB2 receptor and β-Arr2 colocalization detection in our testing conditions.

In our study, we also did not find colocalization of CB2 receptor with either Rab5 (data not shown) or the EEA-1. Because Rab5 and EEA-1 are known markers of early endosomes, these data suggest that the classical (clathrin-mediated) internalization pathway described for other GPCRs is not activated for the CB2 receptor after TLR4 triggering in BMMCs, which was expected because the intracellular location of CB2 receptors in this cell type. However, it is possible that at steady-state, only a tiny fraction of CB2 accumulates in this compartment. By contrast, we demonstrated that CB2 receptor colocalized with Rab 11 after LPS addition. It is known that Rab11 controls the slow recycling pathway of GPCRs back to the plasma membrane and this protein is mainly located at the perinuclear area and in the trans-Golgi network (48, 100). Because we were not able to detect the CB2 receptor at the plasma membrane, it is possible that Rab11-mediated trafficking diverges the receptor to other intracellular locations.

We also found that a sizable fraction of CB2 receptors was located in Rab7-positive compartments (around 20%) and that this fraction further increased after long-term TLR4 activation. In addition, an important fraction of CB2 was distributed in Lamp2-positive lysosomes, suggesting that it is destined for degradation. LPS stimulation significantly increases CB2 receptor location in these compartments and with a delay (7 h) as compared with Rab7-positive compartments (4 h). Because it has been described that Rab7 controls GPCR trafficking to late endosomes and then to lysosomes (100, 101), our data strongly indicate that CB2 receptors undergo degradation in response to LPS-dependent activation of TLR4 in MCs.

MCs have been recognized as important mediators of adaptive immune responses; however, their role in innate protective reactions against pathogens is still under active investigation (102). Their participation in the protective response against Gram-negative bacteria depends on the secretion of TNF (23), and it can be observed principally with low-intensity infections (30). However, they can also amplify deleterious inflammatory reactions (30). Administration of LPS into the peritoneal cavity of mice is a well-recognized murine model to induce endotoxemia and septic shock (103). Under similar conditions, MCs have been found to be responsible for the observed lethal hypothermia in septic shock (104). It is also known that cells become refractory to LPS stimulation, leading to ET. Although this was explained mainly by the inhibition of canonical TLR4 signaling pathways, our data provided in this study show that at least part of the inhibitory action can be explained by an autocrine feedback loop involving the endocannabinoid system (Fig. 8). This includes in vivo effects, as 2-AG promotes a refractory state to LPS challenge in peritoneal MCs. These data constitute, to our knowledge, the first evidence that endocannabinoids can inhibit MC-dependent innate immune responses in vivo, suggesting that the negative role of 2-AG on MCs activation could play a physiological role.

Our study provides evidence that MC-derived 2-AG, produced in response to TLR4 activation, is functionally relevant for the physiological control of inflammatory responses. As MCs are known to participate in several inflammatory processes, the possible contribution of this endocannabinoid to other physiological mechanisms directed to avoid noncontrolled inflammation should be explored. Besides, the CB2 receptor appears as a potential therapeutic target as it seems to be involved in several processes of immune tolerance (105).

We thank Dr. Jorge Fernández Hernández, Ma. Antonieta López-López, Ricardo Glaxiola Centeno, Víctor Manuel García Gómez, and Benjamín E. Chávez-Álvarez for assistance in maintaining the colonies of mice used in this study (Unit for Production and Experimentation with Laboratory Animals, Cinvestav); Dr. Mario E. Cruz Muñoz from the Autonomous University of Morelos State (Mexico) for kindly providing SHIP inhibitor (3-AC); and Samira Benadda for support on the use of the confocal microscope (Paris, France).

This work was supported by the Consejo Nacional de Ciencia y Tecnología-Agence Nationale de la Recherche (Conacyt-ANR) Grant 188565 (to C.G.-E. and U.B.), Conacyt Grant FC-1122 (to C.G.-E.), and Conacyt Grant 220772 (to G.R.-M.). Z.P.E.-R. received a scholarship from Conacyt (277991) for Ph.D. studies. Conacyt-ANR Grant 188565 and Conacyt Grant 291212 supported an academic exchange period for Z.P.E.-R. at the Université Paris VII.

The online version of this article contains supplemental material.

Abbreviations used in this article:

3-AC

3-α-aminocholestane

AEA

anandamide

2-AG

2-arachidonoylglycerol

β-Arr2

β-arrestin 2

BMMC

bone marrow–derived MC

DAG

diacylglycerol

DAGL

DAG lipase

EEA-1

early endosome Ag 1

ET

endotoxin tolerance

GPCR

G protein–coupled receptor

IRAK-M

IL-1R–associated kinase M

MC

mast cell

NOX

NADPH oxidase

Wsh rec

reconstituted Wsh

WT

wild-type.

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The authors have no financial conflicts of interest.

Supplementary data