Identification and in vivo detection of side-chain hydroxylated metabolites of 4ß-hydroxycholesterol.

Oxysterols are oxidized derivatives of cholesterol that are formed by enzymatic processes or through the action of reactive oxygen species. Several of these bioactive lipids have been shown to be affected and/or play a role in inflammatory processes. 4ß-hydroxycholesterol is one of the major oxysterols in mice and humans and its levels are affected by inflammatory diseases. However, apart from its long half-life, little is known about its catabolism. By incubating 4ß-hydroxycholesterol with mouse mitochondria-enriched liver fractions, as well as 25-hydroxycholesterol and 27-hydroxycholesterol with recombinant CYP3A4, we identified 4ß,25-dihydroxycholesterol and 4ß,27-dihydroxycholesterol as 4ß-hydroxycholesterol metabolites. Supporting the biological relevance of this metabolism, we detected both metabolites after incubation of J774, primary mouse peritoneal macrophages and PMA-differentiated THP-1 cells with 4ß-hydroxycholesterol. Across our experiments, the incubation of cells with lipopolysaccharides differentially affected the levels of the 25- and 27-hydroxylated metabolites of 4ß-hydroxycholesterol. Finally, 4ß,27-dihydroxycholesterol was also detected in mice liver and plasma after intraperitoneal administration of 4ß-hydroxycholesterol. To our knowledge, this is the first report of the in vitro and in vivo detection and quantification of 4ß-hydroxycholesterol metabolites.

Dysosmobacter welbionis effects on glucose, lipid, and energy metabolism are associated with specific bioactive lipids.

The newly identified bacterium Dysosmobacter welbionis J115T improves host metabolism in high-fat diet (HFD)-fed mice. To investigate mechanisms, we used targeted lipidomics to identify and quantify bioactive lipids produced by the bacterium in the culture medium, the colon, the brown adipose tissue (BAT), and the blood of mice. In vitro, we compared the bioactive lipids produced by D. welbionis J115T versus the probiotic strain Escherichia coli Nissle 1917. D. welbionis J115T administration reduced body weight, fat mass gain, and improved glucose tolerance and insulin resistance in HFD-fed mice. In vitro, 19 bioactive lipids were highly produced by D. welbionis J115T as compared to Escherichia coli Nissle 1917. In the plasma, 13 lipids were significantly changed by the bacteria. C18-3OH was highly present at the level of the bacteria, but decreased by HFD treatment in the plasma and normalized in D. welbionis J115T-treated mice. The metabolic effects were associated with a lower whitening of the BAT. In the BAT, HFD decreased the 15-deoxy-Δ12,14-prostaglandin J2, a peroxisome proliferator-activated receptor (PPAR-γ) agonist increased by 700% in treated mice as compared to HFD-fed mice. Several genes controlled by PPAR-γ were upregulated in the BAT. In the colon, HFD-fed mice had a 60% decrease of resolvin D5, whereas D. welbionis J115T-treated mice exhibited a 660% increase as compared to HFD-fed mice. In a preliminary experiment, we found that D. welbionis J115T improves colitis. In conclusion, D. welbionis J115T influences host metabolism together with several bioactive lipids known as PPAR-γ agonists.

Lipid nanocapsules for the nose-to-brain delivery of the anti-inflammatory bioactive lipid PGD2-G.

Here, prostaglandin D2-glycerol ester (PGD2-G) was selected to target neuroinflammation. As PGD2-G is reported to have a short plasmatic half-life, we propose to use lipid nanocapsules (LNC) as vehicle to safely transport PGD2-G to the central nervous system (CNS). PGD2-G-loaded LNC (PGD2-G-LNC) reduced pro-inflammatory cytokine expression in activated microglial cells, even so after crossing a primary olfactory cell monolayer. A single nasal administration of PGD2-G-LNC in lipopolysaccharide (LPS)-treated mice reduced pro-inflammatory cytokine expression in the olfactory bulb. Coating LNC's surface with a cell-penetrating peptide, transactivator of transcription (TAT), increased its accumulation in the brain. Although TAT-coated PGD2-G-LNC modestly exerted its anti-inflammatory effect in a mouse model of multiple sclerosis similar to free PGD2-G after nasal administration, TAT-coated LNC surprisingly reduced the expression of pro-inflammatory chemokines in the CNS. These data propose LNC as an interesting drug delivery tool and TAT-coated PGD2-G-LNC remains a good candidate, in need of further work.

Effect of a methanolic extract of Salvadora oleoides Decne. on LPS-activated J774 macrophages, its in vitro and in vivo toxicity study and dereplication of its chemical constituents.

Salvadora oleoides is used in Pakistani traditional medicine to treat inflammatory conditions, piles, boils, and ulcers. To evaluate the anti-inflammatory potential of S. oleoides (a mixture of aerial branches, leaves, and stem bark), we prepared crude extracts in Soxhlet apparatus by successively using different solvents and found the methanolic extract (OLM) to significantly inhibit the LPS-induced expression of pro-inflammatory cytokines and enzymes in J774 macrophages, at 50 µg/mL concentration. We also analysed the chemical constituents of OLM by dereplication, performed by HPLC-MS/MS and molecular networking. The major detected constituents were flavonoids and phenolic acids glycosides, most of them identified for the first time in S. oleoides. We also evaluated the toxicity of OLM against five cell lines, namely Caco-2, HepG2, HeLa, J774, and WI-38 by MTT assay. The IC50 was found to be higher than 100 µg/mL against these five cell lines after 72 h treatment. Furthermore, OLM was tested in mice for acute and sub-acute oral toxicity according to the guidelines of the Organisation for Economic Co-operation and Development (OECD). OLM was found non-toxic, except for some fibrosis observed in the spleens of treated mice in the sub-acute oral toxicity test. Our results confirm the anti-inflammatory potential of OLM and that it could be tested in in vivo inflammatory models, but its effect on the spleen should be considered before designing the experiments.

5'AMP-activated protein kinase: an emerging target of phytochemicals to treat chronic inflammatory diseases.

Inflammation is a defensive response of the organism to traumatic, infectious, toxic, ischemic, and autoimmune injury. Inflammatory mediators are released to effectively eliminate the inflammatory trigger and restore homeostasis. However, failure of these processes can lead to chronic inflammatory conditions and diseases such as inflammatory bowel diseases, rheumatoid arthritis, inflammatory lung diseases, atherosclerosis, and neurodegenerative diseases. The cure of chronic inflammatory diseases remains challenging as current therapies have various limitations, such as pronounced side effects, progressive loss of efficacy, and high cost especially for biologics. In this context, phytochemicals (such as alkaloids, flavonoids, lignans, phenolic acids, saponins, terpenoids, and other classes) are considered as an interesting alternative approach. Among the numerous targets of phytochemicals, AMP-activated protein kinase (AMPK) can be considered as an interesting target in the context of inflammation. AMPK regulates inflammatory response by inhibiting inflammatory pathways (NF-κB, JAK/STAT, and MAPK) and regulating several other processes of the inflammatory response (oxidative stress, autophagy, and apoptosis). In this review, we summarize and discuss the studies focusing on phytochemicals that showed beneficial effects by blocking different inflammatory pathways implicating AMPK activation in chronic inflammatory disease models. We also highlight elements to consider when investigating AMPK in the context of phytochemicals.

Dereplication and Quantification of Major Compounds of Convolvulus arvensis L. Extracts and Assessment of Their Effect on LPS-Activated J774 Macrophages.

Convolvulus arvensis is used in Pakistani traditional medicine to treat inflammation-related disorders. Its anti-inflammatory potential was evaluated on hexane, dichloromethane, ethyl acetate, methanol, and aqueous extracts of whole plant on pro-inflammatory mediators in LPS-activated murine macrophage J774 cells at the non-cytotoxic concentration of 50 µg/mL. Ethyl acetate (ARE) and methanol (ARM) extracts significantly decreased mRNA levels of IL-6, TNF-α, MCP-1, COX-2, and iNOS. Furthermore, both extracts dose dependently decreased IL-6, TNF-α, and MCP-1 secretion. Forty-five compounds were putatively identified in ARE and ARM by dereplication (using HPLC-UV-HRMSn analysis and molecular networking), most of them are reported for the first time in C. arvensis, as for example, nineteen phenolic derivatives. Rutin, kaempferol-3-O-rutinoside, chlorogenic acid, 3,5-di-O-caffeoylquinic acid, N-trans-p-coumaroyl-tyramine, and N-trans-feruloyl-tyramine were main constituents identified and quantified by HPLC-PDA in ARE and ARM. Furthermore, chlorogenic acid, tyramine derivatives, and the mixture of the six identified major compounds significantly decreased IL-6 secretion by LPS-activated J774 cells. The activity of N-trans-p-coumaroyl-tyramine is shown here for the first time. Our results indicate that ARE, ARM and major constituents significantly inhibited the expression of pro-inflammatory mediators, which supports the use of this plant to treat inflammatory diseases.

N-Acylethanolamine-Hydrolyzing Acid Amidase Inhibition, but Not Fatty Acid Amide Hydrolase Inhibition, Prevents the Development of Experimental Autoimmune Encephalomyelitis in Mice.

N-acylethanolamines (NAEs) are endogenous bioactive lipids reported to exert anti-inflammatory and neuroprotective effects mediated by cannabinoid receptors and peroxisome proliferator-activated receptors (PPARs), among others. Therefore, interfering with NAE signaling could be a promising strategy to decrease inflammation in neurological disorders such as multiple sclerosis (MS). Fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase (NAAA) are key modulators of NAE levels. This study aims to investigate and compare the effect of NAAA inhibition, FAAH inhibition, and dual inhibition of both enzymes in a mouse model of MS, namely the experimental autoimmune encephalomyelitis (EAE). Our data show that NAAA inhibition strongly decreased the hallmarks of the pathology. Interestingly, FAAH inhibition was less efficient in decreasing inflammatory hallmarks despite the increased NAE levels. Moreover, the inhibition of both NAAA and FAAH, using a dual-inhibitor or the co-administration of NAAA and FAAH inhibitors, did not show an added value compared to NAAA inhibition. Furthermore, our data suggest an important role of decreased activation of astrocytes and microglia in the effects of NAAA inhibition on EAE, while NAAA inhibition did not affect T cell recall. This work highlights the beneficial effects of NAAA inhibition in the context of central nervous system inflammation and suggests that the simultaneous inhibition of NAAA and FAAH has no additional beneficial effect in EAE.

Effects of R-flurbiprofen and the oxygenated metabolites of endocannabinoids in inflammatory pain mice models.

Pain is one of the cardinal signs accompanying inflammation. The prostaglandins (PGs), synthetized from arachidonic acid by cyclooxygenase (COX)-2, are major bioactive lipids implicated in inflammation and pain. However, COX-2 is also able to metabolize other lipids, including the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA), to give glycerol ester (PG-G) and ethanolamide (PG-EA) derivatives of the PGs. Consequently, COX-2 can be considered as a hub not only controlling PG synthesis, but also PG-G and PG-EA synthesis. As they were more recently characterized, these endocannabinoid metabolites are less studied in nociception compared to PGs. Interestingly R-profens, previously considered as inactive enantiomers of nonsteroidal anti-inflammatory drugs (NSAIDs), are substrate-selective COX inhibitors. Indeed, R-flurbiprofen can selectively block PG-G and PG-EA production, without affecting PG synthesis from COX-2. Therefore, we compared the effect of R-flurbiprofen and S-flurbiprofen in models of inflammatory pain triggered by local administration of lipopolysaccharides (LPS) and carrageenan in mice. Remarkably, the effects of flurbiprofen enantiomers on mechanical hyperalgesia seem to depend on (i) the inflammatory stimuli, (ii) the route of administration, and (iii) the timing of administration. We also assessed the effect of administration of the PG-Gs, PG-EAs, and PGs on LPS-induced mechanical hyperalgesia. Our data support the interest of studying the nonhydrolytic endocannabinoid metabolism in the context of inflammatory pain.

25-Hydroxycholesterol metabolism is altered by lung inflammation, and its local administration modulates lung inflammation in mice.

Inflammation is a critical component of many lung diseases including asthma and acute lung injury (ALI). Using high-performance liquid chromatography-mass spectrometry, we quantified the levels of oxysterols in two different murine models of lung diseases. These are lipid mediators derived from cholesterol and known to modulate immunity and inflammation. Interestingly, 25-hydroxycholesterol (25-OHC) was the only oxysterol with altered levels during lung inflammation, and its levels were differently affected according to the model. Therefore, we sought to assess how this oxysterol would affect lung inflammatory responses. In a model of lipopolysaccharide (LPS)-induced acute lung inflammation, 25-OHC levels were increased, and most of the hallmarks of the model (eg, leukocyte recruitment, mRNA expression, and secretion of inflammatory cytokines) were decreased following its intratracheal administration. We also found that, when administered in the lung, 25-OHC is metabolized locally into 25-hydroxycholesterol-3-sulfate and 7α,25-dihydroxycholesterol. Their administration in the lungs did not recapitulate all the effects of 25-OHC. Conversely, in a model of allergic asthma induced by intranasal administration of house dust mites (HDM), 25-OHC levels were decreased, and when intranasally administered, this oxysterol worsened the hallmarks of the model (eg, leukocyte recruitment, tissue remodeling [epithelium thickening and peribranchial fibrosis], and cytokine expression) and induced changes in leukotriene levels. Ex vivo, we found that 25-OHC decreases LPS-induced primary alveolar macrophage activation while having no effect on neutrophil activation. Its sulfated metabolite, 25-hydroxycholesterol-3-sulfate, decreased neutrophil, but not macrophage activation. Taken together, our data support a differential role of 25-OHC in ALI and allergic inflammation models.

Bioactive lipids in inflammatory bowel diseases - From pathophysiological alterations to therapeutic opportunities.

Inflammatory bowel diseases (IBDs), such as Crohn's disease and ulcerative colitis, are lifelong diseases that remain challenging to treat. IBDs are characterized by alterations in intestinal barrier function and dysregulation of the innate and adaptive immunity. An increasing number of lipids are found to be important regulators of inflammation and immunity as well as gut physiology. Therefore, the study of lipid mediators in IBDs is expected to improve our understanding of disease pathogenesis and lead to novel therapeutic opportunities. Here, through selected examples - such as fatty acids, specialized proresolving mediators, lysophospholipids, endocannabinoids, and oxysterols - we discuss how lipid signaling is involved in IBD physiopathology and how modulating lipid signaling pathways could affect IBDs.

miRNA profile is altered in a modified EAE mouse model of multiple sclerosis featuring cortical lesions.

Cortical lesions represent a hallmark of multiple sclerosis and are proposed as a predictor of disease severity. microRNAs are suggested to be important players in the disease pathogenesis and the experimental autoimmune encephalomyelitis animal model. We implemented a mouse model recapitulating more closely the human pathology as it is characterized by both an autoimmune heterogeneity and the presence of cortical lesions, two parameters missing in experimental autoimmune encephalomyelitis. In our model, mice clustered in two groups displaying high or low clinical scores. Upon cortical cytokine injection, lesions appeared with a specific topography while cortical miRNA profiles were altered. These two features differed according to disease severity. We evidenced changes in miRNA regulators and targets suggesting that miRNA alteration had functional repercussions that could explain the differences in cortical lesions. This model represents a crucial tool for the study of both miRNA involvement and cortical lesion formation in disease pathogenesis.

Effects of orthotopic implantation of rat prostate tumour cells upon components of the N-acylethanolamine and monoacylglycerol signalling systems: an mRNA study.

There is good evidence that the N-acylethanolamine (NAE)/monoacylglycerol (MAG) signalling systems are involved in the pathogenesis of cancer. However, it is not known how prostate tumours affect these systems in the surrounding non-malignant tissue and vice versa. In the present study we have investigated at the mRNA level 11 components of these systems (three coding for anabolic enzymes, two for NAE/MAG targets and six coding for catabolic enzymes) in rat prostate tissue following orthotopic injection of low metastatic AT1 cells and high metastatic MLL cells. The MLL tumours expressed higher levels of Napepld, coding for a key enzyme in NAE synthesis, and lower levels of Naaa, coding for the NAE hydrolytic enzyme N-acylethanolamine acid amide hydrolase than the AT1 tumours. mRNA levels of the components of the NAE/MAG signalling systems studied in the tissue surrounding the tumours were not overtly affected by the tumours. AT1 cells in culture expressed Faah, coding for the NAE hydrolytic enzyme fatty acid amide hydrolase, at much lower levels than Naaa. However, the ability of the intact cells to hydrolyse the NAE arachidonoylethanolamide (anandamide) was inhibited by an inhibitor of FAAH, but not of NAAA. Treatment of the AT1 cells with interleukin-6, a cytokine known to be involved in the pathogenesis of prostate cancer, did not affect the expression of the components of the NAE/MAG system studied. It is thus concluded that in the model system studied, the tumours show different expressions of mRNA coding for key the components of the NAE/MAG system compared to the host tissue, but that these changes are not accompanied by alterations in the non-malignant tissue.

Altered mRNA Expression of Genes Involved in Endocannabinoid Signalling in Squamous Cell Carcinoma of the Oral Tongue.

Little is known about the endocannabinoid (eCB) system in squamous cell carcinoma of the oral tongue (SCCOT). Here we have investigated, at the mRNA level, expression of genes coding for the components of the eCB system in tumour and non-malignant samples from SCCOT patients. Expression of NAPEPLD and PLA2G4E, coding for eCB anabolic enzymes, was higher in the tumour tissue than in non-malignant tissue. Among genes coding for eCB catabolic enzymes, expression of MGLL was lower in tumour tissue while PTGS2 was increased. It is concluded that the eCB system may be dysfunctional in SCCOT.

Endocannabinoid and Prostanoid Crosstalk in Pain.

Interfering with endocannabinoid (eCB) metabolism to increase their levels is a proven anti-nociception strategy. However, because the eCB and prostanoid systems are intertwined, interfering with eCB metabolism will affect the prostanoid system and inversely. Key to this connection is the production of the cyclooxygenase (COX) substrate arachidonic acid upon eCB hydrolysis as well as the ability of COX to metabolize the eCBs anandamide (AEA) and 2-arachidonoylglycerol (2-AG) into prostaglandin-ethanolamides (PG-EA) and prostaglandin-glycerol esters (PG-G), respectively. Recent studies shed light on the role of PG-Gs and PG-EAs in nociception and inflammation. Here, we discuss the role of these complex systems in nociception and new opportunities to alleviate pain by interacting with them.

Role of pannexin-1 in the cellular uptake, release and hydrolysis of anandamide by T84 colon cancer cells.

The large pore ion channel pannexin-1 (Panx1) has been reported to play a role in the cellular uptake and release of anandamide (AEA) in the hippocampus. It is not known whether this is a general mechanism or limited to the hippocampus. We have investigated this pharmacologically using T84 colon cancer cells. The cells expressed Panx1 at the mRNA level, and released ATP in a manner that could be reduced by treatment with the Panx1 inhibitors carbenoxolone and mefloquine and the Panx1 substrate SR101. However, no significant effects of these compounds upon the uptake or hydrolysis of exogenously applied AEA was seen. Uptake by T84 cells of the other main endocannabinoid 2-arachidonoylglycerol and the AEA homologue palmitoylethanolamide was similarly not affected by carbenoxolone or mefloquine. Total release of tritium from [3H]AEA-prelabelled T84 cells over 10 min was increased, rather than inhibited by carbenoxolone and mefloquine. Finally, AEA uptake by PC3 prostate cancer and SH-SY5Y neuroblastoma cells, which express functional Panx1 channels, was not inhibited by carbenoxolone. Thus, in contrast to the hippocampus, Panx1 does not appear to play a role in AEA uptake and release from the cells studied under the conditions used.

Low mRNA expression and activity of monoacylglycerol lipase in human SH-SY5Y neuroblastoma cells.

Relatively little is known about the endocannabinoid system in human neuroblastoma cell lines. In the present study, we have investigated the expression of the genes coding for the enzymes involved in the synthesis and catabolism of endocannabinoids in the SH-SY5Y cell line. The expression of MGLL, the gene coding for the 2-arachidonoylglycerol hydrolytic enzyme monoacylglycerol lipase (MAGL), was found to be 85 and 340 fold lower than the expression levels for the genes coding for alpha/beta-hydrolase domain containing 6 and 12 (ABHD6, ABHD12), which are alternative hydrolytic enzymes for this endocannabinoid. In comparison, mRNA levels of MGLL were 1.5 fold higher than ABHD6 and 2 fold lower than the levels of ABHD12 in DU-145 human prostate cells. In functional assays, the hydrolysis of the 2-arachidonoylglycerol homologue 2-oleoylglycerol by intact SH-SY5Y cells was partially inhibited by the ABHD6 inhibitor WWL70, but not by the MAGL inhibitor JZL184, whereas the reverse was true in DU-145 cells. The combination of JZL184 + WWL70 did, however produce a significantly greater inhibition of 2-OG hydrolysis than seen with WWL70 alone in the SH-SY5Y cells. The low MGLL expression in the SH-SY5Y cells was not due to epigenetic silencing, since levels were not affected by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine and/or the histone acetylase inhibitor trichostatin A. The low MGLL expression in SH-SY5Y cells should be taken into account when using these cells in experiments investigating the involvement of the endocannabinoid system in models of physiological and pathological processes.

The α/ß-hydrolase domain 6 inhibitor WWL70 decreases endotoxin-induced lung inflammation in mice, potential contribution of 2-arachidonoylglycerol, and lysoglycerophospholipids.

Lung inflammation plays a crucial role in the pathogenesis of many respiratory diseases that are in need of new therapeutic strategies. Previously, we showed that inhibition of α/ß-hydrolase domain 6 (ABHD6) decreased macrophage activation and exerted anti-inflammatory effects. Therefore, we thought to assess the effects of ABHD6 inhibition in a mouse model of acute lung injury (ALI) induced by intratracheal administration of lipopolysaccharides. ABHD6 inhibition with N-methyl-N-{[3-(4-pyridinyl)phenyl]methyl}-carbamic acid 4'-(aminocarbonyl)(1,1'-biphenyl)-4-yl ester (WWL70) decreases most of the hallmarks of ALI, including neutrophil infiltration, cytokine secretion, and protein extravasation. mRNA expression of proinflammatory markers in the cells recovered in the bronchoalveolar lavage was also decreased. Interestingly, ABHD6 inhibition was more efficient than monoacylglycerol lipase inhibition by 4-nitrophenyl-4-[dibenzo(d)(14)dioxol-5-yl(hydroxy)methyl]piperidine-1-carboxylate. We also studied ABHD6 inhibition on primary alveolar macrophages and neutrophils to explore their potential implication in the effects of ABHD6 inhibition in vivo. Moreover, we quantified by high-performance liquid chromatography-mass spectrometry the levels of reported substrates of ABHD6 [i.e., 2-arachidonoylglycerol (2-AG) and lysophospholipids]. The potential implication of these lipid mediators in the effects of WWL70 was further investigated on primary alveolar macrophages. Taken together, these data support ABHD6 inhibition as an interesting anti-inflammatory strategy in acute lung inflammation and assess the possible contribution of 2-AG and lysophospholipids in the observed effects.-Bottemanne, P., Paquot, A., Ameraoui, H., Alhouayek, M., Muccioli, G. G. The α/ß-hydrolase domain 6 inhibitor WWL70 decreases endotoxin-induced lung inflammation in mice, potential contribution of 2-arachidonoylglycerol, and lysoglycerophospholipids.

Prostaglandin D2-glycerol ester decreases carrageenan-induced inflammation and hyperalgesia in mice.

Pain is one of the cardinal signs of inflammation and is present in many inflammatory conditions. Therefore, anti-inflammatory drugs such as NSAIDs also have analgesic properties. We previously showed that prostaglandin D2-glycerol ester (PGD2-G), endogenously produced by cyclooxygenase-2 from the endocannabinoid 2-arachidonoylglycerol, has anti-inflammatory effects in vitro and in vivo that are partly mediated by DP1 receptor activation. In this work, we investigated its effect in a model of carrageenan-induced inflammatory pain. PGD2-G decreased hyperalgesia and edema, leading to a faster recovery. Moreover, PGD2-G decreased carrageenan-induced inflammatory markers in the paw as well as inflammatory cell recruitment. The effects of PGD2-G were independent from metabolite formation (PGD2 and 15d-PGJ2-G) or DP1 receptor activation in this model. Indeed PGD2 delayed recovery from hyperalgesia while 15d-PGJ2-G worsened the edema. However, while PGD2-G decreased hyperalgesia in this model of inflammatory pain, it had no effect when tested in the capsaicin-induced pain model. While the targets mediating the effects of this bioactive lipid in inflammatory pain remain to be elucidated, our findings further support the interest of anti-inflammatory lipid mediators in the management of inflammatory pain.

Interferon γ treatment increases endocannabinoid and related N-acylethanolamine levels in T84 human colon carcinoma cells.

BACKGROUND AND PURPOSE: Endocannabinoids and related N-acylethanolamines (NAEs) are involved in regulation of gut function, but relatively little is known as to whether inflammatory cytokines such as IFNγ affect their levels. We have investigated this in vitro using cultures of T84 colon cancer cells. EXPERIMENTAL APPROACH: T84 cells, when cultured in monolayers, differentiate to form adult colonic crypt-like cells with excellent permeability barrier properties. The integrity of the permeability barrier in these monolayers was measured using transepithelial electrical resistance (TEER). NAE levels were determined by ultra-performance liquid chromatography-tandem mass spectrometric analysis. Expression of the enzymes involved in NAE and 2-arachidonoylglycerol (2-AG) turnover were assessed with qPCR. KEY RESULTS: IFNγ treatment for 8 or 24 h increased levels of both endocannabinoids (anandamide and 2-AG) and the related NAEs. The treatment did not affect the rate of hydrolysis of either anandamide or palmitoylethanolamide by intact cells, and in both cases, fatty acid amide hydrolase (FAAH) rather than NAE-hydrolysing acid amidase (NAAA) was mainly responsible for the hydrolysis of these NAEs. IFNγ treatment reduced the TEER of the cells in a manner that was not prevented by inhibition of either FAAH or NAAA but was partially reversed by apical administration of the NAE palmitoylethanolamide. CONCLUSION AND IMPLICATIONS: IFNγ treatment mobilized endocannabinoid and related NAE levels in T84 cells. However, blockade of anandamide or NAE hydrolysis was insufficient to negate the deleterious effects of this cytokine upon the permeability barrier of the cell monolayers. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.