Roles of Toll-like receptor 2/4, monoacylglycerol lipase, and cyclooxygenase in social defeat stress-induced prostaglandin E2 synthesis in the brain and their behavioral relevance

Inflammation in the brain and periphery has been associated with stress-related pathology of mental illness. We have shown that prostaglandin (PG) E2, an arachidonic acid-derived lipid mediator, and innate immune receptors Toll-like receptor (TLR) 2/4 are crucial for repeated stress-induced behavioral changes in rodents. However, how the stress induces PGE2 synthesis in the brain and whether TLR2/4 are involved in the PGE2 synthesis remain unknown. Using mice lacking TLR2 and TLR4 in combination, here we show that social defeat stress (SDS) induced the PGE2 synthesis in subcortical, but not cortical, tissues in a TLR2/4-dependent manner. It is known that PGE2 in the brain is mainly derived by monoacylglycerol lipase (MAGL)-mediated conversion of endocannabinoid 2-arachidonoylglycerol to free-arachidonic acid, a substrate for cyclooxygenase (COX) for PGE2 synthesis. We found that TLR2/4 deletion reduced the mRNA expression of MAGL and COX1 in subcortical tissues after repeated SDS. Perturbation of MAGL and COX1 as well as COX2 abolished SDS-induced PGE2 synthesis in subcortical tissues. Furthermore, systemic administration of JZL184, an MAGL inhibitor, abolished repeated SDS-induced social avoidance. These results suggest that SDS induces PGE2 synthesis in subcortical regions of the brain via the MAGL-COX pathway in a TLR2/4-dependent manner, thereby leading to social avoidance.

Neutral Lipids Are Not a Source of Arachidonic Acid for Lipid Mediator Signaling in Human Foamy Monocytes

Human monocytes exposed to free arachidonic acid (AA), a secretory product of endothelial cells, acquire a foamy phenotype which is due to the accumulation of cytoplasmic lipid droplets with high AA content. Recruitment of foamy monocytes to the inflamed endothelium contributes to the development of atherosclerotic lesions. In this work, we investigated the potential role of AA stored in the neutral lipids of foamy monocytes to be cleaved by lipases and contribute to lipid mediator signaling. To this end, we used mass spectrometry-based lipidomic approaches combined with strategies to generate monocytes with different concentrations of AA. Results from our experiments indicate that the phospholipid AA pool in monocytes is stable and does not change upon exposure of the cells to the external AA. On the contrary, the AA pool in triacylglycerol is expandable and can accommodate relatively large amounts of fatty acid. Stimulation of the cells with opsonized zymosan results in the expected decreases of cellular AA. Under all conditions examined, all of the AA decreases observed in stimulated cells were accounted for by decreases in the phospholipid pool; we failed to detect any contribution of the triacylglycerol pool to the response. Experiments utilizing selective inhibitors of phospholipid or triacylglyerol hydrolysis confirmed that the phospholipid pool is the sole contributor of the AA liberated by stimulated cells. Thus, the AA in the triacylglycerol is not a source of free AA for the lipid mediator signaling during stimulation of human foamy monocytes and may be used for other cellular functions.

Cellular Plasmalogen Content Does Not Influence Arachidonic Acid Levels or Distribution in Macrophages: A Role for Cytosolic Phospholipase A2γ in Phospholipid Remodeling

Availability of free arachidonic acid (AA) constitutes a rate limiting factor for cellular eicosanoid synthesis. AA distributes differentially across membrane phospholipids, which is largely due to the action of coenzyme A-independent transacylase (CoA-IT), an enzyme that moves the fatty acid primarily from diacyl phospholipid species to ether-containing species, particularly the ethanolamine plasmalogens. In this work, we examined the dependence of AA remodeling on plasmalogen content using the murine macrophage cell line RAW264.7 and its plasmalogen-deficient variants RAW.12 and RAW.108. All three strains remodeled AA between phospholipids with similar magnitude and kinetics, thus demonstrating that cellular plasmalogen content does not influence the process. Cell stimulation with yeast-derived zymosan also had no effect on AA remodeling, but incubating the cells in AA-rich media markedly slowed down the process. Further, knockdown of cytosolic-group IVC phospholipase A2γ (cPLA2γ) by RNA silencing significantly reduced AA remodeling, while inhibition of other major phospholipase A2 forms such as cytosolic phospholipase A2α, calcium-independent phospholipase A2β, or secreted phospholipase A2 had no effect. These results uncover new regulatory features of CoA-IT-mediated transacylation reactions in cellular AA homeostasis and suggest a hitherto unrecognized role for cPLA2γ in maintaining membrane phospholipid composition via regulation of AA remodeling.

Cloning, expression, purification of active recombinant human 12R-LOX enzyme and its inhibition by Acalypha indica extracts

12R-LOX over-expression has been reported in various pathologies such as psoriasis, proliferative dermatitis as well as pulmonary obstructive diseases indicating that this enzyme plays significant role in pathogenesis of inflammatory diseases. 12R-LOX, therefore, is a suitable target for therapeutic intervention with potential application of its inhibitors in the treatment of skin and other inflammatory disorders. Identification of such inhibitors requires sufficient quantity of active enzyme to be produced by an easy and cost effective expression systems and further development of a robust assay system to screen inhibitors against the 12R-LOX enzyme. Therefore, in the present study, a prokaryotic expression system was developed to over-express and purify active human 12R-LOX enzyme by a single step purification process. We have further standardized an HPLC based assay system to assess the activity of purified human 12R-LOX enzyme. We show here that purified 12R-LOX preferentially utilizes free arachidonic acid as the substrate but it is also active on methyl ester of arachidonic acid, albeit less efficiently. Additionally, using this assay system we observed the potent inhibition of human 12R-LOX enzyme activity by the ethyl acetate and aqueous sub-fractions of Acalypha indica leaves, which is widely used in traditional medicines for the treatment of various skin ailments

Cloning, expression, purification of active recombinant human 12R-LOX enzyme and its inhibition by Acalypha indica extracts

12R-LOX over-expression has been reported in various pathologies such as psoriasis, proliferative dermatitis as well as pulmonary obstructive diseases indicating that this enzyme plays significant role in pathogenesis of inflammatory diseases. 12R-LOX, therefore, is a suitable target for therapeutic intervention with potential application of its inhibitors in the treatment of skin and other inflammatory disorders. Identification of such inhibitors requires sufficient quantity of active enzyme to be produced by an easy and cost effective expression systems and further development of a robust assay system to screen inhibitors against the 12R-LOX enzyme. Therefore, in the present study, a prokaryotic expression system was developed to over-express and purify active human 12R-LOX enzyme by a single step purification process. We have further standardized an HPLC based assay system to assess the activity of purified human 12R-LOX enzyme. We show here that purified 12R-LOX preferentially utilizes free arachidonic acid as the substrate but it is also active on methyl ester of arachidonic acid, albeit less efficiently. Additionally, using this assay system we observed the potent inhibition of human 12R-LOX enzyme activity by the ethyl acetate and aqueous sub-fractions of Acalypha indica leaves, which is widely used in traditional medicines for the treatment of various skin ailments

An Optimized High Throughput Clean-Up Method Using Mixed-Mode SPE Plate for the Analysis of Free Arachidonic Acid in Plasma by LC-MS/MS

A high throughput sample preparation method was developed utilizing mixed-mode solid phase extraction (SPE) in 96-well plate format for the determination of free arachidonic acid in plasma by LC-MS/MS. Plasma was mixed with 3% aqueous ammonia and loaded into each well of 96-well plate. After washing with water and methanol sequentially, 3% of formic acid in acetonitrile was used to elute arachidonic acid. The collected fraction was injected onto a reversed phase column at 30°C with mobile phase of acetonitrile/water (70 : 30, v/v) and detected by LC-MS/MS coupled with electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The calibration curve ranged from 10 to 2500 ng/mL with sufficient linearity (r2= 0.9999). The recoveries were in the range of 99.38% to 103.21% with RSD less than 6%. The limit of detection is 3 ng/mL.

Effects of short-term infusion of lipid emulsions on pro-inflammatory cytokines and lymphocyte apoptosis in septic and non-septic rats

Long-term administration of PUFA is known to modulate immune functions and apoptotic pathways depending on the respective amount of n-6 and n-3 fatty acids (FA). Data on short-term effects on apoptotic pathways are rare. Apoptosis of splenic lymphocytes is the hallmark of detrimental sepsis. Therefore, we aimed to compare the immediate effects of parenterally administered n-6-enriched soyabean oil (SO)- and n-3-enriched fish oil (FO)-based lipid emulsions after laparotomy (LAP; sham procedure) and after induction of acute, severe sepsis by caecal ligation and incision. After 390 min of observation time, plasma was analysed for IL-1β, IL-6 and NEFA. Apoptosis in splenic lymphocytes was quantified by Annexin-V expression. After LAP, infusion of both FO and SO did not change cytokine concentrations. Sepsis increased both cytokines. FO but not SO further augmented the rise. After LAP, SO increased NEFA, and both lipid emulsions reduced free arachidonic acid (AA). Sepsis resulted in a dramatic decrease in NEFA and AA. The drop in NEFA and AA was prevented by both SO and FO. In addition, FO resulted in an increased concentration of n-3 FA under both conditions. Infusion of both lipid emulsions induced apoptosis in splenic lymphocytes after LAP. Sepsis-induced apoptosis was not further enhanced by FO or SO. The present study shows that short-term administration of FO as opposed to SO caused pro-inflammatory effects during sepsis. Moreover, short-term administration of both SO and FO suffices to induce apoptosis in splenic lymphocytes. Finally, SO and FO do not further enhance sepsis-induced splenic apoptosis.