2-Arachidonyl-lysophosphatidylethanolamine Induces Anti-Inflammatory Effects on Macrophages and in Carrageenan-Induced Paw Edema

2-Arachidonyl-lysophosphatidylethanolamine, shortly 2-ARA-LPE, is a polyunsaturated lysophosphatidylethanolamine. 2-ARA-LPE has a very long chain arachidonic acid, formed by an ester bond at the sn-2 position. It has been reported that 2-ARA-LPE has anti-inflammatory effects in a zymosan-induced peritonitis model. However, it’s action mechanisms are poorly investigated. Recently, resolution of inflammation is considered to be an active process driven by M2 polarized macrophages. Therefore, we have investigated whether 2-ARA-LPE acts on macrophages for anti-inflammation, whether 2-ARA-LPE modulates macrophage phenotypes to reduce inflammation, and whether 2-ARA-LPE is anti-inflammatory in a carrageenan-induced paw edema model. In mouse peritoneal macrophages, 2-ARA-LPE was found to inhibit lipopolysaccharide (LPS)-induced M1 macrophage polarization, but not induce M2 polarization. 2-ARA-LPE inhibited the inductions of inducible nitric oxide synthase and cyclooxygenase-2 in mouse peritoneal macrophages at the mRNA and protein levels. Furthermore, products of the two genes, nitric oxide and prostaglandin E2, were also inhibited by 2-ARA-LPE. However, 1-oleoyl-LPE did not show any activity on the macrophage polarization and inflammatory responses. The anti-inflammatory activity of 2-ARA-LPE was also verified in vivo in a carrageenan-induced paw edema model. 2-ARA-LPE inhibits LPS-induced M1 polarization, which contributes to anti-inflammation and suppresses the carrageenan-induced paw edema in vivo.


Introduction
Lysophosphatidic acid is a representative lyso-type intercellular mediator that acts through G protein-coupled receptors (LPA 1-6 ) [1]. Lysophosphatidylethanolamine (LPE) is another lyso-type phospholipid and has been detected in human serum at concentrations of several hundreds of ng/ml [2,3]. However, its action has not been much studied.
On the other hand, analysis of plasma LPE species using a liquid chromatographytandem mass spectrometry found 7-17 µM of total LPE in patients undergoing coronary angiography [8]. Specifically, plasma levels of polyunsaturated long chain LPEs including 22:6 LPE, 20:4 LPE, and 18:2 LPE were increased about three-fold in acute coronary syndrome subjects compared to levels in patients with normal coronary arteries [8]. Furthermore, 2-polyunsaturated acyl-LPEs, such as 2-arachidonyl LPE (2-ARA-LPE) and 2-docosahexaenoyl LPE, were found to have anti-inflammatory action in a zymosan Ainduced peritonitis model [9]. Reduction of vascular leakage was found in a 2-ARA-LPE-treated group, however, its action mechanisms are not well investigated [9]. Recently, resolution of inflammation is considered to be an active process driven by M2 polarized macrophages. Therefore, we have investigated whether 2-ARA-LPE acts on macrophages for anti-inflammation, whether 2-ARA-LPE modulates macrophage phenotypes to reduce inflammation in comparison with 1-OLE-LPE, and whether 2-ARA-LPE is anti-inflammatory in a carrageenan-induced paw edema model.

Effects of 2-ARA-LPE on COX-2 and iNOS Expression
We also examined the inhibitory effects of 2-ARA-LPE at the protein level. As shown in Figure 3, the expressions of iNOS and COX-2 protein were obviously induced by LPS. 2-ARA-LPE strongly and concentration-dependently inhibited iNOS protein induction and COX-2 protein induction (Figure 3). At concentrations 10 and 50 µM, 2-ARA-LPE had significant inhibitory effects on iNOS protein induction. COX-2 protein expression was also significantly inhibited by 2-ARA-LPE at 50 µM concentration ( Figure 3). Expressions of both proteins, however, were not affected by 1-OLE-LPE ( Figure 4). 2-ARA-LPE-induced suppression of inflammasome-related caspase-1 and 11 expression at mRNA level was not confirmed at protein level (Figures 1 and 3), although there was a suppressive tendency on caspase-1 ( Figure 3). RT-PCR for pro-inflammatory genes and anti-inflammatory genes was performed. The data are representative of three independent experiments. Relative mRNA levels of each gene versus glyceraldehyde 3-phosphate dehydrogenase(GAPDH) are shown as histograms. ** p < 0.01, *** p < 0.001 vs. the none-treated group. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. the LPS-treated group. RT-PCR for pro-inflammatory genes and anti-inflammatory genes was performed. The data are representative of three independent experiments. Relative mRNA levels of each gene versus glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are shown as histograms. ** p < 0.01, *** p < 0.001 vs. the none-treated group. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. the LPS-treated group.
Next, the inhibitory effects of 2-ARA-LPE on the expressions of iNOS and COX-2 were confirmed by measuring the products of iNOS and COX-2, that is, nitric oxide (NO) and prostaglandin E 2 (PGE 2 ), respectively. As shown in Figure 5, 2-ARA-LPE significantly and concentration-dependently inhibited LPS-induced NO production ( Figure 5A), and as shown in Figure 5B

Effects of 2-ARA-LPE on COX-2 and iNOS Expression
We also examined the inhibitory effects of 2-ARA-LPE at the protein level. As shown in Figure 3, the expressions of iNOS and COX-2 protein were obviously induced by LPS. 2-ARA-LPE strongly and concentration-dependently inhibited iNOS protein induction and COX-2 protein induction ( Figure 3). At concentrations 10 and 50 M, 2-ARA-LPE had significant inhibitory effects on iNOS protein induction. COX-2 protein expression was also significantly inhibited by 2-ARA-LPE at 50 M concentration ( Figure 3). Expressions of both proteins, however, were not affected by 1-OLE-LPE ( Figure 4). 2-ARA-LPE-induced suppression of inflammasome-related caspase-1 and 11 expression at mRNA level was not confirmed at protein level ( Figure 1 and Figure 3), although there was a suppressive tendency on caspase-1 ( Figure 3).

Effect of 2-ARA-LPE on Carrageenan-Induced Paw Edema
In a previous study, 2-ARA-LPE was found to reduce vascular leakage in a zymosan A-induced peritonitis model [9]. In order to verify and expand the anti-inflammatory effect of 2-ARA-LPE, carrageenan-induced acute inflammatory paw edema model was employed [11]. Injection of carrageenan into the hind paw of mice resulted in edema which was assessed by paw thickness (Figure 6). After injection of carrageenan, the paw size increased 48% in one hour. Maximal effect of carrageenan was produced at three hours after injection, with 60% thickening of the paw. The swelling sustained for six hours.  Histological analysis of tissue sections revealed reduction of cellular infiltration of immune cells in the 2-ARA-LPE-treated group. Microscopic photographs of the control stained with hematoxylin and eosin showed normal paw tissue with no signs of inflammation ( Figure 7). In the carrageenan-treated group, high infiltration damage was found due to accumulation of immune cells and collection of fluid, as the arrow indicated in Figure 7B shows. However, 2-ARA-LPE ( Figure 7C), and dexamethasone ( Figure 7D)treated groups showed only moderate infiltration damage. The 2-ARA-LPE-treated group showed anti-inflammatory effects similar to that of the positive control dexamethasone. Next, the inhibitory effects of 2-ARA-LPE on the expressions of iNOS and COX-2 were confirmed by measuring the products of iNOS and COX-2, that is, nitric oxide (NO) and prostaglandin E2 (PGE2), respectively. As shown in Figure 5, 2-ARA-LPE significantly and concentration-dependently inhibited LPS-induced NO production ( Figure 5A), and as shown in Figure 5B   Next, the inhibitory effects of 2-ARA-LPE on the expressions of iNOS and COX-2 were confirmed by measuring the products of iNOS and COX-2, that is, nitric oxide (NO) and prostaglandin E2 (PGE2), respectively. As shown in Figure 5, 2-ARA-LPE significantly and concentration-dependently inhibited LPS-induced NO production ( Figure 5A), and as shown in Figure 5B   Histological analysis of tissue sections revealed reduction of cellular infiltration of immune cells in the 2-ARA-LPE-treated group. Microscopic photographs of the control stained with hematoxylin and eosin showed normal paw tissue with no signs of inflammation (Figure 7). In the carrageenan-treated group, high infiltration damage was found due to accumulation of immune cells and collection of fluid, as the arrow indicated in Figure 7B shows. However, 2-ARA-LPE ( Figure 7C), and dexamethasone ( Figure 7D)-

Discussion
Recent analysis of plasma lysophospholipids species using a liquid chromatographytandem mass spectrometry found 7-17 M of total LPE in patients undergoing coronary angiography [8]. Specifically, plasma levels of polyunsaturated very long chain LPEs including 22:6 LPE, 20:4 LPE, and 18:2 LPE were increased about three-fold in acute coronary syndrome subjects compared to levels in patients with normal coronary arteries [8]. However, the meaning of their levels has not been studied. In a previous study, the antiinflammatory effect of 2-ARA-LPE was reported in a zymosan-induced peritonitis model [9]. In this present study, we found the inhibitory effect of 2-ARA-LPE on LPS-induced M1 polarization of macrophages in vitro as a mechanism for the anti-inflammation, although 2-ARA-LPE did not affect M2 polarization. Furthermore, we found an in vivo inhibitory effect of 2-ARA-LPE in a carrageenan-induced edema model.
One interesting finding is that 1-OLE-LPE did not induce suppressive effect on M1 polarization. We previously have studied 1-OLE-LPE as the most potent Ca 2+ -mobilizing agonist on several cell lines [4][5][6][7]. In mouse peritoneal macrophages, however, 2-ARA-LPE is anti-inflammatory but 1-OLE-LPE is not. This implies that the target of 2-ARA-LPE is different from the receptors for 1-OLE-LPE, and that there may be many different receptors for different LPE species. Further studies are necessary to elucidate the action mechanism of LPEs.

Discussion
Recent analysis of plasma lysophospholipids species using a liquid chromatographytandem mass spectrometry found 7-17 µM of total LPE in patients undergoing coronary angiography [8]. Specifically, plasma levels of polyunsaturated very long chain LPEs including 22:6 LPE, 20:4 LPE, and 18:2 LPE were increased about three-fold in acute coronary syndrome subjects compared to levels in patients with normal coronary arteries [8]. However, the meaning of their levels has not been studied. In a previous study, the anti-inflammatory effect of 2-ARA-LPE was reported in a zymosan-induced peritonitis model [9]. In this present study, we found the inhibitory effect of 2-ARA-LPE on LPSinduced M1 polarization of macrophages in vitro as a mechanism for the anti-inflammation, although 2-ARA-LPE did not affect M2 polarization. Furthermore, we found an in vivo inhibitory effect of 2-ARA-LPE in a carrageenan-induced edema model.
One interesting finding is that 1-OLE-LPE did not induce suppressive effect on M1 polarization. We previously have studied 1-OLE-LPE as the most potent Ca 2+ -mobilizing agonist on several cell lines [4][5][6][7]. In mouse peritoneal macrophages, however, 2-ARA-LPE is anti-inflammatory but 1-OLE-LPE is not. This implies that the target of 2-ARA-LPE is different from the receptors for 1-OLE-LPE, and that there may be many different receptors for different LPE species. Further studies are necessary to elucidate the action mechanism of LPEs.

Animals
8-10 week old male C57BL/6 (19-22g) mice and 6 week old male ICR (28-31g) mice were purchased from Daehan Biolink (DBL; Seoul, Korea), housed in a laboratory animal facility at Pusan National University, and provided with food and water ad lib. The animal protocol used in this study was reviewed and approved beforehand by the Pusan National University-Institutional Animal Care Committee (PNU-IACUC) with respect to ethical and scientific care.

Isolation and Culture of Mouse Peritoneal Macrophages
Mouse peritoneal macrophages were isolated from the peritoneal cavity of a 3% thioglycollate-treated C57BL/6 mouse 4 days after treatment and cultured at 37 • C in a 5% CO 2 humidified incubator. Isolated macrophages were maintained in RPMI1640 containing 10% (v/v) heat-inactivated fetal bovine serum, 100 units/mL penicillin, 50 µg/mL streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate for 18 h and then incubated in 0.5% FBScontaining media for 24 h. RNA and protein samples were prepared after 5 h or 24 h of LPS treatment (10 ng/mL or 1 µg/mL), respectively. LPEs were added 1 h before adding LPS [10].

Reverse Transcriptase-PCR
To determine the expressions of marker proteins of M1 or M2 polarization in macrophages by RT-PCR, first strand cDNA was synthesized with total RNA isolated using Trizol reagent (Invitrogen, USA). Synthesized cDNA products and primers for each gene were used for PCR,

Nitrites Measurement
NO production was estimated by measuring the amount of nitrite (a stable metabolite of NO) in medium using Griess reagent, as previously described [14,15]. Cells were pretreated with different concentrations of 2-ARA-LPE for 1 h and subsequently stimulated with LPS (1 µg/ml) for 24 h. Nitrite concentrations in medium were determined using the Griess Reagent System (Promega, Madison, WI, USA).

Carrageenan-Induced Paw Edema Assay
The carrageenan-induced hind paw edema model in mice was used to assess antiinflammatory activity [11]. ICR mice were divided into the 3 groups (n = 5/group). 2-ARA-LPE (1 mg/kg) or dexamethasone (1 mg/kg) dissolved in 1% DMSO in PBS was administered by intraperitoneal injection, and the solvent alone served as a vehicle control. Thirty minutes after the administration of drugs, paw edema was induced by sub-plantar injection of 50 µl of 1% freshly prepared carrageenan suspension in PBS into the left hind paw of each mouse. The right hind paw was injected with 50 µl of PBS. To gauge the extent of inflammation, paw thickness was measured before (0 h) and at intervals of 1, 2, 3, 4, and 6 h after carrageenan injection using a digital vernier caliper (Stainless Steel Digital Caliper, Find it at the Bay, Gaithersburg, MD, USA) [17].

Histology
After 6 h of carrageenan-induced edema, five animals from each group were euthanized. Paw samples were taken for histological examination. Sectioned tissues were stained with hematoxylin and eosin and viewed under a light microscope (Zeiss, Jena, Germany).

Statistical Analysis
Results are expressed as the means ± SDs of the indicated numbers of determinations. The statistical significances of differences were determined by analysis of variance (ANOVA) with turkey's post hoc, and statistical significance was accepted for p values < 0.05. Analyses were performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA).  Informed Consent Statement: Not applicable for not involving humans.

Data Availability Statement:
The study does not report any data.

Conflicts of Interest:
The authors declare no conflict of interest.