Effect Of Lysophosphatidylcholine Research Articles

Dietary Lysophosphatidylcholine Improves the Uptake of Astaxanthin and Modulates Cholesterol Transport in Pacific White Shrimp Litopenaeus vannamei.

Astaxanthin (AST), functioning as an efficient antioxidant and pigment, is one of the most expensive additives in shrimp feeds. How to improve the uptake efficiency of dietary astaxanthin into farmed shrimp is of significance. The present study investigated the effects of lysophosphatidylcholine (LPC), an emulsifier, on dietary astaxanthin efficiency, growth performance, body color, body composition, as well as lipid metabolism of juvenile Pacific white shrimp (average initial body weight: 2.4 g). Three diets were prepared: control group, the AST group (supplemented with 0.02% AST), and the AST + LPC group (supplemented with 0.02% AST and 0.1% LPC). Each diet was fed to triplicate tanks, and each tank was stocked with 30 shrimp. The shrimp were fed four times daily for eight weeks. The AST supplementation improved the growth of white shrimp, while LPC further promoted the final weight of shrimp, but the whole-shrimp proximate composition and fatty acid composition were only slightly affected by AST and LPC. The LPC supplementation significantly increased the astaxanthin deposition in the muscle. The LPC supplementation significantly increased the shell yellowness of both raw and cooked shrimp compared to the AST group. Moreover, the dietary LPC increased the high-density lipoprotein-cholesterol content but decreased the low-density lipoprotein-cholesterol content in the serum, indicating the possible regulation of lipid and cholesterol transport. The addition of astaxanthin significantly up-regulated the expression of npc2 in the hepatopancreas compared to the control group, while the addition of LPC down-regulated the expression of mttp compared to the AST group. In conclusion, the LPC supplementation could facilitate the deposition of dietary astaxanthin into farmed shrimp and further enlarge the beneficial effects of dietary astaxanthin. LPC may also independently regulate shrimp body color and cholesterol transportation. This was the first investigation of the promoting effects of LPC on dietary astaxanthin efficiency.

A41 IDENTIFICATION OF MICROBIAL LYSOPHOSPHATIDYLCHOLINE (LPC) AND LYSOPHOSPHATIDIC ACID (LPA) PRODUCERS AND THEIR EFFECTS ON THE HOST

Abstract Background Gut microbiota plays a key role in shaping our immune system, producing a myriad of molecules with neuro- and immunomodulatory properties. Many of these bioactive molecules are generated by bacterial metabolism of common dietary components. We have recently found that fecal samples of patients with chronic abdominal pain contain high levels of inflammatory phospholipids, lysophosphatidylcholine (LPC) and its active metabolite lysophosphatidic acid (LPA). Both phospholipids have been previously linked to genesis and maintenance of chronic neuropathic pain. In addition, they appear to have pro-inflammatory properties. Aims To investigate whether gut bacteria can produce LPC and LPA from dietary precursor phosphatidylcholine (PC), and whether these phospholipids can induce inflammatory responses in the host. Methods Fecal samples from patients with chronic abdominal pain were analyzed for microbial profiles using 16S rRNA (Illumina) analysis, and abundance of individual bacteria was correlated with fecal LPC and LPA levels as measured by ELISA. To confirm ability to produce these lipids, identified bacteria were incubated with either phosphatidylcholine (PC) or LPC (the immediate precursors of LPC and LPA, respectively) and LPC and LPA levels measured using liquid chromatography-mass spectrometry. To investigate pro-inflammatory effect of LPC and LPA, mouse splenocytes were incubated with LPC and/or LPA in vitro, and tested for cytokine production. Results Higher abundance of several bacterial species was associated with higher fecal levels of LPC and LPA. Among them, a bacterium from family Oscillospiraceae was identified as the most likely producer of both LPA and LPC; its abundance also correlated with high pain scores. Culturing and incubating this bacterium with PC and LPC resulted in a significant production of LPC and LPA. Additionally, LPA, but not LPC, resulted in increased levels of IL-6 production by splenocytes in vitro compared to the PBS only control. Interestingly, LPA induced IL-6 secretion was inhibited in the presence of LPC. Conclusions We have identified several bacterial producers of LPC and LPA, among them a bacterium from family Oscillospiraceae, which was also associated with high abdominal pain scores. LPA, but not LPC, seems to induce proinflammatory responses in the host. Further experiments are needed to identify the specific mechanisms by which these bioactive phospholipids induce abdominal pain and immune responses in the host. Funding Agencies CIHRFarncombe Family Digestive Health Research Institute

Autotaxin-lysophosphatidic acid receptor 5 axis evokes endothelial dysfunction via reactive oxygen species signaling.

Lysophosphatidylcholine (LPC) is a bioactive lipid that has been shown to attenuate endothelium-dependent vasorelaxation contributing to endothelial dysfunction; however, the underlying mechanisms are not well understood. In this study, we investigated the molecular mechanisms involved in the development of LPC-evoked impairment of endothelium-dependent vasorelaxation. In aortic rings isolated from wild-type (WT) mice, a 20-min exposure to LPC significantly reduced the acetylcholine chloride (ACh)-induced vasorelaxation indicating the impairment of normal endothelial function. Interestingly, pharmacological inhibition of autotaxin (ATX) by GLPG1690 partially reversed the endothelial dysfunction, suggesting that lysophosphatidic acid (LPA) derived from LPC may be involved in the effect. Therefore, the effect of LPC was also tested in aortic rings isolated from different LPA receptor knock-out (KO) mice. LPC evoked a marked reduction in ACh-dependent vasorelaxation in Lpar1, Lpar2, and Lpar4 KO, but its effect was significantly attenuated in Lpar5 KO vessels. Furthermore, addition of superoxide dismutase reduced the LPC-induced endothelial dysfunction in WT but not in the Lpar5 KO mice. In addition, LPC increased H2O2 release from WT vessels, which was significantly reduced in Lpar5 KO vessels. Our findings indicate that the ATX-LPA-LPA5 receptor axis is involved in the development of LPC-induced impairment of endothelium-dependent vasorelaxation via LPA5 receptor-mediated reactive oxygen species production. Taken together, in this study, we identified a new pathway contributing to the development of LPC-induced endothelial dysfunction.

Abstract 101: Distinctive Individual Effects of LPC(18:2) and LPC(22:6) on Survival and Neurological Outcomes After Cardiac Arrest: Combination As A Future Therapy

Introduction: Decreased plasma Lysophosphatidylcholine (LPC) was found to be associated with survival after cardiac arrest (CA) in humans and rats. Supplementing LPC species containing docosahexaenoic acid, LPC(22:6), improved rat brain function and survival after CA. Thus, LPC deficiency may be a major cause of CA pathology and LPC supplementation may be a novel therapeutic approach to CA. However, the therapeutic effects of individual LPC species are not fully understood, particularly a species containing linoleic acid, LPC(18:2). While 22:6 is an omega-3 fatty acid and highly abundant in the brain, 18:2 is omega-6 and minimally present in the brain. Therefore, we hypothesized that the protective role of LPC(18:2) may be different from LPC(22:6). Furthermore, LPC(18:2) may provide additive protection when administered together with LPC(22:6). Objective: To investigate the protective role of supplementing LPC(18:2) compared to LPC(22:6) after CA. Methods and Results: We first compared the relationship between plasma LPC(18:2) levels and outcomes of CA patients as compared to LPC(22:6). LPC(22:6) was found to be associated with both survival and neurological outcomes, whereas the levels of LPC(18:2) were associated with survival only. We then tested whether supplementing LPC(18:2) can improve survival and neurological function in rats undergoing 10 minutes of asphyxial CA. Supplementing LPC(18:2) post-resuscitation significantly improved survival as did LPC(22:6) in rats. Although LPC(18:2) supplementation improved in some individual neurofunctional tasks, the overall neurological function of survived rats was not improved. Particularly, while 3 of 7 survived rats treated with LPC(22:6) were able to walk across a ledge, none of 10 survived rats treated with LPC(18:2) were able to cross the ledge. Conclusion: Significantly improved survival without significantly improved brain function in rats and poor correlation with neurological outcomes from humans suggest that LPC(18:2) may protect organs other than the brain. The results further indicate that different LPC species have different mechanisms of improving outcomes, demonstrating the potential of utilizing different combinations of LPC as improved therapeutic treatments in CA.

Effects of Lysophosphatidylcholine on Intestinal Health of Turbot Fed High-Lipid Diets

An 8-week feeding trial was conducted, where turbot were fed four experimental diets, containing different LPC levels (0%, 0.1%, 0.25%, and 0.5%, named LPC0, LPC0.1, LPC0.25, and LPC0.5, respectively). The intestinal morphology results showed that there were no widened lamina propria and mixed inflammatory cells in the LPC-supplemented groups. Dietary LPC remarkably decreased the expression of TLRs (TLR3, TLR8, TLR9, and TLR22), MyD88, and signaling molecules (NF-κB, JNK, and AP-1). Similarly, diets with LPC supplementation markedly depressed the gene expression of NF-κB and JNK signaling pathway downstream genes (TNF-α, IL-1β, Bax, Caspase9, and Caspase-3). Furthermore, dietary LPC modified the intestinal microbial profiles, increasing the relative abundance of short-chain fatty acids-producers, lactic acid bacteria, and digestive enzyme-producing bacteria. Predictive functions of intestinal microbiota showed that turbot fed LPC diets had a relatively higher abundance of functions, such as lipid metabolism and immune system, but a lower abundance of functions, such as metabolic diseases and immune system diseases. The activities of intestinal acid phosphatase and alkaline phosphatase were also increased by dietary LPC. In conclusion, LPC supplementation could regulate the intestinal mucosal barrier via the TLR signaling pathway and alter the intestinal microbiota profile of turbot fed high-lipid diets.

Lysophosphatidylcholine Alleviates Acute Lung Injury by Regulating Neutrophil Motility and Neutrophil Extracellular Trap Formation.

Sepsis is predominantly initiated by bacterial infection and can cause systemic inflammation, which frequently leads to rapid death of the patient. However, this acute systemic inflammatory response requires further investigation from the perspectives of clinical judgment criteria and early treatment strategies for the relief of symptoms. Lysophosphatidylcholine (LPC) 18:0 may relieve septic symptoms, but the relevant mechanism is not clearly understood. Therefore, we aimed to assess the effectiveness of LPC as a therapeutic treatment for acute inflammation in the lung induced by lipopolysaccharide in mice. Systemic inflammation of mice was induced by lipopolysaccharide (LPS) inoculation to investigate the role of LPC in the migration and the immune response of neutrophils during acute lung injury. By employing two-photon intravital imaging of the LPS-stimulated LysM-GFP mice and other in vitro and in vivo assays, we examined whether LPC alleviates the inflammatory effect of sepsis. We also tested the effect of LPC to human neutrophils from healthy control and sepsis patients. Our data showed that LPC treatment reduced the infiltration of innate immune cells into the lung. Specifically, LPC altered neutrophil migratory patterns and enhanced phagocytic efficacy in the damaged lung. Moreover, LPC treatment reduced the release of neutrophil extracellular trap (NET), which can damage tissue in the inflamed organ and exacerbate disease. It also reduced human neutrophil migration under inflammatory environment. Our results suggest that LPC can alleviate sepsis-induced lung inflammation by regulating the function of neutrophils. These findings provide evidence for the beneficial application of LPC treatment as a potential therapeutic strategy for sepsis.

Transcriptomic atlas of GNAT family members in pulmonary epithelia under pathological conditions using single‐cell and bulk cell sequencing

Transcriptomic atlas of GNAT family members in pulmonary epithelia under pathological conditions using single‐cell and bulk cell sequencing

Effects of Lysophosphatidylcholine on Jejuna Morphology and Its Potential Mechanism.

Lysophosphatidylcholine (LPC) plays a vital role in promoting jejuna morphology in broilers. However, the potential mechanism behind LPC improving the chicken jejuna morphology is unclear. Therefore, the present study was designed to reveal the important genes associated with LPC regulation in birds' jejuna. Thus, GSE94622, the gene expression microarray, was obtained from Gene Expression Omnibus (GEO). GSE94622 consists of 15 broiler jejuna samples from two LPC-treated (LPC500 and LPC1000) and the control groups. Totally 98 to 217 DEGs were identified by comparing LPC500 vs. control, LPC1000 vs. control, and LPC1000 vs. LPC500. Gene ontology (GO) analysis suggested that those DEGs were mainly involved in the one-carbon metabolic process, carbon dioxide transport, endodermal cell differentiation, the positive regulation of dipeptide transmembrane transport, cellular pH reduction, and synaptic transmission. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the DEGs were enriched in NOD-like receptor (NLR), RIG-I-like receptor (RILR), Toll-like receptor (TLR), and necroptosis signaling pathway. Moreover, many genes, such as RSAD2, OASL, EPSTI1, CMPK2, IFIH1, IFIT5, USP18, MX1, and STAT1 might be involved in promoting the jejuna morphology of broilers. In conclusion, this study enhances our understanding of LPC regulation in jejuna morphology.

Single Subcutaneous Injection of Lysophosphatidyl-Choline Evokes ASIC3-Dependent Increases of Spinal Dorsal Horn Neuron Activity.

Lysophosphatidyl-choline (LPC), a member of the phospholipid family, is an emerging player in pain. It is known to modulate different pain-related ion channels, including Acid-Sensing Ion Channel 3 (ASIC3), a cationic channel mainly expressed in peripheral sensory neurons. LPC potentiates ASIC3 current evoked by mild acidifications, but can also activate the channel at physiological pH. Very recently, LPC has been associated to chronic pain in patients suffering from fibromyalgia or osteoarthritis. Accordingly, repetitive injections of LPC within mouse muscle or joint generate both persistent pain-like and anxiety-like behaviors in an ASIC3-dependent manner. LPC has also been reported to generate acute pain behaviors when injected intraplantarly in rodents. Here, we explore the mechanism of action of a single cutaneous injection of LPC by studying its effects on spinal dorsal horn neurons. We combine pharmacological, molecular and functional approaches including in vitro patch clamp recordings and in vivo recordings of spinal neuronal activity. We show that a single cutaneous injection of LPC exclusively affects the nociceptive pathway, inducing an ASIC3-dependent sensitization of nociceptive fibers that leads to hyperexcitabilities of both high threshold (HT) and wide dynamic range (WDR) spinal neurons. ASIC3 is involved in LPC-induced increase of WDR neuron’s windup as well as in WDR and HT neuron’s mechanical hypersensitivity, and it participates, together with TRPV1, to HT neuron’s thermal hypersensitivity. The nociceptive input induced by a single LPC cutaneous rather induces short-term sensitization, contrary to previously described injections in muscle and joint. If the effects of peripheral LPC on nociceptive pathways appear to mainly depend on peripheral ASIC3 channels, their consequences on pain may also depend on the tissue injected. Our findings contribute to a better understanding of the nociceptive signaling pathway activated by peripheral LPC via ASIC3 channels, which is an important step regarding the ASIC3-dependent roles of this phospholipid in acute and chronic pain conditions.

GPR55‐Dependent Excitation of Dorsal Root Ganglion Neurons by Lysophosphatidylcholine

Irritable Bowel Syndrome is a functional gastrointestinal (GI) disorder that leads to chronic abdominal pain. The perception of this visceral pain involves peripheral mechanisms, such as nociceptors with cell bodies in dorsal root ganglia (DRG) and axons in the gut wall, and central mechanisms, such as neuroplasticity within the spinal cord and brain. Lysophosphatidylcholine (LPC), which has recently been shown to bind to GPR55 in human prostate carcinoma cells, is released from cell membranes and is elevated in patients with IBS. Dorsal root ganglion (DRG) neurons express GPR55, and activation of GPR55 has been implicated in inflammatory pain. Therefore, we hypothesised that elevated LPC during IBS contributes to pain due to its activation of GPR55 on dorsal root ganglion (DRG) neurons, leading to the excitation of visceral pain pathways. Current clamp recordings revealed that application of LPC (10 µM) to murine DRG neurons depolarised the resting membrane potential (p = 0.0001) by approximately 8 mV and decreased the rheobase (p < 0.05) by approximately 20%. Using ratiometric Ca2+ imaging using FURA‐2 AM, LPC (10 µM) doubled intracellular [Ca2+]i. This effect was significantly reduced by the selective GPR55 antagonist CID16020046 (10 µM) (p < 0.05), suggesting the response to LPC is at least partially mediated by GPR55. The source of the [Ca2+] elevation following LPC application was elucidated using cyclopiazonic acid (CPA; 10 µM), which depletes intracellular Ca2+ stores, and a 0‐Ca2+ external solution to remove the contribution of Ca2+ influx from extracellular sources. While both significantly decreased the Ca2+ influx elicited by LPC, the 0‐Ca2+ external solution almost abolished the effect (p < 0.0001) of LPC. Together, these data suggest that the increased [Ca2+] elicited by LPC activation of GPR55 is partially mediated through the release of Ca2+ from intracellular stores but is mostly due to influx of extracellular Ca2+.

Dietary lysophosphatidylcholine regulates diacylglycerol, cardiolipin and free fatty acid contents in the fillet of turbot.

Lysophosphatidylcholine (LPC) has been widely used as emulsifier in animal feeds to enhance the lipid utilization. However, the effects of LPC on fillet quality has rarely been known. The present study was the first time to investigate the response of fish muscle lipidomics to dietary LPC supplementation. Turbot muscle samples were collected after a 56-day feeding trial where the experimental diet contained 0 or 0.25% LPC. Targeted tandem mass spectrometry was used in the lipidomic analysis. A total of 62 individual lipids (58 up-regulated and 7 down-regulated by LPC) showed significant difference in concentration in response to dietary LPC. Most of these differentially abundant lipids were diacylglycerol, free fatty acid and cardiolipin, and they all were up-regulated by dietary LPC. However, LPC exerted only marginal effects on muscle fatty acid composition and lipid content. The effects of dietary LPC on fillet lipid composition cannot be neglected in fish product evaluation.

Astragaloside IV Inhibits Bleomycin-Induced Ferroptosis in Human Umbilical Vein Endothelial Cells by Mediating LPC.

Ferroptosis, as an iron-dependent programmed cell death pathway, can induce a variety of cardiovascular diseases. Astragaloside IV (AS-IV), which is purified from Astragalus membranaceus, can protect endothelial function and promote vascular regeneration. However, the role played by AS-IV in ferroptosis remains unknown. In this study, the lipid metabolomics in HUVECs treated with/without bleomycin and/or AS-IV were explored using LC/MS. The most differential metabolite between groups was further identified via GO and pathway enrichment analyses. The effects of lysophosphatidylcholine (LPC), AS-IV, and FIN56 on cell viability were explored using the CCK-8 assay, their effects on cell senescence were examined by β-galactosidase staining, and their effects on ferroptosis were detected by a flow cytometric analysis of lipid ROS levels, transmission electron microscopy, and an assay for cellular iron levels. The related mechanisms were investigated by real-time PCR and Western blot assays. Our results showed that LPC, as the most differential metabolite, inhibited cell viability but promoted cell apoptosis and senescence as its concentration increased. Also, the decreased cell activity, increased iron ion and lipid ROS levels, and the enhanced cell senescence induced by LPC treatment were all significantly reversed by AS-IV but further enhanced by FIN56 treatment. The changes in mitochondrial morphology caused by the LPC treatment were significantly alleviated by the AS-IV treatment, while treatment with FIN56 reversed those phenomena. Moreover, AS-IV partially upregulated the levels of SLC7A11 and GPX4 expression which were reduced by LPC. However, those changes were prevented by FIN56 treatment. In conclusion, our data suggested that AS-IV could serve as a novel drug for treating ferroptosis-related diseases.

Lysophosphatidylcholine Offsets the Protective Effects of Bone Marrow Mesenchymal Stem Cells on Inflammatory Response and Oxidative Stress Injury of Retinal Endothelial Cells via TLR4/NF-κB Signaling.

Diabetic retinopathy (DR), as a major cause of blindness worldwide, is one common complication of diabetes mellitus. Inflammatory response and oxidative stress injury of endothelial cells play significant roles in the pathogenesis of DR. The study is aimed at investigating the effects of lysophosphatidylcholine (LPC) on the dysfunction of high glucose- (HG-) treated human retinal microvascular endothelial cells (HRMECs) after being cocultured with bone marrow mesenchymal stem cells (BMSCs) and the underlying regulatory mechanism. Coculture of BMSCs and HRMECs was performed in transwell chambers. The activities of antioxidant-related enzymes and molecules of oxidative stress injury and the contents of inflammatory cytokines were measured by ELISA. Flow cytometry analyzed the apoptosis of treated HRMECs. HRMECs were further treated with 10-50 μg/ml LPC to investigate the effect of LPC on the dysfunction of HRMECs. Western blotting was conducted to evaluate levels of TLR4 and p-NF-κB proteins. We found that BMSCs alleviated HG-induced inflammatory response and oxidative stress injury of HRMECs. Importantly, LPC offsets the protective effects of BMSCs on inflammatory response and oxidative stress injury of HRMECs. Furthermore, LPC upregulated the protein levels of TLR4 and p-NF-κB, activating the TLR4/NF-κB signaling pathway. Overall, our study demonstrated that LPC offsets the protective effects of BMSCs on inflammatory response and oxidative stress injury of HRMECs via TLR4/NF-κB signaling.

Lysophosphatidylcholine induces apoptosis and inflammatory damage in brain microvascular endothelial cells via GPR4-mediated NLRP3 inflammasome activation

Lysophosphatidylcholine (LPC), as the main active component of oxidized low-density lipoproteins (ox-LDLs), has significant effects in cerebrovascular disease. However, the complex mechanism by which LPC functions in brain microvascular endothelial cells (BMECs) is not clearly understood. In this study, BMECs were transfected with G protein-coupled receptor 4 (GPR4) siRNA or an NLRP3-overexpression plasmid, and GPR4 expression was identified by RT-qPCR and western blotting; IL-1β, IL-18, and IL-33 levels were evaluated by ELISA. Apoptosis was monitored by flow cytometry and Hoechst staining, while Caspase 3, ASC, NLRP3, and GPR4 protein expression were examined by western blotting. Our results showed that LPC significantly increased the levels of inflammatory cytokines (IL-1β, IL-18, and IL-33) and markedly induced apoptosis and NLRP3 inflammasome activation in BMECs. Moreover, LPC notably upregulated GPR4 in BMECs, and knockdown of GPR4 significantly attenuated the effects of LPC in BMECs. Above all, we also proved that LPC induced apoptosis and inflammatory injury in BMECs by causing GPR4 to activate NLRP3 inflammasomes. Therefore, GPR4-mediated activation of NLRP3 inflammasomes might be the underlying mechanism by which LPC promotes the progression of cerebrovascular disease. In summary we found that LPC is an important pathogenic factor in cerebrovascular disease, and can induce GPR4 to active NLRP3 inflammasomes.

The Effect of Bioactive Phospholipids on Normal and Malignant Hematopoiesis—Modulation of Their Effects By Downregulating the Activities of Heme Oxygenase 1 (HO-1) and Inducible Nitric Oxygenase Synthase (iNOS)

Background. Bioactive phospholipids, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), lysophosphatidylcholine (LPC), and its derivative lysophosphatidic acid (LPA), haveemerged as important mediators regulating the trafficking of several types of normal and malignant cells. As we have demonstrated, these factors are upregulated in tissues, including in the bone marrow microenvironment after radio/chemotherapy [Leukemia 2010; 24: 976-985] . While the role of S1P in regulating the trafficking of normal and malignant hematopoietic cells is well established, in this work we focused on the biological effects of C1P, LPC, and LPA. Hypothesis. We hypothesized that not only S1P but also other bioactive phospholipids are involved in regulating hematopoietic cell trafficking and expansion within hematopoietic tissues. Moreover, based on our results showing negative effects of heme oxygenase 1 (HO-1) [ Stem Cell Rev . 2015, 11, 110-118 ] and inducible nitric oxygenase synthase (iNOS) [ Stem Cell Rev . 2017, 13, 92-103 ] on the trafficking of normal hematopoietic cells, we hypothesized that this process could be inhibited by upregulating HO-1 and iNOS in malignant hematopoietic cells.Materials and Methods. We employed eight human myeloid and lymphoid cell lines as well as cells from patients diagnosed with acute myeloid leukemia. We phenotyped these cells for expression of bioactive lipid receptors and tested the functionality of these receptors by assaying for activation of p42/44 MAPK and AKT after stimulation with S1P, C1P, LPC, and LPA. We also tested the effect of bioactive phospholipids on cell migration, adhesion, and proliferation. In parallel experiments, we evaluated the effect of bioactive phospholipids on the expression of HO-1 and iNOS in human leukemic cells and the involvement of p38 MAPK in these phenomena. Furthermore, in in vivo experiments the expression of HO-1 and iNOS was upregulated by administration of the p38 MAPK inhibitor SB203580. We also performed in vivo seeding efficiency studies in immunodeficient mice with cells stimulated by bioactive phospholipids in the presence or absence of SB203580. In parallel, we also tested the chemotactic responsiveness of normal human CD34+ cells and clonogenic blasts from AML patients to bioactive phospholipids in chemotaxis and adhesion experiments. Results. We demonstrate for the first time that, besides S1P receptors, human leukemic cells express functional receptors for other bioactive lipids and that C1P, LPC, and LPA are, as has been reported for S1P, important chemoattractants and pro-adhesive factors for human leukemic cells. In contrast only S1P and C1P, but not LPC or LPA stimulated migration and adhesion of normal human clonogeneic CD34+ cells. At the same time, we report that all of the tested phospholipids did not stimulate proliferation of human clonogeneic CD34+ or leukemic cells. We also demonstrate that these pro-migratory effects of bioactive phospholipids can be explained, at least partially, by downregulation HO-1 and iNOS activities in a p38 MAPK-dependent manner. Stimulation of leukemic cells before injection into immunodeficient mice with bioactive phospholipids increased their seeding efficiency to BM, liver, and lungs. This effect was significantly inhibited after exposure of these cells to SB203580. Conclusions. We demonstrate for the first time that, besides S1P receptors, human leukemic cells express other functional receptors that enable chemotaxis in response to, as well as adhesion to, several bioactive phospholipids. Since this effect is mediated, at least partially, by downregulating HO-1 and iNOS expression in a p38 MAPK-dependent manner, inhibitors of p38 MAPK may find application in inhibiting the spread of leukemic cells in situations in which S1P, C1P, LPC, and LPA become upregulated in the tissues. This may occur, for example, after radio- and chemotherapy, leading to the unwanted spread of leukemic cells that have escaped therapy. DisclosuresNo relevant conflicts of interest to declare.

Lysophosphatidylcholine aggravates contact hypersensitivity by promoting neutrophil infiltration and IL17 expression

Lysophosphatidylcholine (LPC) is a bioactive lysolipid known to contribute to the development of lung allergic diseases. However, it remains unknown whether LPC possesses proinflammatory properties in the skin as well. Here, we investigated this issue by injection of LPC into the murine contact hypersensiti-vity (CHS) model induced by 2,4-dinitrofluorobenzene (DNFB). LPC increased the expression of IL17, recruited more neutrophils, and eventually aggravated the CHS in the skins. Moreover, the effects of LPC diminished after neutralizing IL17 or depleting neutrophils. Mechanistically, LPC upregulated not only IL17 but also CXCL1 and CXCL2 in a G2A-dependent manner. Taken together, our study demonstrated that the upregulation of LPC could contribute to allergic skin inflammation by increasing IL17 expression and neutrophil recruitment via G2A receptor.

Uptake of Vitamins D2, D3, D4, D5, D6, and D7 Solubilized in Mixed Micelles by Human Intestinal Cells, Caco-2, an Enhancing Effect of Lysophosphatidylcholine on the Cellular Uptake, and Estimation of Vitamins D' Biological Activities.

Vitamins D have various biological activities, as well as intestinal calcium absorption. There has been recent concern about insufficient vitamin D intake. In addition to vitamins D2 and D3, there are lesser-known vitamins D4–D7. We synthesized vitamins D5–D7, which are not commercially available, and then evaluated and compared the mixed micelles-solubilized vitamins D uptake by Caco-2 cells. Except for vitamin D5, the uptake amounts of vitamins D4–D7 by differentiated Caco-2 cells were similar to those of vitamins D2 and D3. The facilitative diffusion rate in the ezetimibe inhibited pathway was approximately 20% for each vitamin D type, suggesting that they would pass through the pathway at a similar rate. Lysophosphatidylcholine enhanced each vitamin D uptake by approximately 2.5-fold. Lysophosphatidylcholine showed an enhancing effect on vitamin D uptake by reducing the intercellular barrier formation of Caco-2 cells by reducing cellular cholesterol, suggesting that increasing the uptakes of vitamins D and/or co-ingesting them with lysophosphatidylcholine, would improve vitamin D insufficiency. The various biological activities in the activated form of vitamins D4–D7 were estimated by Prediction of Activity Spectra for Substances (PASS) online simulation. These may have some biological activities, supporting the potential as nutritional components.

Abstract 155: Identification of Decreased Plasma Lysophosphatidylcholine Using Phospholipidomics in Cardiac Arrest: A Novel Therapeutic Application

Introduction: Cardiac arrest (CA) is a significant public health burden with few effective therapies available. Plasma phospholipids are important for proper organ function. We hypothesized that alterations in plasma phospholipids may play an important role in the pathophysiology of CA and normalizing these alterations may be a novel therapeutic application for the treatment of CA. Objective: The aim of this study was to test the phospholipid therapy as a novel therapeutic approach in CA. Methods and Results: We performed phospholipidomics on plasma samples from CA patients and controls from North Shore University Hospital to identify plasma phospholipids that correlate with injury severity. We then confirmed the finding using our rat model of 10 and 14 min of asphyxia-induced CA. Finally, we tested the effect of administering LPC using the same model. From the phospholipids analyzed, lysophosphatidylcholine (LPC) levels were found to be the most significantly decreased in CA patients, which was also associated with survival of patients. The same trend was observed in the rat model where rats after 14 min CA had significantly lower plasma LPC levels than rats after 10 min CA. We also found that LPC levels began to decrease continuously following resuscitation, but not during the ischemic phase of CA. Finally, we found that LPC administration substantially increased rat survival compared to the control group. Rats who survived in the LPC-treated group displayed significantly improved brain function and the neuroprotective effect of LPC is supported by improved brain histology. We also found LPC treatment increased plasma IL-10 levels and decreased IL-6 levels post-CA. Conclusion: Plasma LPC levels inversely correlated with survival and LPC treatment significantly improved brain function and rat survival. This data indicates that the decrease of plasma LPC is a major mechanism responsible for brain damage demonstrating the potential of LPC as a novel therapeutic in CA.

Lysophosphatidylcholines inhibit human eosinophil activation and suppress eosinophil migration in vivo

Eosinophils are important multifaceted effector cells involved in allergic inflammation. Following allergen challenge, eosinophils and other immune cells release secreted phospholipases, generating lysophosphatidylcholines (LPCs). LPCs are potent lipid mediators, and serum levels of LPCs associate with asthma severity, suggesting a regulatory activity of LPCs in asthma development. As of yet, the direct effects of LPCs on eosinophils remain unclear. In the present study, we tested the effects of the major LPC species (16:0, 18:0 and 18:1) on eosinophils isolated from healthy human donors. Addition of saturated LPCs in the presence of albumin rapidly disrupted cholesterol-rich nanodomains on eosinophil cell membranes and suppressed multiple eosinophil effector responses, such as CD11b upregulation, degranulation, chemotaxis, and downstream signaling. Furthermore, we demonstrate in a mouse model of allergic cell recruitment, that LPC treatment markedly reduces immune cell infiltration into the lungs. Our observations suggest a strong modulatory activity of LPCs in the regulation of eosinophilic inflammation in vitro and in vivo.

A Model for Myomerger Function in Myoblast Fusion

Myomerger, an integral membrane protein, has been recently established to play a crucial role in myoblast fusion by driving the transition from the hemifusion to the full fusion stage of the reaction. To accomplish this task, the ectodomains of myomerger molecules located at the proximal (outer) leaflets of the fusing plasma membranes must be able to mediate formation of a fusion pore in the hemifusion diaphragm (HD), which consist of the distal (inner) membrane leaflets. We now report that, similarly to myomerger, the phospholipid lysophosphatidylcholine (LPC) promotes hemifusion-to-fusion transition, if added to the proximal leaflets of the hemifused membranes. An apparent similarity between LPC and myomerger consists in their ability to generate a positive spontaneous curvature of lipid monolayers. This effect of LPC is known to be driven by its effective molecular shape, while myomerger could act through insertion of its N-terminal amphipathic domain into the membrane matrix. Here we propose a mechanism of indirect promotion of the fusion pore formation. The essence of the mechanism is a geometrical and mechanical coupling of the proximal and distal leaflets of the fusing membranes mediated by the HD rim. The elastic stresses generated by LPC and/or myomerger in the distal membrane leaflets are transmitted through the HD rim into the leaflets of the hemifusion diaphragm, which facilitates the fusion pore generation and growth. To substantiate the mechanism, using the theory of the membrane bending, splay and tilt elasticity, we computed the elastic stresses propagating into the HD as functions of LPC concentration in the proximal membrane leaflets, determined the effective values of HD line tension and calculated the kinetic barriers of the fusion pore formation. We predicted, quantitatively, the degree of acceleration of the fusion pore formation by relevant LPC amounts in the membrane.