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Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype.

ABSTRACT: Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1-6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed "LPA" throughout our study) in mediating a motile and pro-inflammatory microglial phenotype via LPAR5 that couples to protein kinase D (PKD)-mediated pathways.Using the xCELLigence system and time-lapse microscopy, we investigated the migrational response of microglial cells. Different M1 and M2 markers were analyzed by confocal microscopy, flow cytometry, and immunoblotting. Using qPCR and ELISA, we studied the expression of migratory genes and quantitated the secretion of pro-inflammatory cytokines and chemokines, respectively. Different transcription factors that promote the regulation of pro-inflammatory genes were analyzed by western blot. Reactive oxygen species (ROS) and nitric oxide (NO) production, phagocytosis, and microglial cytotoxicity were determined using commercially available assay kits.LPA induces MAPK family and AKT activation and pro-inflammatory transcription factors' phosphorylation (NF-?B, c-Jun, STAT1, and STAT3) that were inhibited by both LPAR5 and PKD family antagonists. LPA increases migratory capacity, induces secretion of pro-inflammatory cytokines and chemokines and expression of M1 markers, enhances production of ROS and NO by microglia, and augments cytotoxicity of microglial cell-conditioned medium towards neurons. The PKD family inhibitor blunted all of these effects. We propose that interference with this signaling axis could aid in the development of new therapeutic approaches to control neuroinflammation under conditions of overshooting LPA production.In the present study, we show that inflammatory LPA levels increased the migratory response of microglia and promoted a pro-inflammatory phenotype via the LPAR5/PKD axis. Interference with this signaling axis reduced microglial migration, blunted microglial cytotoxicity, and abrogated the expression and secretion of pro-inflammatory mediators.

SUBMITTER: Plastira I

PROVIDER: S-EPMC5735906 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype.

Plastira I I Bernhart E E Goeritzer M M DeVaney T T Reicher H H Hammer A A Lohberger B B Wintersperger A A Zucol B B Graier W F WF Kratky D D Malle E E Sattler W W

Journal of neuroinflammation 20171219 1

<h4>Background</h4>Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1-6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed "LPA" throughout our study) in mediating a moti ...[more]

PMID: 29258556

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Project description:Epithelial ovarian carcinoma tissues express high levels of tumor necrosis factor-alpha (TNF-α) and other inflammatory cytokines. The underlying mechanism leading to the abnormal TNF-α expression in ovarian cancer remains poorly understood. In the current study, we demonstrated that lysophosphatidic acid (LPA), a lipid mediator present in ascites of ovarian cancer patients, induced expression of TNF-α mRNA and release of TNF-α protein in ovarian cancer cells. LPA also induced expression of interleukin-1β (IL-1β) mRNA but no significant increase in IL-1β protein was detected. LPA enhanced TNF-α mRNA through NF-κB-mediated transcriptional activation. Inactivation of ADAM17, a disintegrin and metalloproteinase, with a specific inhibitor TMI-1 or by shRNA knockdown prevented ovarian cancer cells from releasing TNF-α protein in response to LPA, indicating that LPA-mediated TNF-α production relies on both transcriptional upregulations of the TNF-α gene and the activity of ADAM17, the membrane-associated TNF-α-converting enzyme. Like many other biological responses to LPA, induction of TNF-α by LPA also depended on the transactivation of the epidermal growth factor receptor (EGFR). Interestingly, our results revealed that ADAM17 was also the shedding protease responsible for the transactivation of EGFR by LPA in ovarian cancer cells. To explore the biological outcomes of LPA-induced TNF-α, we examined the effects of a TNF-α neutralizing antibody and recombinant TNF-α soluble receptor on LPA-stimulated expression of pro-tumorigenic cytokines and chemokines overexpressed in ovarian cancer. Blockade of TNF-α signaling significantly reduced the production of IL-8, IL-6, and CXCL1, suggesting a hierarchy of mechanisms contributing to the robust expression of the inflammatory mediators in response to LPA in ovarian cancer cells. In contrast, TNF-α inhibition did not affect LPA-dependent cell proliferation. Taken together, our results establish that the bioactive lipid LPA drives the expression of TNF-α to regulate an inflammatory network in ovarian cancer.

Project description:Microglia, the resident immune cells of the brain, can exhibit a broad range of activation phenotypes, many of which have been implicated in several diseases and disorders of the central nervous system including those related to alcohol abuse. Given the complexity of global-scale molecular changes that define microglial activation, accurate phenotypic classification in the context of alcohol exposure is still lacking. We employed an optimized method for deep, quantitative proteome profiling of primary microglia in order to characterize their response to acute exposure to alcohol (ethanol) as well as the pro-inflammatory driver and TLR4 agonist, LPS. From this analysis, 5,062 total proteins were identified where 4,857 and 4,928 of those proteins were quantifiable by label-free quantitation in ethanol and LPS treatment groups, respectively. This study highlights the subtle, yet significant proteomic changes that occur in ethanol-treated microglia, which do not align with the robust pro-inflammatory phenotype induced by TLR4 activation. Specifically, our results indicate inhibition of several upstream regulators associated with inflammation, opposing effects on pathways such as phagocytosis upon comparison to TLR4-mediated pro-inflammatory phenotype, and a potential metabolic shift associated with increased expression of proteins related to OXPHOS and lipid homeostasis. Data are available via ProteomeXchange with identifier PXD14466. SIGNIFICANCE: Alcohol abuse has a significant impact on the central nervous system, which includes the pathophysiological mechanisms resulting from glial cell activation. Microglia, in particular, are the resident immune cells of the brain and exhibit a broad range of activation phenotypes. The molecular changes that drive microglial activation phenotype are complex and have yet to be fully characterized in the context of alcohol exposure. Our study highlights the first and most comprehensive characterization of alcohol-induced proteomic changes in primary microglia to date and has shed light on novel immune-related and metabolic pathways that are altered due to alcohol exposure. The results from this study provide an important foundation for future work aimed to understand the complexity of alcohol-induced microglial activation in vivo and other translational models of acute and chronic alcohol exposure.

Project description:BackgroundLysophosphatidic acid receptor 1 (LPA1) is in the spotlight because its synthetic antagonist has been under clinical trials for lung fibrosis and psoriasis. Targeting LPA1 might also be a therapeutic strategy for cerebral ischemia because LPA1 triggers microglial activation, a core pathogenesis in cerebral ischemia. Here, we addressed this possibility using a mouse model of transient middle cerebral artery occlusion (tMCAO).MethodsTo address the role of LPA1 in the ischemic brain damage, we used AM095, a selective LPA1 antagonist, as a pharmacological tool and lentivirus bearing a specific LPA1 shRNA as a genetic tool. Brain injury after tMCAO challenge was accessed by determining brain infarction and neurological deficit score. Role of LPA1 in tMCAO-induced microglial activation was ascertained by immunohistochemical analysis. Proinflammatory responses in the ischemic brain were determined by qRT-PCR and immunohistochemical analyses, which were validated in vitro using mouse primary microglia. Activation of MAPKs and PI3K/Akt was determined by Western blot analysis.ResultsAM095 administration immediately after reperfusion attenuated brain damage such as brain infarction and neurological deficit at 1 day after tMCAO, which was reaffirmed by LPA1 shRNA lentivirus. AM095 administration also attenuated brain infarction and neurological deficit at 3 days after tMCAO. LPA1 antagonism attenuated microglial activation; it reduced numbers and soma size of activated microglia, reversed their morphology into less toxic one, and reduced microglial proliferation. Additionally, LPA1 antagonism reduced mRNA expression levels of proinflammatory cytokines and suppressed NF-κB activation, demonstrating its regulatory role of proinflammatory responses in the ischemic brain. Particularly, these LPA1-driven proinflammatory responses appeared to occur in activated microglia because NF-κB activation occurred mainly in activated microglia in the ischemic brain. Regulatory role of LPA1 in proinflammatory responses of microglia was further supported by in vitro findings using lipopolysaccharide-stimulated cultured microglia, showing that suppressing LPA1 activity reduced mRNA expression levels of proinflammatory cytokines. In the ischemic brain, LPA1 influenced PI3K/Akt and MAPKs; suppressing LPA1 activity decreased MAPK activation and increased Akt phosphorylation.ConclusionThis study demonstrates that LPA1 is a new etiological factor for cerebral ischemia, strongly indicating that its modulation can be a potential strategy to reduce ischemic brain damage.

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Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype.

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Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype.

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