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

Abstract

BackgroundExtracellular 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.MethodsUsing 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.ResultsLPA induces MAPK family and AKT activation and pro-inflammatory transcription factors’ phosphorylation (NF-κB, 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.ConclusionsIn 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.Electronic supplementary materialThe online version of this article (10.1186/s12974-017-1024-1) contains supplementary material, which is available to authorized users.

Lysophosphatidic acid up-regulates IL-10 production to inhibit TNF-α synthesis in Mϕs stimulated with LPS

“…Earlier studies have indicated an ability of LPA to induce p38 phosphorylation in J774 and other cell types, e.g., murine microglia, embryonic carcinoma cells, and rat fibroblasts . In our studies, a role of p38 in the IL‐10 production in LPA/LPS‐stimulated cells was indicated by its abrogation by the p38 inhibitor SB 202190 and also by the analysis of the dynamics of p38 phosphorylation and IL‐10 secretion in LPA and/or LPS‐treated cells.…”

“…Until now, the immunomodulatory role of LPA 5 and LPA 6 has been addressed in few studies only. The pro‐inflammatory responses induced by LPA in microglia, e.g., expression of pro‐inflammatory M1‐type markers and secretion of pro‐inflammatory cytokines, were attenuated by an LPA 5 inhibitor . Inhibition of B‐cell receptor signaling was observed following activation of LPA 5 in murine B cells .…”

“…Recent studies indicate that LPA alone has some pro‐inflammatory activity, e.g., it induces polarization of murine microglia to M1 type and enhances expression of TNF‐α in THP‐1 monocytes, however, the role played by LPA in the modulation of LPS‐induced inflammation is less clear . Thus, in rodents, injection of LPA decreased LPS‐induced organ injury and increased survival during endotoxemia .…”

Lysophosphatidic Acid Receptors: Biochemical and Clinical Implications in Different Diseases

“…This indicates that LPA 5 signaling is related to neuropathic pain mediated by multiple sclerosis [96]. It was found that intravenous LPA and GGPP (LPA 5 agonists) induced allodynia, but GGPP-induced allodynia did not show up in LPA 5 -KO mice, indicating pain signals in the spinal cord by LPA 5 transfer [94][95][96].…”

“…LPA may activate macrophages/microglia via LPAR1 and LPAR3 and promote self-amplification of LPA, increasing microglial migration and pro-inflammatory phenotype through the LPAR5/protein kinase D (PKD) axis. Activation and migration of microglia are associated with demyelination in the spinal cord after injury, and microglia are more involved in the initiation of neuropathic pain (NP) [94,95]. In the cuprizone (CPZ)-induced multiple sclerosis (MS) model, LPA5 signaling mediates pain allergy induced by A-delta fibers and demyelination produced by CPZ.…”