MAPK signaling determines lysophosphatidic acid (LPA)-induced inflammation in microglia

Abstract

Background: In the extracellular environment, lysophosphatidic acid (LPA) species are generated via autotaxin (ATX)-mediated hydrolysis of lysophospholipid precursors. Members of the LPA family are potent lipid mediators transmitting signals via six different G protein-coupled LPA receptors (LPAR1-6). The LPA signaling axis is indispensable for brain development and function of the nervous system; however, during damage of the central nervous system, LPA levels can increase and aberrant signaling events counteract brain function. Here, we investigated regulation of the ATX/LPA/LPAR axis in response to lipopolysaccharide-induced systemic inflammation in mice and potential neurotoxic polarization programs in LPA-activated primary murine microglia. Methods: In vivo, LPAR1-6 expression was established by qPCR in whole murine brain homogenates and in FACSsorted microglia. ELISAs were used to quantitate LPA concentrations in the brain and cyto-/chemokine secretion from primary microglia in vitro. Transcription factor phosphorylation was analyzed by immunoblotting, and plasma membrane markers were analyzed by flow cytometry. We used MAPK inhibitors to study signal integration by the JNK, p38, and ERK1/2 branches in response to LPA-mediated activation of primary microglia. Results: Under acute and chronic inflammatory conditions, we observed a significant increase in LPA concentrations and differential regulation of LPAR, ATX (encoded by ENPP2), and cytosolic phospholipase A2 (encoded by PLA2G4A) gene expression in the brain and FACS-sorted microglia. During pathway analyses in vitro, the use of specific MAPK antagonists (SP600125, SB203580, and PD98059) revealed that JNK and p38 inhibition most efficiently attenuated LPA-induced phosphorylation of proinflammatory transcription factors (STAT1 and-3, p65, and c-Jun) and secretion of IL-6 and TNFα. All three inhibitors decreased LPA-mediated secretion of IL-1β, CXCL10, CXCL2, and CCL5. The plasma membrane marker CD40 was solely inhibited by SP600125 while all three inhibitors affected expression of CD86 and CD206. All MAPK antagonists reduced intracellular COX-2 and Arg1 as well as ROS and NO formation, and neurotoxicity of microglia-conditioned media.

Lysophosphatidic Acid Induces Aerobic Glycolysis, Lipogenesis, and Increased Amino Acid Uptake in BV-2 Microglia

“…In the brain, LPA mediates diverse biological actions on different cell types including neurons, astrocytes, oligodendrocytes, and microglia [ 52 ]. In microglia LPA can have protective [ 38 , 53 ] or detrimental effects [ 6 , 27 ], the outcome presumably depending on the cellular pre-activation state. BV2 microglia that were used throughout the present study are a well-characterized and widely used in vitro system for microglia studies [ 54 ].…”

“…LPA treatment of BV-2 and primary murine microglia induces activation of pro-inflammatory signaling cascades, secretion of chemo-/cytokines, induction of ROS production and M1 plasma membrane marker expression indicating polarization towards a neurotoxic phenotype [ 6 , 27 , 28 ]. To investigate whether these alterations are accompanied by the induction of a specific immunometabolic phenotype we investigated specific aspects of energy metabolism in LPA-treated BV-2 microglia.…”

“…During the inflammatory response, microglia can adopt different polarization states ranging from a pro-inflammatory to an immunosuppressive/anti-inflammatory phenotype [ 4 ]. Depending on the outcome of the polarization program, microglia can aggravate disease or support neuronal survival [ 5 , 6 ]. Combination of sophisticated analytical methods, such as RNAseq, transcriptomic, and proteomic analysis of microglia at a single-cell level revealed unique phenotypic signatures in physiological and neurodegenerative settings [ 7 , 8 , 9 ].…”

“…Aberrant LPA signaling contributes to multiple disease states, including neuropathic pain, neurodegenerative, neurodevelopmental, and neuropsychiatric disorders [ 30 ]. In microglia many inflammatory effects of LPA are transmitted via LPAR5 [ 6 , 28 , 36 ], which was identified as a member of the microglia sensome [ 11 ].…”

Experimental Pulmonary Tuberculosis in the Absence of Detectable Brain Infection Induces Neuroinflammation and Behavioural Abnormalities in Male BALB/c Mice

“…Moreover, the activity of coordinate MAPK in microglia and astrocytes has a regulatory role in the synthesis of inflammatory cytokines mediators. The JNK and p38 pathways regulate the mRNA stability of cyclooxygenase 2 (COX-2), TNF-α, IL-3, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1α), granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), urokinase-type plasminogen activator (uPA), and iNOS genes [ 45 ] and contributes to microglia-induced neuronal cell death [ 46 , 47 ]. Thus, we evaluated the activation of these proteins during pulmonary infection with M. tb in the same regions that we evaluated the immune response.…”