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S1P/S1P₂ Signaling Axis Regulates Both NLRP3 Upregulation and NLRP3 Inflammasome Activation in Macrophages Primed with Lipopolysaccharide.

ABSTRACT: The activation of NLRP3 inflammasome is a key factor for various inflammatory diseases. Here, we provide experimental evidence supporting the regulatory role of sphingosine-1-phosphate (S1P) in NLRP3 inflammasome activation in mouse bone-marrow-derived macrophages (BMDMs), along with the S1P receptor subtype involved and underlying regulatory mechanisms. During the priming stage, S1P induced NLRP3 upregulation in BMDMs only when primed with lipopolysaccharide (LPS). In this event, S1P₂, but not S1P₁, was involved based on the attenuated NLRP3 upregulation with JTE013 (S1P₂ antagonist) or S1P₂ knockdown. During the activation stage, S1P induced NLRP3 inflammasome activation in LPS-primed BMDMs via caspase-1 activation, interleukin 1β maturation, apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, and IL-1β secretion. Such NLRP3 inflammasome activation was blocked by either pharmacological inhibition or genetic knockdown of S1P₂. NF-κB, PI3K/Akt, and ERK1/2 were identified as effector pathways underlying S1P/S1P₂ signaling in the regulation of NLRP3 upregulation in LPS-primed BMDMs. Further, reactive oxygen species (ROS) production was dependent on the S1P/S1P₂ signaling axis in these cells, and the ROS generated regulate NLRP3 inflammasome activation, but not NLRP3 priming. Collectively, our findings suggest that S1P promotes NLRP3 upregulation and NLRP3 inflammasome activation in LPS-primed BMDMs via S1P₂ and subsequent effector pathways.

SUBMITTER: Lee CH

PROVIDER: S-EPMC8614891 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The free-radical-operated mechanism of death of activated macrophages at sites of inflammation is unclear, but it is important to define it in order to find targets to prevent further tissue dysfunction. A well-defined model of macrophage activation at sites of inflammation is the treatment of RAW 264.7 cells with lipopolysaccharide (LPS), with the resulting production of reactive oxygen species (ROS). ROS and other free radicals can be trapped with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), a cell-permeable probe with antioxidant properties, which thus interferes with free-radical-operated oxidation processes. Here we have used immuno-spin trapping to investigate the role of free-radical-operated protein oxidation in LPS-induced cytotoxicity in macrophages. Treatment of RAW 264.7 cells with LPS resulted in increased ROS production, oxidation of proteins, cell morphological changes and cytotoxicity. DMPO was found to trap protein radicals to form protein-DMPO nitrone adducts, to reduce protein carbonyls, and to block LPS-induced cell death. N-Acetylcysteine (a source of reduced glutathione), diphenyleneiodonium (an inhibitor of NADPH oxidase), and 2,2'-dipyridyl (a chelator of Fe(2+)) prevented LPS-induced oxidative stress and cell death and reduced DMPO-nitrone adduct formation, suggesting a critical role of ROS, metals, and protein-radical formation in LPS-induced cell cytotoxicity. We also determined the subcellular localization of protein-DMPO nitrone adducts and identified some candidate proteins for DMPO attachment by LC-MS/MS. The LC-MS/MS data are consistent with glyceraldehyde-3-phosphate dehydrogenase, one of the most abundant, sensitive, and ubiquitous proteins in the cell, becoming labeled with DMPO when the cell is primed with LPS. This information will help find strategies to treat inflammation-associated tissue dysfunction by focusing on preventing free radical-operated proteotoxic stress and death of macrophages.

Dataset Information

S1P/S1P₂ Signaling Axis Regulates Both NLRP3 Upregulation and NLRP3 Inflammasome Activation in Macrophages Primed with Lipopolysaccharide.

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OmicsDI is part of the ELIXIR infrastructure

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S1P/S1P2 Signaling Axis Regulates Both NLRP3 Upregulation and NLRP3 Inflammasome Activation in Macrophages Primed with Lipopolysaccharide.

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OmicsDI is part of the ELIXIR infrastructure

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S1P/S1P₂ Signaling Axis Regulates Both NLRP3 Upregulation and NLRP3 Inflammasome Activation in Macrophages Primed with Lipopolysaccharide.