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Extracellular low pH modulates phosphatidylserine-dependent phagocytosis in macrophages by increasing stabilin-1 expression.

ABSTRACT: Microenvironmental acidosis is a common feature of inflammatory loci, in which clearance of apoptotic cells is necessary for the resolution of inflammation. Although it is known that a low pH environment affects immune function, its effect on apoptotic cell clearance by macrophages has not been fully investigated. Here, we show that treatment of macrophages with low pH medium resulted in increased expression of stabilin-1 out of several receptors, which are known to be involved in PS-dependent removal of apoptotic cells. Reporter assays showed that the -120/-1 region of the mouse stabilin-1 promoter was a low pH-responsive region and provided evidence that extracellular low pH mediated transcriptional activation of stabilin-1 via Ets-2. Furthermore, extracellular low pH activated JNK, thereby inducing translocation of Ets-2 into the nucleus. When macrophages were preincubated with low pH medium, phagocytosis of phosphatidylserine-exposed red blood cells and phosphatidylserine-coated beads by macrophages was enhanced. Blockade of stabilin-1 in macrophages abolished the enhancement of phagocytic activity by low pH. Thus, our results demonstrate that a low pH microenvironment up-regulates stabilin-1 expression in macrophages, thereby modulating the phagocytic capacity of macrophages, and suggest roles for stabilin-1 and Ets-2 in the maintenance of tissue homeostasis by the immune system.

SUBMITTER: Park SY

PROVIDER: S-EPMC3322863 | biostudies-other | 2012 Mar

REPOSITORIES: biostudies-other

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Extracellular low pH modulates phosphatidylserine-dependent phagocytosis in macrophages by increasing stabilin-1 expression.

Park Seung-Yoon SY Bae Dong-Jun DJ Kim Mi-Jin MJ Piao Mei Lan ML Kim In-San IS

The Journal of biological chemistry 20120210 14

Microenvironmental acidosis is a common feature of inflammatory loci, in which clearance of apoptotic cells is necessary for the resolution of inflammation. Although it is known that a low pH environment affects immune function, its effect on apoptotic cell clearance by macrophages has not been fully investigated. Here, we show that treatment of macrophages with low pH medium resulted in increased expression of stabilin-1 out of several receptors, which are known to be involved in PS-dependent r ...[more]

PMID: 22334667

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Project description:Inflammation and metabolism are intricately linked during inflammatory diseases in which activation of the nucleotide-binding domain-like receptors Family Pyrin Domain Containing 3 (NLRP3) inflammasome, an innate immune sensor, is critical. Several factors can activate the NLRP3 inflammasome, but the nature of the link between NLRP3 inflammasome activation and metabolism remains to be thoroughly explored. This study investigates whether the small molecule inhibitor of the NLRP3 inflammasome, MCC950, modulates the lipopolysaccharide (LPS) -and amyloid-β (Aβ)-induced metabolic phenotype and inflammatory signature in macrophages. LPS + Aβ induced IL-1β secretion, while pre-treatment with MCC950 inhibited this. LPS + Aβ also upregulated IL-1β mRNA and supernatant concentrations of TNFα, IL-6 and IL-10, however these changes were insensitive to MCC950, confirming that MCC950 specifically targets inflammasome activation in BMDMs. LPS + Aβ increased glycolysis and the glycolytic enzyme, PFKFB3, and these effects were decreased by MCC950. These findings suggest that NLRP3 inflammasome activation may play a role in modulating glycolysis. To investigate this further, the effect of IL-1β on glycolysis was assessed. IL-1β stimulated glycolysis and PFKFB3, mimicking the effect of LPS + Aβ and adding to the evidence that inflammasome activation impacts on metabolism. This contention was supported by the finding that the LPS + Aβ-induced changes in glycolysis and PFKFB3 were attenuated in BMDMs from NLRP3-deficient and IL-1R1-deficient mice. Consistent with a key role for PFKFB3 is the finding that the PFKFB3 inhibitor, 3PO, attenuated the LPS + Aβ-induced glycolysis. The data demonstrate that activation of the NLRP3 inflammasome, and the subsequent release of IL-1β, play a key role in modulating glycolysis via PFKFB3. Reinstating metabolic homeostasis by targeting the NLRP3 inflammasome-PFKFB3 axis may provide a novel therapeutic target for treatment of acute and chronic disease.

Project description:Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10-20?min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3- ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar bicarbonate solution. For that reason, regulating NETosis by pH with specific buffers such as THAM could be more effective than bicarbonate in managing NET-related diseases.

Dataset Information

Extracellular low pH modulates phosphatidylserine-dependent phagocytosis in macrophages by increasing stabilin-1 expression.

Publications

Extracellular low pH modulates phosphatidylserine-dependent phagocytosis in macrophages by increasing stabilin-1 expression.

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