Skewing cPLA ₂ α activity toward oxoeicosanoid production promotes neutrophil N2 polarization, wound healing, and the response to sepsis

Abstract

Uncontrolled inflammation is linked to poor outcomes in sepsis and wound healing, both of which proceed through distinct inflammatory and resolution phases. Eicosanoids are a class of bioactive lipids that recruit neutrophils and other innate immune cells. The interaction of ceramide 1-phosphate (C1P) with the eicosanoid biosynthetic enzyme cytosolic phospholipase A 2 (cPLA 2 ) reduces the production of a subtype of eicosanoids called oxoeicosanoids. We investigated the effect of shifting the balance in eicosanoid biosynthesis on neutrophil polarization and function. Knockin mice expressing a cPLA 2 mutant lacking the C1P binding site ( cPLA 2 α KI/KI mice) showed enhanced and sustained neutrophil infiltration into wounds and the peritoneum during the inflammatory phase of wound healing and sepsis, respectively. The mice exhibited improved wound healing and reduced susceptibility to sepsis, which was associated with an increase in anti-inflammatory N2-type neutrophils demonstrating proresolution behaviors and a decrease in proinflammatory N1-type neutrophils. The N2 polarization of cPLA 2 α KI/KI neutrophils resulted from increased oxoeicosanoid biosynthesis and autocrine signaling through the oxoeicosanoid receptor OXER1 and partially depended on OXER1-dependent inhibition of the pentose phosphate pathway (PPP). Thus, C1P binding to cPLA 2 α suppresses neutrophil N2 polarization, thereby impairing wound healing and the response to sepsis.

Ceramide-1-phosphate is a regulator of Golgi structure and is co-opted by the obligate intracellular bacterial pathogen Anaplasma phagocytophilum

“…How CERK-derived C1P activates PKCα to facilitate subsequent activation of Cdc42 and JNK is not known, but PKCα possesses a C2-domain ( 114 , 115 ). C1P binds and activates group IVA phospholipase A 2 via this enzyme’s C2 domain facilitating its translocation to the Golgi ( 49 – 52 , 57 , 58 , 61 , 116 , 117 ). This gives rise to the hypothesis that C1P analogously binds PKCα at its C2-domain to translocate PKCα to the Golgi.…”

“…Total cell lipids were harvested, sphingolipids extracted, and UPLC-ESI-MS/MS analyses were performed to quantitate sphingolipids as described ( 49 – 52 , 54 , 55 , 109 ).…”

Role of Polyunsaturated Fatty Acids (PUFAs) and Eicosanoids on Dry Eye Symptoms and Signs

“…Schirmer strips were stored in −80 °C until analysis. Eicosanoids were extracted and analyzed by UPLC ESI-MS/MS, as previously described by us and others [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. Briefly, tear strips were placed in tubes with 4 mL of water and an internal standard (IS) mixture comprising 10% methanol (400 μL) and glacial acetic acid (20 μL) and an internal standard (20 μL) containing the following deuterated eicosanoids (1.5 pmol/μL, 30 pmol total) (all standards purchased from Cayman Chemicals, Ann Arbor, MI, USA): ( d 4 ) 6keto-prostaglandin F 1 α, ( d 4 ) prostaglandin F 2 α, ( d 4 ) prostaglandin E 2 , ( d 4 ) prostaglandin D 2 , ( d 8 ) 5-hydroxyeicosatetraenoic acid (5-HETE), ( d 8 ) 12-hydroxyeicosatetraenoic acid (12-HETE), ( d 8 ) 15-hydroxyeicosatetraenoic acid (15-HETE), ( d 6 ) 20-hydroxyeicosatetraenoic acid (20-HETE), ( d 11 ) 8,9 epoxyeicosa-trienoic acid, ( d 8 ) 14,15 epoxyeicosa-trienoic acid, ( d 8 ) arachidonic acid, ( d 5 ) Eicosapentaenoic acid, ( d 5 ) docosahexaenoic acid, ( d 4 ) prostaglandin A2, ( d 4 ) leukotriene B 4 , ( d 4 ) leukotriene C4, ( d 4 ) leukotriene D4, ( d 4 ) leukotriene E4, ( d 5 ) 5(S),6(R)-lipoxin A4, ( d 11 ) 5-iPF2α-VI, ( d 4 ) 8-iso prostaglandin F2α, ( d 11 ) (±)14,15-DHET, ( d 11 ) (±)8,9-DHET, ( d 11 ) (±)11,12-DHET, ( d 4 ) prostaglandin E1, ( d 4 ) thromboxane B2, ( d 6 ) dihomo gamma linoleic acid, ( d 5 ) resolvin D2, ( d 5 ) resolvin D1 (RvD1), ( d 5 ) Maresin2, ( d 7 ) 5-OxoETE, and ( d 5 ) resolvin D3.…”

“…Eicosanoids were monitored using precursor → product MRM pairs. The mass spectrometer parameters used were as follows: curtain gas: 20 psi; CAD: medium; ion spray voltage: −4500 V; temperature: 300 °C; gas 1: 40 psi; gas 2: 60 psi; declustering potential, collision energy, and cell exit potential vary per transition as reported [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ].…”

“…Schirmer strips were stored in −80 • C until analysis. Eicosanoids were extracted and analyzed by UPLC ESI-MS/MS, as previously described by us and others [22][23][24][25][26][27][28][29]. Briefly, tear strips were placed in tubes with 4 mL of water and an internal standard (IS) mixture comprising 10% methanol (400 µL) and glacial acetic acid (20 µL) and an internal standard (20 µL) containing the following deuterated eicosanoids (1.5 pmol/µL, 30 pmol total) (all standards purchased from Cayman Chemicals, Ann Arbor, MI, USA): (d 4 ) 6ketoprostaglandin Eicosanoids were separated using a Shimadzu Nexera X2 LC-30AD (Shimadzu, Kyoto, Japan) coupled with a SIL-30AC auto injector (Shimadzu, Kyoto, Japan) and a DGU-20A5R (Shimadzu, Kyoto, Japan) degassing unit in the following way: A 14 min, reversed-phase LC method utilizing an Ascentis Express C18 column (150 mm × 2.1 mm, 2.7 µm) was used to separate the eicosanoids at a 0.5 mL/min flow rate at 40 • C. The column was equilibrated with 100% Solvent A (acetonitrile/water/formic acid (20:80:0.02, v/v/v)) for 5 min and then 10 µL of sample was injected.…”