NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A

Abstract

Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lysoPS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91 phox؊/؊ ). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91 phox؊/؊ mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation.Neutrophils are often robustly recruited early in inflammation. Within hours of their activation in tissues, they are removed by phagocytes, an event required for resolution of inflammation and the return to normalcy of tissue function. It is known that neutrophils undergoing apoptosis drive the production of anti-inflammatory mediators such as transforming growth factor-␤ that actively suppress production of inflammatory cytokines, chemokines, eicosanoids, and nitric oxide (1, 2). Indeed, enhanced induction of neutrophil apoptosis in vivo is potently anti-inflammatory (3, 4). However, if recognition and clearance fail, activated and dying neutrophils ultimately disintegrate releasing injurious intracellular constituents (e.g. serine proteases) (5). Failure of timely cell clearance is associated with both autoimmunity and enhanced inflammation (6, 7).Phosphatidylserine (PS) 2 exposed in the plasma membrane outer leaflet of apoptotic cells has long been known as a key ligand important for their recognition and removal. Interaction with various PS receptors, including the recently identified TIM4 (8, 9), BAI1 (10), and stabilin 2 (11) or PS-recognizing bridge molecule-receptor combinations (e.g. MFG-E8 and ␣ v integrins or Gas6 and Mer (12)), have been demonstrated. In many, bu...

CD36, but not G2A, modulates efferocytosis, inflammation, and fibrosis following bleomycin-induced lung injury

“…Nevertheless, in this present study, we were able to provide important confi rmatory data supporting a key role for G2A in apoptotic neutrophil clearance in the zymosan-induced peritoneal infl ammation model ( Fig. 7 ) as originally reported by Frasch et al ( 37,38 ). This, in conjunction with data from the bleomycin-induced lung injury model, further underscores the potential degree of context-dependent functional redundancy between these receptors in terms of their requirement for efferocytosis in vivo.…”

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ABHD12 controls brain lysophosphatidylserine pathways that are deregulated in a murine model of the neurodegenerative disease PHARC

“…LPS has been shown to be a bioactive lipid that produces several cellular effects (14), including mast cell degranulation (15)(16)(17), macrophage clearance of apoptotic cells (18,19), leukemic cell stimulation (20), and chemotactic migration of human glioma cells (21) and murine fibroblasts (22). Although the bioactivity of LPS in the nervous system has not been well studied, our findings support a model where LPS exhibits similar immunomodulatory activities to stimulate microglial function in the brain.…”

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“…Prior studies demonstrated impaired macrophage clearance of apoptotic neutrophils in NADPH oxidase deficiency, reflecting in part deficient generation of lysophosphatidylserine, which enhances their uptake by macrophages, and in part decreased macrophage efferocytosis. [60][61][62] We also observed a higher fraction of uningested apoptotic neutrophils, at a time when peritoneal neutrophil counts were falling, although a similar fraction of efferocytosing macrophages in WT and X-CGD mice. However, our results suggest that the predominant mechanism underlying increased neutrophilic inflammation in DAMP-associated injury in CGD is amplified IL-1a/G-CSF production, leading to an enhanced systemic response that delivers increased numbers of neutrophils to the peritoneum; treatments that normalized excessive early neutrophil accumulation to WT levels were sufficient to promote resolution at a later stage.…”