Impaired clearance of apoptotic cells by phagocytes (efferocytosis) contributes to chronic inflammation and the development of large necrotic cores in atherosclerotic plaques. Many chronic inflammatory states are characterized by excessive lipid peroxidation, which produces reactive lipid aldehydes capable of covalently modifying phospholipids. The authors investigated whether reactive aldehyde adduction to phosphatidylethanolamines generates N‑aldehyde–modified phosphatidylethanolamines (NAMPs), whether NAMPs accumulate under oxidative conditions, and whether exposure to NAMPs inhibits macrophage efferocytosis.
The functional effects of NAMPs on efferocytosis were assessed in real time using the Incucyte Live‑Cell Analysis System to quantify ingestion of CypHer‑labeled apoptotic cells by cultured bone marrow–derived macrophages. Chemical and biochemical analyses of NAMP formation were performed using liquid chromatography coupled with mass spectrometry (LC–MS). Recombinant N‑acyl phosphatidylethanolamine hydrolyzing phospholipase D (NAPE‑PLD) was used to enzymatically hydrolyze NAMPs in vitro, and genetic deletion approaches targeted macrophage NAPE‑PLD to test its role in cellular responses. Pharmacologic blockade of glutathione peroxidase 4 (GPX4) was used to increase lipid peroxidation in macrophages. Effects of modified versus unmodified high‑density lipoprotein (HDL) on efferocytosis were evaluated, and HDL isolated from subjects with familial hypercholesterolemia was analyzed for NAMP content.
The investigators used LC–MS to examine the extent of NAMP formation under multiple oxidative conditions. Reactive lipid aldehydes produced by lipid peroxidation were shown to generate NAMP species on phosphatidylethanolamines. Exposure of HDL to reactive aldehydes or to peroxidizing agents increased levels of multiple NAMP species on the lipoprotein. Similarly, pharmacologic inhibition of GPX4 in macrophages increased cellular NAMP concentrations, indicating that conditions that favor lipid peroxidation promote NAMP accumulation.
Several representative synthetic NAMP species were tested for functional effects on macrophage clearance of apoptotic cells. These included N‑isolevuglandin‑phosphatidylethanolamine (N‑IsoLG‑PE), N‑4‑hydroxynonenal‑phosphatidylethanolamine (N‑HNE‑PE), and N‑azeloyl‑phosphatidylethanolamine (N‑Aze‑PE). Each of these NAMPs inhibited efferocytosis in a concentration‑dependent manner when applied to bone marrow–derived macrophages in the Incucyte efferocytosis assay, demonstrating that NAMPs are sufficient to reduce macrophage ingestion of apoptotic cells.
Hydrolysis of NAMPs using recombinant NAPE‑PLD abolished the inhibitory activity of NAMPs on efferocytosis, indicating that intact N‑aldehyde modifications on phosphatidylethanolamines are required for their effect. In contrast, genetic deletion of macrophage NAPE‑PLD enhanced the inhibitory effect of NAMPs on efferocytosis, consistent with a role for cellular NAPE‑PLD in processing and detoxifying NAMPs and thereby protecting efferocytic capacity.
Blocking glutathione peroxidase 4 (GPX4), an enzyme that inhibits lipid peroxidation, led to increased macrophage NAMP concentrations and impaired efferocytosis. These observations link a loss of cellular antioxidant protection against lipid peroxidation to the accumulation of NAMP species and reduced macrophage clearance of apoptotic cells.
Unmodified HDL promoted efferocytosis in the experimental system. However, when HDL was exposed to reactive lipid aldehydes or subjected to peroxidizing conditions that increased NAMP content, the modified HDL inhibited efferocytosis. Thus, oxidative modification of HDL with NAMP formation converts an efferocytosis‑promoting lipoprotein into an inhibitor of macrophage apoptotic cell clearance.
The authors report that HDL isolated from subjects with familial hypercholesterolemia contained elevated levels of multiple NAMP species. In addition to impairing efferocytosis directly, NAMPs also reduced macrophage cholesterol efflux, a process required for repeated rounds of efferocytosis. Together these findings indicate that NAMP accumulation can both blunt immediate efferocytic activity and interfere with cellular lipid handling needed to sustain continued clearance of apoptotic cells.
The study concludes that N‑aldehyde‑modified phosphatidylethanolamines (NAMPs) accumulate under conditions associated with lipid peroxidation and inhibit macrophage efferocytosis. Enzymatic hydrolysis by NAPE‑PLD negates NAMP activity, while loss of NAPE‑PLD or inhibition of GPX4 increases NAMP levels and worsens efferocytic impairment. Oxidative modification of HDL generates NAMPs and converts HDL from a promoter of efferocytosis into an inhibitor; HDL from persons with familial hypercholesterolemia shows elevated NAMP content. By also impairing macrophage cholesterol efflux, NAMPs may contribute to defective efferocytosis and the formation of necrotic cores in atherosclerotic plaques, linking lipid peroxidation to processes that promote chronic inflammation.
Note: This article is a preprint and has not been peer reviewed. Experimental details, numerical values, and additional methodological specifics were reported in the source but are not restated here beyond the summarized findings.