Lipidomics Suggests a New Role for Ceramide Synthase in Phagocytosis

Abstract

Phagocytosis is an evolutionarily conserved biological process where pathogens or cellular debris are cleared by engulfing them in a membrane-enclosed cellular compartment called the phagosome. The formation, maturation, and subsequent degradation of a phagosome is an important immune response essential for protection against many pathogens. Yet, the global lipid profile of phagosomes remains unknown, especially as a function of their maturation in immune cells. Here, we show using mass spectrometry based quantitative lipidomics that the ceramide class of lipids, especially very long chain ceramides, are enriched on maturing phagosomes with a concomitant decrease in the biosynthetic precursors of ceramides. We thus posit a new function for the enzyme ceramide synthase during phagocytosis in mammalian macrophages. Biochemical assays, cellular lipid feeding experiments, and pharmacological blockade of ceramide synthase together show that this enzyme indeed controls the flux of ceramides on maturing phagosomes. We also find similar results in the primitive eukaryote Dictyostelium discoideum, suggesting that ceramide enrichment may be evolutionarily conserved and likely an indispensible step in phagosome maturation.

A guide to measuring phagosomal dynamics

“…It is important to stress that the use of mass spectrometry as a platform to examine phagosomes carries the potential to fill important gaps in the field. While the phagosomal proteome has been widely investigated, other phagosomal 'omics' such as the lipidome and glycome have been studied to a significantly lesser extent [57,58]. Because of the increasingly recognized contribution of lipids, carbohydrates and metabolites to phagosomal dynamics, defining changes in their molecular landscapes in phagosomes, will likely render important advances in the field, especially in the understanding of host-pathogen interactions [59].…”

C24:0 and C24:1 sphingolipids in cholesterol-containing, five- and six-component lipid membranes

“…Law et al 27 found that VLC Cer were toxic and induced mithophagy in cardiomyocytes. Studies on phagocytes 28 revealed that VLC Cer could be essential for phagosome maturation in immune cells. VLC sphingolipids were found to be crucial in the survival of invariant natural killer T cells in thymus and liver 29 .…”

SOD1 activity threshold and TOR signalling modulate VAP(P58S) aggregation via ROS-induced proteasomal degradation in aDrosophilamodel of Amyotrophic Lateral Sclerosis

“…To confirm whether feeding of paraquat and loss of SOD1 function led to an increase in ROS levels in the larval brain, we measured the levels of oxidized phospholipids, using quantitative mass spectrometry (MS)-based lipidomics (Kamat et al, 2015; Kory et al, 2017; Pathak et al, 2018; Tyurina et al, 2000). On feeding C155-GAL4/+ larvae with 5 mM paraquat, we enriched and detected nine oxidized polyunsaturated fatty acids (PUFAs), belonging to the phosphatidylserine (PS) and phosphatidylethanolamine (PE) (Fig.…”

“…All the phospholipid species analysed in this study were quantified using the multiple reaction monitoring (MRM) high-resolution scanning method on a Sciex X500R QTOF LC-MS with an Exion-LC series quaternary pump. All data were acquired and analysed using SciexOS software as described before (Pathak et al, 2018). LC separation was achieved using a Gemini 5U C-18 column (Phenomenex, 5 μm, 50×4.6 mm) coupled to a Gemini guard column (Phenomenex, 4×3 mm, Phenomenex security cartridge).…”