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.
Highlights
I n 1884, the phagocyte, a type of immune cell, was discovered and found to engulf, digest, and clear bacterial particles.[1]
We show referred to as the lipidome of early phagosome (EP) and late phagosome (LP), we first extracted by biochemical assays, fatty acid feeding, and cellular phar- lipids from EPs and LPs and established a LC-MS/MS based macological studies that the enzyme ceramide synthase method to exhaustively analyze >400 unique lipid species from controls the flux of ceramides during phagosomal maturation 22 lipid classes in tandem
Our findings taken together provide compelling evidence that mature phagosomes (LPs) are enriched with ceramides, especially very long chain (VLC) fatty acid containing ceramides, which are biosynthesized from sphingosine and fatty acyl-CoA by the action of CerS2, a major ceramide synthase, in mammalian macrophages (Figures 1, 2)
Summary
I n 1884, the phagocyte, a type of immune cell, was discovered and found to engulf, digest, and clear bacterial particles.[1]. This universally conserved process has been extensively studied and shown to be a critical component of both the innate and adaptive mammalian immune system.[2] Once internalized, these particles acquire membranes from the cellular plasma membrane and undergo a choreographed sequence of events broadly referred to as “phagosomal maturation,” culminating in fusion of the phagosomes with lysosomes. Over the past two decades, several elegant proteomics studies have shown the role of specific proteins in the maturation process, and these in turn have served as unique markers for the different stages in the phagosomal maturation process.[2] For example, the GTPase Rab[5] and early endosome antigen 1 (EEA1) localize to the EP and drive the EP to mature into a LP. The GTPase Rab[7] and lysosomal associated membrane protein 1 (LAMP1) are found enriched on LPs and are critical for the formation of the phago-lysosome. We find that cholesterol is enriched on LPs, where it forms membrane lipid raft domains, and deregulated cholesterol metabolism impairs phagosomal maturation.[9−11] Recently, we have shown that the motor protein dynein is enriched and clustered on these cholesterol rich lipid microdomains
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