Mapping Sphingolipid Metabolism Pathways during Phagosomal Maturation

Abstract

Phagocytosis is an important physiological process, which, in higher organisms, is a means of fighting infections and clearing cellular debris. During phagocytosis, detrimental foreign particles (e.g. pathogens and apoptotic cells) are engulfed by phagocytes (e.g. macrophages), enclosed in membrane-bound vesicles called phagosomes, and transported to the lysosome for eventual detoxification. During this well-choreographed process, the nascent phagosome (also called early phagosome, EP) undergoes a series of spatiotemporally regulated changes in its protein and lipid composition and matures into a late phagosome (LP), which subsequently fuses with the lysosomal membrane to form the phagolysosome. While several elegant proteomic studies have identified the role of unique proteins during phagosomal maturation, the corresponding lipidomic studies are sparse. Recently, we reported a comparative lipidomic analysis between EPs and LPs and showed that ceramides are enriched on the LPs. Further, we found that this ceramide accumulation on LPs was orchestrated by ceramide synthase 2, inhibition of which hampers phagosomal maturation. Following up on this study, here, using biochemical assays, we first show that the increased ceramidase activity on EPs also significantly contributes to the accumulation of ceramides on LPs. Next, leveraging lipidomics, we show that de novo ceramide synthesis does not significantly contribute to the ceramide accumulation on LPs, while concomitant to increased ceramides, glucosylceramides are substantially elevated on LPs. We validate this interesting finding using biochemical assays and show that LPs indeed have heightened glucosylceramide synthase activity. Taken together, our studies provide interesting insights and possible new roles of sphingolipid metabolism during phagosomal maturation.

Sphingoid Bases Regulate the Sigma-1 Receptor—Sphingosine and N,N’-Dimethylsphingosine Are Endogenous Agonists

“…It is generally accepted, for example, that ceramidases play significant roles in regulating ceramide and sphingosine concentrations in cellular membranes [ 90 , 91 ]. Ceramides contribute mainly, but not exclusively, to the bilayers of MAM, ER, and Golgi [ 92 ]; for example, even in late phagosomes, glucosylceramides formed by glucosylceramide synthase (GCase) and UDP glucose are elevated for ceramide processing [ 93 ]. A generic signature binding sequence, VAMTLGQIYY, for sphingomyelin [ 94 , 95 , 96 ] (structure in Figure 2 B), has been identified within the TM helix of the Golgi coat protein, COPI p24 [ 97 ].…”

Nanosecond pulsed electric field ablation‐induced modulation of sphingolipid metabolism is associated with Ly6c2+ mononuclear phagocyte differentiation in liver cancer

“…The phagocytic and antigen‐presenting functions of MNPs are known to rely on sphingolipid metabolism [ 44 , 45 ]. The accumulation of ceramide in the phagosomes of MNPs has been reported to promote phagosomal maturation [ 46 ]. Indeed, a large body of studies have shown that sphingolipid metabolism could drive the migration, activation, and polarization of monocytes/macrophages.…”

Functional roles of sphingolipids in immunity and their implication in disease

“…Analysis of phagocytosis with quantitative lipidomics during the process of phagosomal maturation revealed that early phagosomes have enhanced activity of neutral ceramidase compared to CerS under neutral (pH 7.0) conditions, resulting in decreased ceramide levels. However, ceramides and glucosylceramides with long hydrocarbon chains (16:0, 18:0, 20:0, 22:0, and 24:0 but not 12:0 and 14:0) are gradually enriched on mature phagosomes of Raw264.7 cells, a murine macrophage cell line, with enhanced glucosylceramide synthase (GCS) activity, while sphingosines and the expression/activity of CerS2 are decreased 96 . During phagosomal maturation, the levels of sphingomyelin and C1P are comparable in early and late phagosomes, but activation of CERK and C1P can facilitate Fc receptor-mediated phagocytosis, collectively implying the delicate roles of sphingolipids in macrophage function 82 , 96 .…”

“…However, ceramides and glucosylceramides with long hydrocarbon chains (16:0, 18:0, 20:0, 22:0, and 24:0 but not 12:0 and 14:0) are gradually enriched on mature phagosomes of Raw264.7 cells, a murine macrophage cell line, with enhanced glucosylceramide synthase (GCS) activity, while sphingosines and the expression/activity of CerS2 are decreased 96 . During phagosomal maturation, the levels of sphingomyelin and C1P are comparable in early and late phagosomes, but activation of CERK and C1P can facilitate Fc receptor-mediated phagocytosis, collectively implying the delicate roles of sphingolipids in macrophage function 82 , 96 . Although some bacteria and hosts have evolved to live together by symbiosis, other bacteria called pathogens have evolved to exploit and hijack host metabolites for their own purposes 53 , 62 .…”

“…Even worse, several kinds of toxins produced by pathogens (e.g., the α-toxin produced by S. aureus ) can bind to surface metalloproteinases (e.g., ADAM10) of the host and induce the release of acid ceramidase-mediated ceramide outside of the inflamed walls, which eventually leads to degradation of tight junctions 99 . Moreover, the produced chemokines and S1P recruit innate/adaptive immune cells, and recruited macrophages can wisely utilize the SMase/ceramidase strategy of the pathogen to rearrange its cytoskeleton, facilitating the process of phagocytosis 96 , 103 .…”