Abstract
The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.
Highlights
As a first line of defense against pathogens, the innate immune system relies on phagocytic cells that recognize and internalize foreign particulates
Inhibition of sphingolipid biosynthesis reduces phagocytosis of C. albicans by macrophages and dendritic cells
Since the turnover of sphingolipids, especially that of sphingomyelin, is slow [33], cells were grown in the continuous presence of myriocin or fumonisin B1 (FB1) for 4 days to obtain a significant reduction in sphingolipid levels
Summary
As a first line of defense against pathogens, the innate immune system relies on phagocytic cells that recognize and internalize foreign particulates. Phagocytosis of the fungal pathogen Candida albicans involves extensive membrane reorganization and actin remodeling at the plasma membrane for successful formation of a phagocytic cup [1,2,3,4]. The extent to which membrane lipids contribute to the proper operation of innate immune receptors remains largely unknown. Formation of the phagocytic cup involves receptor clustering and cytoskeletal rearrangements at the site where the particle is initially bound. This step is highly coordinated and relies on modulation of phosphoinositide metabolism [9, 11]
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