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Scavenger receptor MARCO contributes to cellular internalization of exosomes by dynamin-dependent endocytosis and macropinocytosis

ABSTRACT: Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor class-A protein that is expressed on the cell surface of macrophages. MARCO mediates binding and ingestion of unopsonized environmental particles, including nano-sized materials. Exosomes are cell-derived, nano-sized vesicles (40–150 nm) that can contain lipids, RNA, DNA, and various proteins. Exosomes play an essential role in cell-to-cell communication via body fluids. However, mechanisms for the recognition and internalization of exosomes by recipient cells remain poorly characterized. In this study, cellular association of serum-derived fluorescent exosomes and 20-nm fluorescent nanoparticles (positive control) was compared between MARCO-expressing (CHO-MARCO) and control (CHO-CT) CHO-K1 cells to examine whether MARCO expression by recipient cells mediates the cellular uptake of exosomes and environmental nanoparticles. Fluorescence microscopic studies and quantitative analyses revealed that the cellular associations of both exosomes and 20-nm nanoparticles were greater in CHO-MARCO cells than in CHO-CT cells. Exosomes and nanoparticles colocalized with green fluorescent protein (GFP)-MARCO in cells transfected with GFP-MARCO-encoding constructs . Furthermore, inhibitory studies showed that actin reorganization and dynamin are involved in the MARCO-mediated cellular internalization of exosomes. These results indicated that MARCO plays a role in the uptake of exosomes.

SUBMITTER: Kanno S

PROVIDER: S-EPMC7733512 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes serious diarrhea in suckling piglets and has the potential for cross-species transmission. Although extensive studies have been reported on the biology and pathogenesis of PDCoV, the mechanisms by which PDCoV enters cells are not well characterized. In this study, we investigated how PDCoV enters IPI-2I cells, a line of porcine intestinal epithelial cells derived from pig ileum. Immunofluorescence assays, small interfering RNA (siRNA) interference, specific pharmacological inhibitors, and dominant negative mutation results revealed that PDCoV entry into IPI-2I cells depended on clathrin, dynamin, and a low-pH environment but was independent of caveolae. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) and the Na+/H+ exchanger (NHE) revealed that PDCoV entry involves macropinocytosis and depends on NHE rather than on PI3K. Additionally, Rab5 and Rab7, but not Rab11, regulated PDCoV endocytosis. This is the first study to demonstrate that PDCoV uses clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter porcine intestinal epithelial cells. We also discussed the entry pathways of PDCoV into other porcine cell lines. Our findings reveal the entry mechanisms of PDCoV and provide new insight into the PDCoV life cycle. IMPORTANCE An emerging enteropathogenic coronavirus, PDCoV, has the potential for cross-species transmission, attracting extensive attenuation. Characterizing the detailed process of PDCoV entry into cells will deepen our understanding of the viral infection and pathogenesis and provide clues for therapeutic intervention against PDCoV. With the objective, we used complementary approaches to dissect the process in PDCoV-infected IPI-2I cells, a line of more physiologically relevant intestinal epithelial cells to PDCoV infection in vivo. Here, we demonstrate that PDCoV enters IPI-2I cells via macropinocytosis, which does not require a specific receptor, and clathrin-mediated endocytosis, which requires a low-pH environment and dynamin, while a caveola-mediated endocytic pathway is used by PDCoV to enter swine testicular (ST) cells and porcine kidney (LLC-PK1) cells. These findings provide a molecular detail of the cellular entry pathways of PDCoV and may direct us toward novel antiviral drug development.

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Scavenger receptor MARCO contributes to cellular internalization of exosomes by dynamin-dependent endocytosis and macropinocytosis

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