Abstract
Alveolar macrophages (AMs) can phagocytose unopsonized pathogens such as S. aureus via innate immune receptors, such as scavenger receptors (SRs). Cytoskeletal events and signaling pathways involved in phagocytosis of unopsonized bacteria likely govern the fate of ingested pathogens, but are poorly characterized. We have developed a high-throughput scanning cytometry-based assay to quantify phagocytosis of S. aureus by adherent human blood-derived AM-like macrophages in a 96-well microplate format. Differential fluorescent labeling of internalized vs. bound bacteria or beads allowed automated image analysis of collapsed confocal stack images acquired by scanning cytometry, and quantification of total particles bound and percent of particles internalized. We compared the effects of the classic SR blocker polyinosinic acid, the cytoskeletal inhibitors cytochalasin D and nocodazole, and the signaling inhibitors staurosporine, Gö 6976, JNK Inhibitor I and KN-93, on phagocytosis of a panel of live unopsonized S. aureus strains, (Wood, Seattle 1945 (ATCC 25923), and RN6390), as well as a commercial killed Wood strain, heat-killed Wood strain and latex beads. Our results revealed failure of the SR inhibitor polyinosinic acid to block binding of any live S. aureus strains, suggesting that SR-mediated uptake of a commercial killed fluorescent bacterial particle does not accurately model interaction with viable bacteria. We also observed heterogeneity in the effects of cytoskeletal and signaling inhibitors on internalization of different S. aureus strains. The data suggest that uptake of unopsonized live S. aureus by human macrophages is not mediated by SRs, and that the cellular mechanical and signaling processes that mediate S. aureus phagocytosis vary. The findings also demonstrate the potential utility of high-throughput scanning cytometry techniques to study phagocytosis of S. aureus and other organisms in greater detail.
Highlights
Staphylococcus aureus is an increasingly frequent and dangerous cause of both community and hospital-acquired pneumonia [1]
We have developed and demonstrated the utility of a high throughput assays for quantifying phagocytosis of fluorescent or non-fluorescent bacteria by adherent macrophages in a 96-well microplate format
Consistent with earlier studies, our assay showed that the uptake of IgG-opsonized (FccRmediated) uptake of S. aureus RN6390 was blocked by inhibition of cytochalasin D, but not by nocodazole or staurosporine
Summary
Staphylococcus aureus is an increasingly frequent and dangerous cause of both community and hospital-acquired pneumonia [1]. SRs are a diverse group of receptors with broad and overlapping specificities that mediate binding and internalization of many polyanionic ligands, including bacteria and their cell wall components, oxidized low density lipoproteins, environmental dusts, apoptotic cells, and CpG DNA. SR-A deficient mice have diminished clearance of S. aureus and survival following IP infection, and macrophages from SR-A deficient mice exhibit diminished phagocytosis of S. aureus [5] Another class A SR, MARCO, binds E. coli and S. aureus, and this binding is blocked by anti-MARCO antibody, as well as by the general SR binding inhibitors polyinosinic acid and polyguanylic acid [2,8,12]. Other SRs that have been implicated as potential mediators of unopsonized pathogen phagocytosis by macrophages include the class A scavenger receptor with C-type lectin (SRCL), known as collectin placenta-1 (CL-P1) [13,14,15], the class E lectin-like oxidized low-density lipoprotein receptor (LOX-1) [16,17,18,19], and the class G scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX), known as CXC chemokine ligand 16 (CXCL16) [20,21]
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