A Multiparametric and High-Throughput Assay to Quantify the Influence of Target Size on Phagocytosis

Abstract

Phagocytosis by macrophages represents a fundamental process essential for both immunity and tissue homeostasis. It consists in the uptake of pathogenic or cellular targets larger than 0.5 mm. For the biggest particles, the phagocytic process involves a massive reorganization of membrane and actin cytoskeleton as well as an important intracellular deformation all in a matter of minutes. The study of the role of the size of objects in their phagocytosis has led to contradictory results in the last decades. We designed a method using confocal microscopy, automated image analysis, and databases for fast quantitative analysis of phagocytosis assays. It yields comprehensive data on the cells and targets geometric and fluorescence intensity parameters, automatically discriminates internalized from external targets, and stores the relationship between a cell and the targets it has engulfed. We used two types of targets (solid polystyrene beads and liquid lipid droplets) to investigate the influence of size on the phagocytic uptake of macrophages. The method made it possible not only to perform phagocytic assays with functionalized droplets and beads of different sizes but to use polydisperse particles to further our understanding of the role of size in phagocytosis. The use of monodisperse and polydisperse objects shows that whereas smaller monodisperse objects are internalized in greater numbers, objects of different sizes presented simultaneously are internalized without preferred size. The total surface engulfed by the cell is thus the main factor limiting the uptake of particles, regardless of their nature or size. A meta-analysis of the literature reveals that this dependence in surface is consistently conserved throughout cell types, targets' nature, or activated receptors.

Antibody-Dependent Cellular Phagocytosis of HIV-1-Infected Cells Is Efficiently Triggered by IgA Targeting HIV-1 Envelope Subunit gp41

“…Indeed, in ADCC, one effector cell can contact several opsonized targets, each by one or the two Ab isotypes, in turn triggering their lysis. In contrast in ADCP, following these multiple contacts with the IgA and/or IgG-opsonized target cells, the effector in turn engulfs the opsonized target, which, for geometrical reason, cannot be more than one per effector cell (50). Accordingly, we could not observe the phagocytosis of CEM-NKR cells which size is much larger than the monocyte one ( Supplementary Figure 1).…”

Mannose-Coated Fluorescent Lipid Microparticles for Specific Cellular Targeting and Internalization via Glycoreceptor-Induced Phagocytosis

“…As can be seen on Figure A, mannolipid-coated droplets were internalized, however, fluorescence from the mannolipid can be seen in all macrophages regardless of the internalization of droplets. An average of 1.3 saturated droplets/cell were internalized, a number similar to the uptake of saturated IgG-coated 8 μm diameter droplets by the same cells . Unsaturated droplets were less internalized, with a phagocytic index of 0.3 droplet/cell, and the uptake of uncoated droplets was significantly lower than both coated droplet assays, under 0.05 droplet/cell, as summarized on Figure A.…”

“…The phagocytic uptake of mannolipid-coated 8 μm diameter oil droplets at two surface densities (0.1 and 1 surface equivalent) was assessed and compared to the uptake of uncoated droplets, using a method described previously . As can be seen on Figure A, mannolipid-coated droplets were internalized, however, fluorescence from the mannolipid can be seen in all macrophages regardless of the internalization of droplets.…”

“…An average of 1.3 saturated droplets/cell were internalized, a number similar to the uptake of saturated IgGcoated 8 μm diameter droplets by the same cells. 53 To assess whether soluble mannolipid can activate the macrophages and indirectly increase the phagocytic uptake of droplets, cells were exposed to soluble mannolipid (72 μM) for 10 minutes.…”

Phenotyping Polarization Dynamics Of Immune Cells Using A Lipid Droplet - Cell Pairing Microfluidic Platform

“…Accordingly, when the cell pulls on mushroom-like PEG, it might induce a conformational change and bring it into a brush-like conformation, which effectively increases PEG mobility. Of note, cells known to exert stronger forces, such as macrophages (15, 65), are able to form a cluster of antigens on both types of droplets (23) independently of PEG conformation, confirming that the antigen cluster is driven by cell forces. Further molecular dynamic simulations or FRAP experiments out of equilibrium are required to elucidate the physical mechanisms linking pulling forces and active antigen clustering.…”