Disentangling phagocytosis complexity using chemically multiresponsive and deformable particles – PhagoChemiForce

Phagocytosis is a mechanism of internalization and digestion of objects larger than 0.5 microns that relies on receptor triggering leading to actin polymerization and membrane deformation. Partners 1 and 2 have contributed to describe these mechanisms. How multiple receptors simultaneously recognize microbes, pathogens or debris both through direct binding and opsonization, leading to a complex interplay between the signaling pathways and a fine tuning of the fate of the internalised material, is still not well understood.

In particular, phagocytosis by C-type lectin receptors that bind carbohydrates residues on the surface of various microorganisms has been overlooked so far. Mannose receptors for instance importantly bind glycoconjugates with terminal mannose, fucose and N-Acetylglucosamine present in bacterial and yeast walls. Partners 2 and 3 developed functionalized lipid droplets coated with tailor-made fluorescent mannolipids to study C type lectin receptors-induced phagocytosis. The complement receptor 3 (CR3) is an integrin that binds microorganisms directly or a complement opsonized-target indirectly. Partner 1 has contributed to dissect signaling associated with CR3. The integrin CR3 was reported to cooperate with Immunoglobulins receptors to ensure efficient phagocytosis, but how different receptors cooperate with each other during signaling, force generation, phagosome formation and maturation, remains to be investigated.

This proposal aims at untangling critical steps of phagocytosis taking advantage of a multi-disciplinary approach and unique deformable emulsion droplets coated with fluorescent receptor-targeted ligands as targets for phagocytosis by human macrophages to :
- determine by FRET receptors binding and clustering during phagocytosis
- monitor directly the forces generated by the phagocyte and identify important regulators of force generation
analyse the fate of the internalized material and phagosome maturation upon various receptor engagement, taking advantage of novel functional fluorescent probes.

To this end, the complementary expertise of three groups, who have separately made important contributions in their fields and have already collaborated, will be brought together.
They will take advantage of a new class of materials, oil-in-water emulsion droplets, developed by Partner 2, which are deformable particles that can be functionalized with biological ligands freely-diffusing at the interface. The interaction and clustering of different receptors in the contact zone between the phagocytic cell and the droplet will be investigated with high spatial resolution using FRET between fluorescent carbohydrate ligands prepared by Partner 3.
The deformable droplets are unique tools to directly measure mechanical stresses. The intimate relations between MR and CR3 will be analyzed and the role of potential regulators of the CR3 previously identified by Partner 1 will be tested both on receptor clustering and force generation.
To study how receptors influence the fate of the internalized material during phagocytosis, we will combine new probes developed by Partner 3 in various colors allowing multiplexing with the lipid particles.
The phagocytosis assays will be performed in primary human macrophages by Partner 1.

With this project, we will extend the toolkit to address unsolved questions on phagocytic receptors dynamics in relevant phagocytic cells. We will be able to monitor the underlying mechanobiology of the phagocytosis process, with a novel set of combined expertise and techniques: ligand design, particle formulation, mechanical measurements, time-lapse microscopy and force-dependent integrin partners.
Importantly, the receptors of interest play a crucial role in clearance of pathogens as well as neuron pruning, and the phagocytic properties of macrophages can be hijacked in some pathological conditions, which increases the relevance of a better understanding of phagocytosis.