Cues for phagosome maturation deciphered using a tuneable particulate system

Abstract

Phagocytosis, an essential component of the innate immune response, is followed by the process of phagosome maturation inside the cell. Maturation ensures that the engulfed target is delivered to the acidic lysosome for digestion by hydrolytic enzymes, thereby enabling phagocytic immune cells to contain and neutralise any threats from the target. Immune signalling initiated by pathogens upon engaging various pattern recognition receptors on professional phagocytes like macrophages has been proposed to regulate maturation kinetics, but this has not been clearly established yet. Discrepancies exist, for instance, in the role of lipopolysaccharide (LPS) induced toll-like receptor 4 (TLR4) signalling in influencing phagosome maturation, largely arising from a limitation of experimental methods used to address this question. Here, we have employed confocal microscopy, bespoke automated image analysis methods, and sterile, fluorescent polystyrene particles amenable to various modifications as a model phagocytic target in macrophages to delineate intracellular cues that influence phagosome maturation. By modifying individual particle properties, we discern the role of individual factors in directing the process.

In experiments with RAW264.7 macrophages as well as primary murine macrophages, we find that 500 nm-sized sterile, non-modified polystyrene particles localise to the lysosome in a stochastic manner. Upon either treating cells with or conjugating to the particle surface a suite of different ligands, we discovered that ligands engaging specific pattern recognition receptors and scavenger receptors, viz. TLR4, TLR5, SR-A1, and FcγR enhance delivery rates to the lysosome, whereas no differences were observed with the folate ligand.

From a survey of literature, signals enhancing the lysosomal localisation of particles were found to converge at the stress-activated kinase p38 MAPK. Thus, we hypothesized that p38 MAPK activity enhances phagosome maturation and demonstrated that chemical inhibition or siRNA-mediated knockdown of the enzyme abrogates LPS-induced enhancement of lysosomal localisation of particles. LPS treatments were also found to enhance phagosomal acidification kinetics. We quantified this in a collaborative study by conjugating a novel self-ratiometric pH sensor to the particle surface to obtain pH measurements of the maturing phagosome using a live-imaging setup. We observe that within the first 10 minutes of uptake, phagosomes in LPS-activated cells are more acidic compared to control, and inhibition of p38 MAPK activity in these cells changes acidification kinetics back to control levels.

Additionally, we tested the effect of surface charge and particle size upon phagosome maturation. While the former does not affect the extent of lysosomal delivery, we find that size plays an important role - larger particles (3 µm diameter) show enhanced lysosomal accumulation and lower pH in the associated phagosomal compartment compared to the uptake of sub-micron-sized particles. Inhibition of p38 MAPK activity in cells that take up larger particles reduces phagosomal LAMP1 accumulation, further indicating that p38 MAPK influences phagosome maturation.

Together, these data establish a role for LPS-induced signalling and subsequent p38 MAPK activation in regulating phagosome maturation. Real-time pH measurements indicate that differences in acidification under LPS-signalling are triggered early in the maturation process. The size of the particles is also a determinant, wherein larger particles show enhanced delivery to the lysosomes. Such systematic studies bring us closer to understanding the molecular decision-making process ensuing inside a phagocytic cell to govern phagosome maturation and hold significance for the rational design of particle-based systems for drug and vaccine delivery.