Particulate β-glucan activates early and delayed phagosomal maturation and autophagy within macrophage in a NOX-2 dependent manner.

Aims: Macrophage is known to readily engulf any particulate material they encounter, including invading microbes and nano- or micro-particles. While recent studies show that some microparticles (MP) are immunogenic even without drug-cargo, the mechanism underlying this phenomenon is yet unclear. Phagocytosis induces NADPH oxidase-2 (NOX-2) mediated ROS generation that is reported to regulate antibacterial autophagy. We therefore, investigated the role of NOX-2 derived ROS in phagosomal maturation and autophagy induction in response to phagocytic uptake of two kinds of polymeric biodegradable and biocompatible microparticles: yeast-derived β-glucan particles (YDGP) and poly-(D, L-Lactic Acid) microparticles (PMP).

Main Methods: J774A.1 macrophage wereas exposed to polymeric particles and the immune responses: ROS, phagosomal maturation and autophagy induction, were examined by assays including NBT, DCFH-DA, NADPH-Oxidase activity, Lysotracker and Acridine Orange. Further, the LC3 and NOX-2 expression were validated by RT-PCR, immunofluorescence assay and Western blotting. Antimicrobial activity of both MP was examined by CFU counting after administration to Mycobacterium tuberculosis and Salmonella typhimurium infected macrophage.

Key Findings: YDGP induces phagosomal maturation and acidic vesicle accumulation at 30 min and 24 h post-exposure, much more proficiently than that by PMP. YDGP exposure also induced NOX-2 dependent expression of light chain 3 (LC3-II), further confirmed as autophagy activation via autophagic flux assay with autophagolysosome inhibitor bafilomycin A1. Additionally, YDGP displayed superior anti-microbial activity than that by PMP.

Significance: The induction of NOX-2-dependent autophagy and antimicrobial activity exhibited by particulate glucans has significant implications in harnessing these drug delivery vehicles as potential 'value-added' autophagy-mediated therapeutics in future.