Development of ligand-coated beads to measure macrophage antimicrobial activities.

BACKGROUND INFORMATION:After macrophage recognises and phagocytoses the microorganism, their phagosome undergoes a maturation process, which creates a hostile environment for the bacterium. The lumen is acidified, and proteolysis occurs to kill and degrade pathogen for further antigen presentation. It is important to understand the association between the macrophage intracellular activities and the outcome of infection. Different methods have been developed to measure the phagosome dynamics of macrophages, but there are still limitations. RESULTS:We used Mycobacterium tuberculosis (Mtb) antigens, the causative agent of tuberculosis (TB), as a model of infectious disease. Adopting a fluorescent bead-based assay, we developed beads coated with trehalose 6,6'dimycolate (TDM) from Mtb cell wall and β-glucan from yeast cell wall to measure the macrophage phagosomal activities using a microplate reader. We examined the consistency of the assay using J774 cells and validated it using human monocyte-derived macrophages (hMDM) from healthy volunteers and TB patients. There was a decreased pH and increased proteolysis in the lumen of J774 cells after phagocytosing the ligand-coated beads. J774 macrophage showed no difference in the acidification and proteolysis in response to control IgG beads, TDM and β-glucan beads. hMDM from healthy volunteers or TB patients showed heterogeneity in the intracellular activities when treated with ligand-coated beads. CONCLUSIONS AND SIGNIFICANCE:The beads coated with specific ligands from Mtb worked well in both macrophage cell line and human primary macrophages, which can be exploited to further study the phagosomal function of macrophage in TB. Our bead model can be applied to different ligands from other pathogens, which could extend the understanding of the associations between macrophage antimicrobial functions and outcomes of infectious diseases and the possible cellular mechanisms involved.