Analyzing ARF6 and Non-Exosomal Extracellular Vesicles in the Context of Macrophages Infected with M. tuberculosis

Mycobacteria tuberculosis (M.tb), the causative agent of TB, although largely a curable disease, still remains a major cause of morbidity and mortality worldwide, resulting in approximately 1.5 million deaths per year. A better understanding of the host response to M.tb is needed to develop the next generation of therapeutics and vaccines. The objective of this project centers around two major aims: 1) characterizing the role of ARF6 in host signaling and phagosome-lysosome fusion in macrophages upon M.tb infection and 2) characterizing both composition and function of microvesicles after infection. Previous studies have shown that M.tb interferes with trafficking by preventing phagosome-lysosome fusion. ARF6, a small GDP binding protein, is involved in regulating various host-signaling pathways as well as functioning in promoting actin cytoskeleton rearrangements and membrane trafficking. Therefore, this project focuses on the biological processes, such as secretion, phagocytosis, and cytokinesis, previously shown in the literature, to be regulated by ARF6 and also necessary for a successful M.tb infection. In our present studies we have determined that ARF6 is activated in macrophages following an infection with complement opsonized M.tb and that there is an accumulation of ARF6 with the mycobacteria containing phagosomes. Interestingly, we showed a diminished activation of ERK in M.tb infected macrophages when cells expressed an ARF6 mutant defective in GTP hydrolysis (Q67L). However, this diminished activation of ERK did not result in measurable differences in NF-ƙB nuclear localization or production of various cytokines including TNF-α. Our data suggest that ARF6 is not playing a significant role in driving the pro-inflammatory responses induced in macrophages upon an M.tb infection. Previous work in the laboratory has shown that there are mycobacterial specific proteins present in exosomes released from M.tb-infected cells and that these exosomes have a multi-functional immunological role. Further, it has been shown across several infection models (i.e. tissue culture infections, mouse infections, and human M.tb patient) that several of these proteins are conserved, suggesting they could serve as biomarkers for disease. Exosomes have been well characterized in the context of mycobacterial infection; however, macrophages also release microvesicles, which are derived from the budding of the plasma membrane, and these vesicles have not been characterized in this manner. Our findings suggest that exosomes released from M.tb infected macrophages are more enriched in mycobacterial proteins than observed with microvesicles released from the infected cells. These studies also suggest that this exosome population produces a more robust stimulation of macrophages and pre-sensitized T-cells compared to microvesicles. Altogether, our data support a more functional role for exosomes then microvesicles in the pathobiology of an M.tb infection.