The effects of macrophage polarization and granuloma microenvironment on the macrophage response to Mycobacterium tuberculosis

Macrophages display considerable functional and phenotypic plasticity because the cytokine and biochemical environment affects their differentiation and maturation. Macrophages are the primary niche for Mycobacterium tuberculosis inside granulomas. This thesis examines the different macrophage subsets M1-like, M2-like, Mox macrophages and lipid loaded foamy macrophages and how they interact with Mycobacterium tuberculosis complex infection. Human monocyte-derived macrophages were differentiated in the presence of either granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). By examining production of the pro-inflammatory cytokine TNF-α and the anti-inflammatory cytokine IL-10 in response to LPS it was established that these GM- and M-macrophages formed distinct subsets with characteristics similar to M1-like and M2-like macrophages. GM-macrophages and M-macrophages were also matured in the presence of the lipid OxPAPC; these macrophages termed "Mox" have been previously characterized in lipid rich murine atherosclerosis plaques that resemble lipid rich tuberculosis granuloma. The characterization of the generated macrophages subsets was examined and it was found in the presence of Mycobacterium bovis BCG infection, Mox M-macrophages became extremely elongated and spindle shaped. CD14 expression was significantly higher in Mycobacterium bovis BCG infected and uninfected M-macrophages and Mox M-macrophages compared to all GM-macrophages. There were trends towards lower expression of CD14 in M and Mox M macrophages after BCG infection. Also there was a trend towards higher expression of CD209 in M-macrophages and also reduced expression in cells infected with BCG. M-macrophages and Mox M-macrophages displayed significantly higher phagocytic abilities against latex beads and Mycobacterium bovis BCG. To examine lipid loaded foamy macrophages, U937 monocytes were used and differentiated into macrophages using phorbol 13-myristate 13-acetate (PMA). In vitro generated foamy macrophages with oleic acid-BSA were able to retain their lipid droplets up to 7 days. OA-BSA foamy macrophages were infected with Mycobacterium bovis BCG, Mycobacterium tuberculosis H37Rv and Mycobacterium tuberculosis GC1237 and it was observed that Mycobacterium tuberculosis GC1237 caused the highest level of host cell death. Both Mycobacterium tuberculosis H37Rv and Mycobacterium tuberculosis GC1237 induced more apoptosis in foamy macrophages compared to non-foamy macrophages. Mycobacterium bovis BCG infection of OA-BSA foamy cells decreased lipid content significantly after 48 hours infection. While looking at the ability of OA-BSA treated foamy macrophages to take up latex beads, dextran and Mycobacterium bovis BCG it was found that foamy macrophages had similar phagocytic capability to non-foamy cells but had a reduced ability to take up dextran. The induction of necrosis in the TB granuloma is an essential component for the pathogenesis and transmission of TB. While investigating the relative ability of macrophage subsets to undergo programmed necrosis in response to inflammatory stimuli, TNF-α or IFN-γ, it was found that programmed necrosis, necroptosis, could be induced in GM-macrophages and M-macrophages by TNF-α and to a lesser extent by IFN-γ. Necroptosis could be induced as well using TNF-α in OA-BSA treated and untreated macrophages. Necroptosis was more prominent in OA-BSA treated foamy macrophages compared to non-foamy macrophages. These data give and understanding and insight into the roles of macrophage polarization and lipid milieu in tuberculosis infection.