Effects of cytokines on mycobacterial phagosome maturation

Abstract

One of the major mechanisms permitting intracellular pathogens to parasitize macrophages is their ability to alter maturation of the phagosome or affect its physical integrity. These processes are opposed by the host innate and adaptive immune defenses, and in many instances mononuclear phagocytes can be stimulated with appropriate cytokines to restrict the growth of the microorganisms within the phagosomal compartment. Very little is known about the effects that cytokines have on phagosome maturation. Here we have used green fluorescent protein (GFP)-labeled mycobacteria and a fixable acidotropic probe, LysoTracker Red DND-99, to monitor maturation of the mycobacterial phagosome. The macrophage compartments that stained with the LysoTracker probe were examined first. This dye was found to colocalize preferentially with the late endosomal and lysosomal markers rab7 and Lamp1, and with a fluid phase marker chased into the late endosomal compartments. In contrast, LysoTracker showed only a minor overlap with the early endosomal marker rab5. Pathogenic mycobacteria are believed to reside in nonacidified vacuoles sequestered away from late endosomal compartments as a part of their intracellular survival strategy. We examined the status of mycobacterial phagosomes in macrophages from IL-10 knockout mice, in quiescent cells, and in mononuclear phagocytes stimulated with the macrophage-activating cytokine IFN-(gamma). When macrophages were derived from the bone marrow of transgenic IL-10 mice lacking this major deactivating cytokine, colocalization of GFP-fluorescing mycobacteria with the LysoTracker staining appeared enhanced, suggestive of increased acidification of the mycobacterial phagosome relative to macrophages from normal mice. When bone marrow-derived macrophages from normal mice or a J774 murine macrophage cell line were stimulated with IFN-(gamma) and LPS, this resulted in increased colocalization of mycobacteria and LysoTracker, but no statistically significant enhancement was observed in IL-10 transgenic animals. These studies are consistent with the interpretation that proinflammatory and anti-inflammatory cytokines affect maturation of mycobacterial phagosomes. Although multiple mechanisms are likely to be at work, we propose the existence of a direct link between cytokine effects on the host cell and phagosome maturation in the macrophage.

Interleukin-27 impairs BCG antigen clearance and T cell stimulatory potential by neonatal dendritic cells

“…To further address the relevance of these findings in a whole animal model, we vaccinated neonatal WT or KO pups with BCG. Although BCG is an attenuated strain of Mycobacterium bovis and does not cause disease in immune-competent children, BCG is capable of similar phagolysosomal resistance as MTB ( Via et al., 1998 ). Persistence of vaccine antigen has been shown to drive effector responses ( Orme 2010 ).…”

TcrXY is an acid-sensing two-component transcriptional regulator of Mycobacterium tuberculosis and a novel target for enhanced TB therapy

“…1b). Maximum activity was observed at pH 5.4, the pH corresponding to matured phagosomes of activated macrophages 21,22 . Reporter activity was diminished in highly acidic culture media (pH < 5), albeit remained above baseline.…”

Human Tuberculosis I. Epidemiology, Diagnosis and Pathogenetic Mechanisms

“…Studies have shown that immune-activation of murine bone marrow derived macrophages with IFN-γ and/or LPS releases the arrest of phagosome maturation by mycobacteria [167,168]. TDM coated beads are capable of slowing down phagosome maturation, thus suggesting that TDM is contributing to mycobacterial survival in bone marrow-derived macrophages; TDM effect is abolished in immune-activated (by IFN-γ and/or LPS) wild-type, but not in activated nitric oxide synthase deficient macrophages.…”