CD40 induces macrophage anti–Toxoplasma gondii activity by triggering autophagy-dependent fusion of pathogen-containing vacuoles and lysosomes
Rosa M. Andrade, … , Boris Striepen, Carlos S. Subauste
Rosa M. Andrade, … , Boris Striepen, Carlos S. Subauste
Published September 1, 2006
Citation Information: J Clin Invest. 2006;116(9):2366-2377. https://doi.org/10.1172/JCI28796.
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Research Article Infectious disease

CD40 induces macrophage anti–Toxoplasma gondii activity by triggering autophagy-dependent fusion of pathogen-containing vacuoles and lysosomes

Abstract

Many intracellular pathogens, including Toxoplasma gondii, survive within macrophages by residing in vacuoles that avoid fusion with lysosomes. It is important to determine whether cell-mediated immunity can trigger macrophage antimicrobial activity by rerouting these vacuoles to lysosomes. We report that CD40 stimulation of human and mouse macrophages infected with T. gondii resulted in fusion of parasitophorous vacuoles and late endosomes/lysosomes. Vacuole/lysosome fusion took place even when CD40 was ligated after the formation of parasitophorous vacuoles. Genetic and pharmacological approaches that impaired phosphoinositide-3-class 3 (PIK3C3), Rab7, vacuolar ATPase, and lysosomal enzymes revealed that vacuole/lysosome fusion mediated antimicrobial activity induced by CD40. Ligation of CD40 caused colocalization of parasitophorous vacuoles and LC3, a marker of autophagy, which is a process that controls lysosomal degradation. Vacuole/lysosome fusion and antimicrobial activity were shown to be dependent on autophagy. Thus, cell-mediated immunity through CD40 stimulation can reroute an intracellular pathogen to the lysosomal compartment, resulting in macrophage antimicrobial activity.

Authors

Rosa M. Andrade, Matthew Wessendarp, Marc-Jan Gubbels, Boris Striepen, Carlos S. Subauste

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Figure 1

CD40 stimulation induces vacuole/lysosome fusion in human and mouse macrophages infected withT. gondii .

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CD40 stimulation induces vacuole/lysosome fusion in h...
(A) Control and CD154-stimulated human macrophages were incubated with LysoTracker Red, then infected with T. gondii–YFP. Macrophages incubated with opsonized T. gondii were used as controls. Cells were examined by confocal microscopy 6 hours after infection or 2 hours after addition of opsonized parasites. Macrophages shown contain 1 tachyzoite of T. gondii. (B–D) Control and CD40-activated human macrophages were infected with T. gondii–YFP. Macrophages incubated with opsonized T. gondii were used as controls. Macrophages were incubated with anti–LAMP-1 (B), anti-CD63 (C), or anti-cathepsin D (D) Abs; this was followed by addition of secondary antibodies. Cells were examined by confocal microscopy at 6 hours after infection or 1 hour after addition of opsonized T. gondii. CD40-activated macrophages show colocalization of LAMP-1, CD63, and cathepsin D (rings) around T. gondii–containing vacuoles (arrowheads). Cath, cathepsin. Scale bars: 5 μm. (E and F) Quantification of colocalization of late endosomal/lysosomal markers around vacuoles containing T. gondii within human (E) or mouse (F) primary macrophages. Monolayers were examined at 6 and 8 hours after challenge in the case of human and mouse macrophages, respectively. In the case of macrophages incubated with opsonized T. gondii, M6PR was assessed at 15 minutes while LAMP-1, LAMP-2, CD63, and cathepsin D expression were assessed at 1 hour. Percentages indicate the mean ± SD. Results shown are representative of 3–4 independent experiments. Cath, cathepsin; ctr, control; DIC, differential interface contrast; ops, opsonized.