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 7

Blockade of vacuole/lysosome fusion ablates CD40-induced antimicrobial activity.

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Blockade of vacuole/lysosome fusion ablates CD40-induced antimicrobial a...
The effects of knockdown of PIK3C3 (A–C) and Rab7 dominant-negative mutant (D–F) on vacuole/lysosome fusion and antimicrobial activity were examined. (A) hmCD40–RAW 264.7 cells were mock transfected (M) or were transfected with sense (S) or antisense (AS) ODN against PIK3C3. Protein expression of PIK3C3 and actin were analyzed 48 hours after transfection. (B) Transfected cells were incubated with or without hCD154, then infected with T. gondii–YFP. Vacuole/lysosomal fusion was assessed by cathepsin D staining 8 hours after infection. (C) Transfected cells were treated with medium alone, hCD154, or IFN-γ/LPS; this was followed by T. gondii infection. Cells were examined by light microscopy 18 hours after infection. (D) CD40-activated and control mouse peritoneal macrophages were infected with T. gondii–YFP followed by assessment of Rab7 expression by immunofluorescence. Arrowhead denotes colocalization of Rab7 (ring) around T. gondii–containing vacuole in CD40-activated macrophage. Scale bar: 5 μm. (E) hmCD40–RAW 264.7 cells were incubated with medium with or without hCD154 followed by transfection with Rab7 WT or Rab7(T22N). Cells were infected with T. gondii–RFP. Vacuole/lysosomal fusion was assessed by cathepsin D staining 8 hours after infection. (F) Transfected hmCD40–RAW 264.7 cells treated with medium alone, CD154, or IFN-γ/LPS were challenged with T. gondii. Cells were examined by light microscopy 18 hours after challenge. Results are shown as the mean ± SD and are representative of 4 independent experiments. DN, dominant negative.