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.

CD40, autophagy and Toxoplasma gondii

“…Therefore, CD154 + T. gondii-reactive activated CD4 + T cells likely trigger CD40 signaling in macrophages and resident microglia. CD40 stimulation of macrophages allows them to acquire toxoplasmacidal activity (Andrade et al 2003(Andrade et al , 2005a(Andrade et al , b, 2006. This effect is not only mediated by recombinant CD154 or an agonistic anti-CD40 mAb but also by CD154 expressed on the membrane of activated CD4 + T cells (Andrade et al 2005a).…”

“…CD40 causes fusion of parasite-containing vacuoles with late endosomes-lysosomes (Andrade et al 2006, Subauste et al 2007a. A critical question was whether parasitophorous vacuoles, which by definition have been considered non-fusogeneic, fused with late endosomeslysosomes.…”

“…The formation of parasitophorous vacuoles involves secretion of contents of parasite organelles located in the apex of the organism (Archbarou et al 1991, Cesbron-Delauw 1994, Carruthers & Sibley 1997. The use of transgenic parasites that express a fluorescent protein targeted to the dense granules allowed to demonstrate that vacuoles where contents of the dense granules are secreted into the vacuolar lumen fuse with late endosomes-lysosomes (Andrade et al 2006). Moreover, preformed parasitophorous vacuoles still fuse with late endosomes-lysosomes even if CD40 is engaged 18 h after infection (Andrade et al 2006).…”

“…The use of transgenic parasites that express a fluorescent protein targeted to the dense granules allowed to demonstrate that vacuoles where contents of the dense granules are secreted into the vacuolar lumen fuse with late endosomes-lysosomes (Andrade et al 2006). Moreover, preformed parasitophorous vacuoles still fuse with late endosomes-lysosomes even if CD40 is engaged 18 h after infection (Andrade et al 2006). These studies demonstrated that the immune system through CD40 alters a fundamental aspect of the biology of T. gondii: avoidance of lysosomal degradation.…”

“…This question is not only relevant to T. gondii but also to the large number of pathogens that survive by avoiding phago-lysosomal fusion. We recently demonstrated that this is achieved through CD40 stimulation (Andrade et al 2006, Subauste et al 2007a …”