The P2X7 Receptor Mediates Toxoplasma gondii Control in Macrophages through Canonical NLRP3 Inflammasome Activation and Reactive Oxygen Species Production

Abstract

<i>Toxoplasma gondii (T. gondii)</i> is the protozoan parasite that causes toxoplasmosis, a potentially fatal disease to immunocompromised patients, and which affects approximately 30% of the world's population. Previously, we showed that purinergic signaling <i>via</i> the P2X7 receptor contributes to <i>T. gondii</i> elimination in macrophages, through reactive oxygen species (ROS) production and lysosome fusion with the parasitophorous vacuole. Moreover, we demonstrated that P2X7 receptor activation promotes the production of anti-parasitic pro-inflammatory cytokines during early <i>T. gondii</i> infection <i>in vivo</i>. However, the cascade of signaling events that leads to parasite elimination <i>via</i> P2X7 receptor activation remained to be elucidated. Here, we investigated the cellular pathways involved in <i>T. gondii</i> elimination triggered by P2X7 receptor signaling, during early infection in macrophages. We focused on the potential role of the inflammasome, a protein complex that can be co-activated by the P2X7 receptor, and which is involved in the host immune defense against <i>T. gondii</i> infection. Using peritoneal and bone marrow-derived macrophages from knockout mice deficient for inflammasome components (NLRP3^-/-, Caspase-1/11^-/-, Caspase-11^-/-), we show that the control of <i>T. gondii</i> infection <i>via</i> P2X7 receptor activation by extracellular ATP (eATP) depends on the canonical inflammasome effector caspase-1, but not on caspase-11 (a non-canonical inflammasome effector). Parasite elimination <i>via</i> P2X7 receptor and inflammasome activation was also dependent on ROS generation and pannexin-1 channel. Treatment with eATP increased IL-1β secretion from infected macrophages, and this effect was dependent on the canonical NLRP3 inflammasome. Finally, treatment with recombinant IL-1β promoted parasite elimination <i>via</i> mitochondrial ROS generation (as assessed using Mito-TEMPO). Together, our results support a model where P2X7 receptor activation by eATP inhibits <i>T. gondii</i> growth in macrophages by triggering NADPH-oxidase-dependent ROS production, and also by activating a canonical NLRP3 inflammasome, which increases IL-1β production (<i>via</i> caspase-1 activity), leading to mitochondrial ROS generation.