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

Abstract

Toxoplasma gondii (T. gondii) 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 via the P2X7 receptor contributes to T. gondii 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 T. gondii infection in vivo. However, the cascade of signaling events that leads to parasite elimination via P2X7 receptor activation remained to be elucidated. Here, we investigated the cellular pathways involved in T. gondii 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 T. gondii 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 T. gondii infection via 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 via 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 via mitochondrial ROS generation (as assessed using Mito-TEMPO). Together, our results support a model where P2X7 receptor activation by eATP inhibits T. gondii growth in macrophages by triggering NADPH-oxidase-dependent ROS production, and also by activating a canonical NLRP3 inflammasome, which increases IL-1β production (via caspase-1 activity), leading to mitochondrial ROS generation.

The Nrf2 pathway is required for intracellular replication ofToxoplasma gondiiin activated macrophages

“…Growing evidence suggests that accumulation of intracellular ROS activates many effector molecules or adaptor proteins in host cells infected with T gondii to eliminate the intracellular parasites . More recent studies provide direct evidence that ROS is toxic to T gondii . Antioxidative genes expressed by T gondii are important for overcoming oxidative stress; this also requires down‐regulation of ROS‐producing enzymes in host cells.…”

“…[30][31][32] More recent studies provide direct evidence that ROS is toxic to T gondii. 10,13 Antioxidative genes expressed by T gondii are important for overcoming oxidative stress; this also requires downregulation of ROS-producing enzymes in host cells. However, it is still not known whether the cytoprotective pathway contributes to parasite survival.…”

“…Previous studies in mice show that infection by T gondii increases ROS and NADPH oxidase levels . Higher levels of ROS increase inherent cellular oxidative stress, thereby contributing to resistance against invading T gondii . To overcome the effects of oxidative stress, T gondii has developed its own antioxidant machinery, which neutralizes excess ROS .…”

“…[5][6][7] Higher levels of ROS increase inherent cellular oxidative stress, thereby contributing to resistance against invading T gondii. [8][9][10][11] To overcome the effects of oxidative stress, T gondii has developed its own antioxidant machinery, which neutralizes excess ROS. 12-14 A previous report suggests that regulating ROS-producing pathways in T gondii -infected cells is required for intracellular redox homeostasis.…”

Treatment of mice with IL2-complex enhances inflammasome-driven IFN-γ production and prevents lethal toxoplasmosis

“…Toxoplasma gondii appears to activate both NLRP1 and NLRP3 [24], yet the specificity of this activation remains elusive. While the activation of NLRP3 in response to T. gondii appears to be influenced by P2X7 receptor-dependent potassium efflux and the induction of reactive oxygen species [47,[49][50][51], the exact mechanisms of how T. gondii activates multiple inflammasomes remain enigmatic. In this context it is also interesting to note that in vitro infection of mouse macrophages and human monocytes with T. gondii only leads to the secretion of IL-1β, but not IL-18 [24,52].…”