Toxoplasma 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 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-/-, Caspase-11-/- and Caspase-1/11-/-), we show that the control of T. gondii infection via P2X7 receptor activation by 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 NADP 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.

Leishmaniasis is a neglected tropical disease affecting millions of individuals worldwide. P2X7 receptor has been linked to the elimination of Leishmania amazonensis. Biological responses evoked by P2X7 receptor activation have been well-documented, including apoptosis, phagocytosis, cytokine release, such as IL-1β. It was demonstrated that NLRP3 inflammasome activation and IL-1β signaling participated in resistance against L. amazonensis. Furthermore, our group has shown that L. amazonensis elimination through P2X7 receptor activation depended on leukotriene B₄ (LTB₄) production and release. Therefore, we investigated whether L. amazonensis elimination by P2X7 receptor and LTB₄ involved NLRP3 inflammasome activation and IL-1β signaling. We showed that macrophages from NLRP3^-/-, ASC^-/-, Casp-1/11^-/-, gp91^{phox-/- ,} and IL-1R^-/- mice treated with ATP or LTB₄ did not decrease parasitic load as was observed in WT mice. When ASC^-/- macrophages were treated with exogenous IL-1β, parasite killing was noted, however, we did not see parasitic load reduction in IL-1R^-/- macrophages. Similarly, macrophages from P2X7 receptor-deficient mice treated with IL-1β also showed decreased parasitic load. In addition, when we infected Casp-11^-/- macrophages, neither ATP nor LTB₄ were able to reduce parasitic load, and Casp-11^-/- mice were more susceptible to L. amazonensis infection than were WT mice. Furthermore, P2X7^-/- L. amazonensis-infected mice locally treated with exogenous LTB₄ showed resistance to infection, characterized by lower parasite load and smaller lesions compared to untreated P2X7^-/- mice. A similar observation was noted when infected P2X7^-/- mice were treated with IL-1β, i.e., lower parasite load and smaller lesions compared to P2X7^-/- mice. These data suggested that L. amazonensis elimination mediated by P2X7 receptor and LTB₄ was dependent on non-canonical NLRP3 inflammasome activation, ROS production, and IL-1β signaling. Leishmania spp. is a protozoan parasite that infects human and causes several diseases. Leishmania amazonensis causes cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL). Leishmania parasites preferentially infect macrophages. In macrophages, several mechanisms have been described as controlling L. amazonensis infection. Here, we showed that P2X7 receptor and LTB₄ eliminated L. amazonensis in macrophages by a pathway dependent on non-canonical NLRP3 inflammasome activation and IL-1β signaling.

Esophageal cancer is an aggressive tumor and is the sixth leading cause of cancer death worldwide. ATP is well known to regulate cancer progression in a variety of models by different mechanisms, including P2X7R activation. This study aimed to evaluate the role of P2X7R in esophageal squamous cell carcinoma (ESCC) proliferation. Our results show that treatment with high ATP concentrations induced a decrease in cell number, cell viability, number of polyclonal colonies, and reduced migration of ESCC. The treatment with the selective P2X7R antagonist A740003 or siRNA for P2X7 reverted this effect in the KYSE450 cell line. In addition, results showed that P2X7R is highly expressed, at mRNA and protein levels, in KYSE450 lineage. Additionally, KYSE450, KYSE30, and OE21 cells express P2X3R, P2X4R, P2X5R, P2X6R, and P2X7R genes. P2X1R is expressed by KYSE30 and KYSE450, and only KYSE450 expresses the P2X2R gene. Furthermore, esophageal cancer cell line KYSE450 presented higher expression of E-NTPDases 1 and 2 and of Ecto-5′-NT/CD73 when compared to normal cells. This cell line also exhibits ATPase, ADPase, and AMPase activity, although in different levels, and the co-treatment of apyrase was able to revert the antiproliferative effects of ATP. Moreover, results showed high immunostaining for P2X7R in biopsies of patients with esophageal carcinoma, indicating the involvement of this receptor in the growth of this type of cancer. The results suggest that P2X7R may be a potential pharmacological target to treat ESCC and can lead us to further investigate the effect of this receptor in cancer cell progression.