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 B 4 (LTB 4 ) production and release. Therefore, we investigated whether L . amazonensis elimination by P2X7 receptor and LTB 4 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 LTB 4 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 LTB 4 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 LTB 4 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 LTB 4 was dependent on non-canonical NLRP3 inflammasome activation, ROS production, and IL-1β signaling.

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Leishmania amazonensis is the etiological agent of diffuse cutaneous leishmaniasis. The immunopathology of leishmaniasis caused by L. amazonensis infection is dependent on the pathogenic role of effector CD4 + T cells. Purinergic signalling has been implicated in resistance to infection by different intracellular parasites. In this study, we evaluated the role of the P2X7 receptor in modulating the immune response and susceptibility to infection by L. amazonensis. We found that P2X7-deficient mice are more susceptible to L. amazonensis infection than wild-type (WT) mice. P2X7 deletion resulted in increased lesion size and parasite load. Our histological analysis showed an increase in cell infiltration in infected footpads of P2X7-deficient mice. Analysis of the cytokine profile in footpad homogenates showed increased levels of IFN-γ and decreased TGF-β production in P2X7-deficient mice, suggesting an exaggerated pro-inflammatory response. In addition, we observed that CD4 + and CD8 + T cells from infected P2X7-deficient mice exhibit a higher proliferative capacity than infected WT mice. These data suggest that P2X7 receptor plays a key role in parasite control by regulating T effector cells and inflammation during L. amazonensis infection.

The release of damage-associated molecular patterns, including uridine triphosphate (UTP) and adenosine triphosphate (ATP) to the extracellular milieu is a key component of innate immune response to infection. Previously, we showed that macrophage infection by the protozoan parasite Leishmania amazonensis—the etiological agent of cutaneous leishmaniasis—can be controlled by ATP- and UTP-mediated activation of P2Y and P2X7 receptors (activated by UTP/ATP and ATP, respectively), which provided comparable immune responses against the parasite. Interestingly, in context of Leishmania amazonensis infection, UTP/P2Y triggered apoptosis, reactive oxygen species, and oxide nitric (NO) production, which are characteristic of P2X7 receptor activation. Here, we examined a possible “cross-talk” between P2Y2 and P2X7 receptors, and the requirement for pannexin-1 (PANX-1) in the control of L. amazonensis infection in mouse peritoneal macrophages and in vivo. UTP treatment reduced L. amazonensis parasite load, induced extracellular ATP release [which was pannexin-1 (PANX-1) dependent], and triggered leukotriene B4 (LTB4) production in macrophages. UTP-induced parasite control was blocked by pharmacological antagonism of P2Y2 or P2X7 receptors and was absent in macrophages lacking P2X7 or PANX-1. In addition, ATP release induced by UTP was also inhibited by PANX-1 blocker carbenoxolone, and partially reversed by inhibitors of vesicle traffic and actin cytoskeleton dynamics. In vivo, UTP treatment reduced footpad and popliteal lymph node parasite load, and the lesion in wild-type (WT) mice; fact not observed in P2X7−/− mice. Our data reveal that P2Y2 and P2X7 receptors cooperate to trigger potent innate immune responses against L. amazonensis infection.