Purinergic Cooperation Between P2Y2 and P2X7 Receptors Promote Cutaneous Leishmaniasis Control: Involvement of Pannexin-1 and Leukotrienes

Abstract

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.

P2Y2 Receptor Induces L. amazonensis Infection Control in a Mechanism Dependent on Caspase-1 Activation and IL-1β Secretion

“…Previously, we showed that antiparasite effects attributed by UTP and ATP involve P2X7R and PANX-1 channels in infected macrophages [ 19 ]. We also reported that antiparasitic immune responses triggered by the ATP/P2X7R/PANX-1 axis require NLRP3 inflammasome activation in macrophages infected with L. amazonensis [ 14 ].…”

“…Nevertheless, the host defense mechanisms that are modulated to control parasite replication and treat the disease are not thoroughly characterized, and several aspects of the disease remain poorly understood [ 25 ]. We previously reported the involvement of purinergic receptors, including P2Y 2 R and P2X7R, in the control of L. amazonensis infection in vitro and in vivo [ 4 , 19 ]. These receptors induce the activation of several microbicidal mechanisms in host cells during infection (i.e., NO, ROS, LTB 4 production) [ 20 , 21 , 26 , 27 ].…”

“…Intracellular parasite loads were analyzed as previously described [ 19 ]. Briefly, cells were infected and treated with nucleotides and were fixed onto slides, stained using a panoptic stain (Laborclin®, PR, Brazil), and counted using a Primo Star light microscope (Zeiss, Germany), with a 40x objective (100x for representative pictures).…”

“…The parasite load of L. amazonensis in infected tissues was determined using a limiting dilution assay, as previously described [ 19 , 23 ]. Mice were euthanized in a CO 2 chamber, followed by cervical dislocation.…”

“…ATP/P2X7R signaling during L. amazonensis infection has been partially elucidated during the last decade; it is assumed to be the most potent canonical activator of the NLRP3 inflammasome [ 16 ] and more recently in noncanonical activation of NLRP3 inflammasome assembly [ 14 , 17 , 18 ]. We previously demonstrated that the antiparasite immune response attributed to the P2YR agonist UTP involves paracrine activation of the P2X7 receptor (P2X7R) and PANX-1 channels in macrophages from mice infected with L. amazonensis [ 19 ]; UTP induces production and release of reactive oxygen species (ROS), nitrite oxide (NO) [ 20 , 21 ], and leukotriene B 4 (LTB 4 ) [ 19 ] that is assumed to be crucial for parasite death. In addition to these inflammatory mediators, IL-1 β mediates the control of intracellular parasite infections [ 4 ] [ 22 ].…”