Intralesional uridine-5'-triphosphate (UTP) treatment induced resistance to Leishmania amazonensis infection by boosting Th1 immune responses and reactive oxygen species production.

Leishmania amazonensis is the etiologic agent of cutaneous leishmaniasis, an immune-driven disease causing a range of clinical symptoms. Infections caused by L. amazonensis suppress the activation and function of immune cells, including macrophages, dendritic cells, and CD4+ T cells. In this study, we analyzed the course of infection as well as the leishmanicidal effect of intralesional UTP treatment in L. amazonensis-infected BALB/c mice. We found that UTP treatment reduced the parasitic load in both footpad and lymph node sites of infection. UTP also boosted Th1 immune responses, increasing CD4+ T cell recruitment and production of IFN-γ, IL-1ß, IL-12, and TNF-α. In addition, the role of UTP during innate immune response against L. amazonensis was evaluated using the air pouch model. We observed that UTP augmented neutrophil chemoattraction and activated microbicidal mechanisms, including ROS production. In conclusion, our data suggested an important role for this physiological nucleotide in controlling L. amazonensis infection, and its possible use as a therapeutic agent for shifting immune responses to Th1 and increasing host resistance against L. amazonensis infection.

CD39 limits P2X7 receptor inflammatory signaling and attenuates sepsis-induced liver injury.

BACKGROUND & AIMS: The severity of sepsis can be linked to excessive inflammatory responses resulting in hepatic injury. P2X7 receptor activation by extracellular ATP (eATP) exacerbates inflammation by augmenting cytokine production; while CD39 (ENTPD1) scavenges eATP to generate adenosine, thereby limiting P2X7 activation and resulting in A2A receptor stimulation. We aim to determine how the functional interaction of P2X7 receptor and CD39 control the macrophage response, and consequently impact on sepsis and liver injury. METHODS: Sepsis was induced by cecal ligation and puncture in C57BL/6 wild-type (WT) and CD39-/- mice. Several in vitro assays were performed using peritoneal or bone marrow derived macrophages to determine CD39 ectonucleotidase activity and its role in sepsis-induced liver injury. RESULTS: CD39 expression in macrophages limits ATP-P2X7 receptor pro-inflammatory signaling. P2X7 receptor paradoxically boosts CD39 activity. Inhibition and/or deletion of P2X7 receptor in LPS-primed macrophages attenuates cytokine production and inflammatory signaling as well as preventing ATP-induced increases in CD39 activity. Septic CD39-/- mice exhibit higher levels of inflammatory cytokines and show more pronounced liver injury than WT mice. Pharmacological P2X7 blockade largely prevents tissue damage, cell apoptosis, cytokine production, and the activation of inflammatory signaling pathways in the liver from septic WT, while only attenuating these outcomes in CD39-/- mice. Furthermore, the combination of P2X7 blockade with adenosine A2A receptor stimulation completely inhibits cytokine production, the activation of inflammatory signaling pathways, and protects septic CD39-/- mice against liver injury. CONCLUSIONS: CD39 attenuates sepsis-associated liver injury by scavenging eATP and ultimately generating adenosine. We propose boosting of CD39 would suppress P2X7 responses and trigger adenosinergic signaling to limit systemic inflammation and restore liver homeostasis during the acute phase of sepsis. Lay summary: CD39 expression in macrophages limits P2X7-mediated pro-inflammatory responses, scavenging extracellular ATP and ultimately generating adenosine. CD39 genetic deletion exacerbates sepsis-induced experimental liver injury. Combinations of a P2X7 antagonist and adenosine A2A receptor agonist are hepatoprotective during the acute phase of abdominal sepsis.

P2X7 receptor promotes intestinal inflammation in chemically induced colitis and triggers death of mucosal regulatory T cells.

P2X7 receptor activation contributes to inflammation development in different pathologies. We previously reported that the P2X7 receptor is over-expressed in the gut mucosa of patients with inflammatory bowel disease, and that P2X7 inhibition protects against chemically induced colitis. Here, we investigated in detail the role of the P2X7 receptor in inflammatory bowel disease development, by treating P2X7 knockout (KO) and WT mice with two different (and established) colitis inductors. P2X7 KO mice were protected against gut inflammation induced by 2,4,6-trinitrobenzenesulfonic acid or oxazolone, with no weight loss or gut histological alterations after treatment. P2X7 receptor knockout induced regulatory T cell accumulation in the colon, as evaluated by qRT-PCR for FoxP3 expression and immunostaining for CD90/CD45RBlow. Flow cytometry analysis of mesenteric lymph node cells showed that P2X7 activation (by ATP) triggered regulatory T cell death. In addition, such cells from P2X7 KO mice expressed more CD103, suggesting increased migration of regulatory T cells to the colon (relative to the WT). Our results show that the P2X7 has a key role during inflammation development in inflammatory bowel disease, by triggering the death and retention in the mesenteric lymph nodes of regulatory T cells that would otherwise promote immune system tolerance in the gut.

Overexpression of ATP-activated P2X7 receptors in the intestinal mucosa is implicated in the pathogenesis of Crohn's disease.

BACKGROUND: Extracellular nucleotides released in conditions of cell stress alert the immune system from tissue injury or inflammation. We hypothesized that the P2X7 receptor (P2X7-R) could regulate key elements in inflammatory bowel disease pathogenesis. METHODS: Colonoscopy samples obtained from patients with Crohn's disease (CD), ulcerative colitis, and controls were used to analyze P2X7-R expression by RT and real-time PCR, immunohistochemistry, and confocal microscopy. Inflammatory response was determined by the levels of cytokines by enzyme-linked immunosorbent assay in cultures of intestinal explants. Apoptosis was determined by the TUNEL assay. P2X7-R C57BL/6 mice were treated with trinitrobenzene sulfonic acid or dextran sulfate sodium (DSS) for inducing colitis. RESULTS: P2X7-R was expressed in higher levels in inflamed CD epithelium and lamina propria, where it colocalizes more with dendritic cells and macrophages. Basal levels of P2X7-R mRNA were higher in CD inflamed mucosa compared with noninflamed CD and controls and were upregulated after interferon-γ in controls. Apoptotic rates were higher in CD epithelium and lamina propria compared with ulcerative colitis and controls. Levels of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-17 were higher, whereas IL-10 was lower in CD compared with controls. Levels of tumor necrosis factor-α-α and interleukin-1ß increased after adenosine-triphosphate and decreased after KN62 treatment in CD. P2X7-R animals did not develop trinitrobenzene sulfonic acid or DSS colitis. CONCLUSIONS: The upregulation of P2X7-R in CD inflamed mucosa is consistent with the involvement of purinoceptors in inflammation and apoptosis. These observations may implicate purinergic signaling in the pathogenesis of intestinal inflammation, and the P2X7-R may represent a novel therapeutic target in CD.

Extracellular ATP induces cell death in human intestinal epithelial cells.

BACKGROUND: Extracellular ATP is an endogenous signaling molecule released by various cell types and under different stimuli. High concentrations of ATP released into the extracellular medium activate the P2X7 receptor in most inflammatory conditions. Here, we seek to characterize the effects of ATP in human intestinal epithelial cells and to evaluate morphological changes in these cells in the presence of ATP. METHODS: We treated human intestinal epithelial cells with ATP and evaluated the effects of this nucleotide by scanning and transmission electron microscopy analysis and calcium measurements. We used flow cytometry to evaluate apoptosis. We collected human intestinal explants for immunohistochemistry, apoptosis by the TUNEL approach and caspase-3 activity using flow cytometry analyses. We also evaluated the ROS production by flow cytometry and NO secretion by the Griess technique. RESULTS: ATP treatment induced changes characteristic of cell death by apoptosis and autophagy but not necrosis in the HCT8 cell line. ATP induced apoptosis in human intestinal explants that showed TUNEL-positive cells in the epithelium and in the lamina propria. The explants exhibited a significant increase of caspase-3 activity when the colonic epithelial cells were incubated with IFN-gamma followed by ATP as compared to control cells. In addition, it was found that antioxidants were able to inhibit both the ROS production and the apoptosis induced by ATP in epithelial cells. GENERAL SIGNIFICANCE: The activation of P2X7 receptors by ATP induces apoptosis and autophagy in human epithelial cells, possibly via ROS production, and this effect might have implications for gut inflammatory conditions.