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.

Microscopy images as interactive tools in cell modeling and cell biology education.

The advent of genomics, proteomics, and microarray technology has brought much excitement to science, both in teaching and in learning. The public is eager to know about the processes of life. In the present context of the explosive growth of scientific information, a major challenge of modern cell biology is to popularize basic concepts of structures and functions of living cells, to introduce people to the scientific method, to stimulate inquiry, and to analyze and synthesize concepts and paradigms. In this essay we present our experience in mixing science and education in Brazil. For two decades we have developed activities for the science education of teachers and undergraduate students, using microscopy images generated by our work as cell biologists. We describe open-air outreach education activities, games, cell modeling, and other practical and innovative activities presented in public squares and favelas. Especially in developing countries, science education is important, since it may lead to an improvement in quality of life while advancing understanding of traditional scientific ideas. We show that teaching and research can be mutually beneficial rather than competing pursuits in advancing these goals.

Evidence for a perforin-mediated mechanism controlling cardiac inflammation in Trypanosoma cruzi infection.

CD8+ T lymphocytes are considered an important cell population involved in the control of parasitaemia and mortality after Trypanosoma cruzi infection. However, despite recent developments in this field, the mechanism whereby this control is exerted is still not completely understood. Here we have used perforin knockout (-/-) mice infected with Y strain T. cruzi in order to evaluate specifically the participation of the perforin-based cytotoxic pathway in the destruction of cardiomyocytes, cellular inflammatory infiltration, and control of parasitaemia and mortality. We observed that although parasitaemia was equivalent in perforin (+/+) and (-/-) groups, survival rate and spontaneous physical performance were significantly lower in the perforin deficient mice. The cardiac inflammatory cell infiltration, mostly composed of CD8+ cells, was more evident in perforin (-/-) mice. Ultrastructural and immunofluorescence analysis, as well as plasma creatine kinase activity, revealed cardiomyocyte damage and necrosis, more evident in perforin (-/-) mice. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays performed in heart samples revealed similar and modest levels of apoptosis in both perforin (+/+) and (-/-) mice. These results indicate that perforin does not play a pivotal role in the control of parasitaemia and direct lysis of cardiomyocytes, but seems to be an important molecule involved in the control of cardiac inflammation and pathology induced by a highly virulent strain of T. cruzi.