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      Microvesicles released from fat-laden cells promote activation of hepatocellular NLRP3 inflammasome: A pro-inflammatory link between lipotoxicity and non-alcoholic steatohepatitis

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          Abstract

          Non-Alcoholic Fatty Liver Disease (NAFLD) is a major form of chronic liver disease in the general population in relation to its high prevalence among overweight/obese individuals and patients with diabetes type II or metabolic syndrome. NAFLD can progress to steatohepatitis (NASH), fibrosis and cirrhosis and end-stage of liver disease but mechanisms involved are still incompletely characterized. Within the mechanisms proposed to mediate the progression of NAFLD, lipotoxicity is believed to play a major role. In the present study we provide data suggesting that microvesicles (MVs) released by fat-laden cells undergoing lipotoxicity can activate NLRP3 inflammasome following internalization by either cells of hepatocellular origin or macrophages. Inflammasome activation involves NF-kB-mediated up-regulation of NLRP3, pro-caspase-1 and pro-Interleukin-1, then inflammasome complex formation and Caspase-1 activation leading finally to an increased release of IL-1β. Since the release of MVs from lipotoxic cells and the activation of NLRP3 inflammasome have been reported to occur in vivo in either clinical or experimental NASH, these data suggest a novel rational link between lipotoxicity and increased inflammatory response.

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          Most cited references31

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          NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice.

          Inflammasome activation plays a central role in the development of drug-induced and obesity-associated liver disease. However, the sources and mechanisms of inflammasome-mediated liver damage remain poorly understood. Our aim was to investigate the effect of NLRP3 inflammasome activation on the liver using novel mouse models. We generated global and myeloid cell-specific conditional mutant Nlrp3 knock-in mice expressing the D301N Nlrp3 mutation (ortholog of D303N in human NLRP3), resulting in a hyperactive NLRP3. To study the presence and significance of NLRP3-initiated pyroptotic cell death, we separated hepatocytes from nonparenchymal cells and developed a novel flow-cytometry-based (fluorescence-activated cell sorting; FACS) strategy to detect and quantify pyroptosis in vivo based on detection of active caspase 1 (Casp1)- and propidium iodide (PI)-positive cells. Liver inflammation was quantified histologically by FACS and gene expression analysis. Liver fibrosis was assessed by Sirius Red staining and quantitative polymerase chain reaction for markers of hepatic stellate cell (HSC) activation. NLRP3 activation resulted in shortened survival, poor growth, and severe liver inflammation; characterized by neutrophilic infiltration and HSC activation with collagen deposition in the liver. These changes were partially attenuated by treatment with anakinra, an interleukin-1 receptor antagonist. Notably, hepatocytes from global Nlrp3-mutant mice showed marked hepatocyte pyroptotic cell death, with more than a 5-fold increase in active Casp1/PI double-positive cells. Myeloid cell-restricted mutant NLRP3 activation resulted in a less-severe liver phenotype in the absence of detectable pyroptotic hepatocyte cell death. Our data demonstrate that global and, to a lesser extent, myeloid-specific NLRP3 inflammasome activation results in severe liver inflammation and fibrosis while identifying hepatocyte pyroptotic cell death as a novel mechanism of NLRP3-mediated liver damage. © 2014 by the American Association for the Study of Liver Diseases.
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            Characterization and comprehensive proteome profiling of exosomes secreted by hepatocytes.

            Exosomes represent a discrete population of vesicles that are secreted from various cell types to the extracellular media. Their protein and lipid composition are a consequence of sorting events at the level of the multivesicular body, a central organelle which integrates endocytic and secretory pathways. Characterization of exosomes from different biological samples has shown the presence of common as well as cell-type specific proteins. Remarkably, the protein content of the exosomes is modified upon pathological or stress conditions. Hepatocytes play a central role in the body response to stress metabolizing potentially harmful endogenous substances as well as xenobiotics. In the present study, we described and characterized for the first time exosome secretion in nontumoral hepatocytes, and with the use of a systematic proteomic approach, we establish the first extensive proteome of a hepatocyte-derived exosome population which should be useful in furthering our understanding of the hepatic function and in the identification of components that may serve as biomarkers for hepatic alterations. Our analysis identifies a significant number of proteins previously described among exosomes derived from others cell types as well as proteins involved in metabolizing lipoproteins, endogenous compounds and xenobiotics, not previously described in exosomes. Furthermore, we demonstrated that exosomal membrane proteins can constitute an interesting tool to express nonexosomal proteins into exosomes with therapeutic purposes.
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              Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome.

              Dying cells are capable of activating the innate immune system and inducing a sterile inflammatory response. Here, we show that necrotic cells are sensed by the Nlrp3 inflammasome resulting in the subsequent release of the proinflammatory cytokine IL-1beta. Necrotic cells produced by pressure disruption, hypoxic injury, or complement-mediated damage were capable of activating the Nlrp3 inflammasome. Nlrp3 inflammasome activation was triggered in part through ATP produced by mitochondria released from damaged cells. Neutrophilic influx into the peritoneum in response to necrotic cells in vivo was also markedly diminished in the absence of Nlrp3. Nlrp3-deficiency moreover protected animals against mortality, renal dysfunction, and neutrophil influx in an in vivo renal ischemic acute tubular necrosis model. These findings suggest that the inhibition of Nlrp3 inflammasome activity can diminish the acute inflammation and damage associated with tissue injury.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                1 March 2017
                2017
                : 12
                : 3
                : e0172575
                Affiliations
                [1 ]Department of Clinical and Biological Sciences, University of Torino, Torino, Italy
                [2 ]Department of Drug Science and Technology, University of Torino, Torino, Italy
                [3 ]Department of Pediatrics, University of California San Diego (UCSD), La Jolla, CA, United States of America
                [4 ]Department of Health Sciences and Interdisciplinary Research Center for Autoimmune Diseases, University “Amedeo Avogadro” of East Piedmont, Novara, Italy
                [5 ]Department of Medical Sciences, Division of Gastroenterology, University of Torino, Torino, Italy
                Medizinische Fakultat der RWTH Aachen, GERMANY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: SC EM EA AF MC MP.

                • Formal analysis: SC EM SS E. Benetti DP.

                • Funding acquisition: MP.

                • Investigation: SC EM CB BF E. Benetti EN FC MR RF DP SS E. Bugianesi.

                • Methodology: DP SC AF.

                • Project administration: MP EA MC AF.

                • Resources: E. Bugianesi EA SS DP AF.

                • Supervision: SC E. Bugianesi DP AF EA MC MP.

                • Writing – original draft: SC EM EA MC MP.

                Article
                PONE-D-16-30918
                10.1371/journal.pone.0172575
                5331985
                28249038
                855fcee7-771c-4cec-8409-bc33e563c4df
                © 2017 Cannito et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 2 August 2016
                : 7 February 2017
                Page count
                Figures: 8, Tables: 0, Pages: 22
                Funding
                Funded by: AIRC - IG-2014
                Award ID: 15274
                Award Recipient :
                This study has been funded by: i) Associazione Italiana per la Ricerca sul Cancro (AIRC, Milano, Italy), IG-2014 grant, project code 15274, (to MP); ii) the University of Torino (Fondo di Ateneo ex 60%, to EN and MP).
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