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Microparticles released as a consequence of lipid-induced toxicity promote NLRP3 inflammasome activation in HepG2 cells
Digestive and Liver Disease 46 (2014) e1–e17
Contents lists available at ScienceDirect
Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Abstracts of the 47th A.I.S.F. – Italian Association for the Study of the Liver – Annual Meeting 2014. Oral Communications
OC-01 MICROPARTICLES RELEASED AS A CONSEQUENCE OF LIPID-INDUCED TOXICITY PROMOTE NLRP3 INFLAMMASOME ACTIVATION IN HEPG2 CELLS C. Paternostro 1 , E. Benetti 2 , S. Cannito 1 , E. Novo 1 , F. Chiazza 2 , M. Rogazzo 2 , C. Bocca 1 , R. Fantozzi 2 , D. Povero 3 , A. Feldstein 3 , M. Collino 2 , M. Parola 1 1
Conclusions: Fat-laden cells, by releasing MPs in a paracrine way, can efficiently trigger inflammasome activation in surrounding hepatic cells, thus identifying an additional new molecular mechanism of inflammation in NASH pathogenesis. Reference [1] Povero D, et al. Science Signaling 2013;6(296):ra88.
http://dx.doi.org/10.1016/j.dld.2014.01.006
Department Clinical and Biological Sciences, University of Torino, Turin, Italy 2 Department Drug Science and Technology, University of Torino, Turin, Italy 3 Department of Pediatrics, University of California San Diego (UCSD), La Jolla, CA, USA
OC-02
Introduction: Hepatocytes or HepG2 cells overloaded with saturated lipo-toxic free fatty acids, a condition that mimick lipid accumulation occurring in the liver in some forms of steatohepatitis, have been recently reported to release proangiogenic microparticles (MPs) in a caspase 3-dependent manner, an event which occurs also in vivo and may have a role in the pathogenesis of NAFLD/NASH [1]. Aims: In the present study we investigated whether MPs released from fat-laden cells may affect in a paracrine way NLRP3 inflammasome, which is known to be activated in vivo in NAFLD/NASH conditions. Methods: MPs were collected and purified as released by fatladen HepG2 (i.e., HepG2 exposed for 24 h to 0.25 mM palmitic acid or PA), as recently described [1]. HepG2 resting cells were then incubated (15 min–24 h) with MPs, LPS (100 ng/mL–1 g/mL) or PA (150–500 M), the latter known to induce NLRP3 inflammasome in hepatocytes. Expression of NLRP-3, pro-caspase and cleaved caspase 1, pro-IL-1 and cleaved IL-1 was evaluated by Western blot analysis in cell lysates, whereas ELISA assays were used to measure IL-1 and IL-18 levels released by resting HepG2. Results: MPs induced a time-dependent increase in NLRP3 expression in resting HepG2 cells starting from 6 h and then reaching a plateau at 16–24 h, with a kinetics that overlapped the one exerted by PA and was delayed as compared to LPS (1–3 h). Interestingly, both MPs and PA, but not LPS, significantly induced caspase-1 activation and consequent release of IL-1 and IL-18 in a timedependent manner.
A. Cappon 1 , R. Bruno 2 , S. Gessani 3 , A. Masotti 4 , F. Marra 1
1590-8658/$36.00
PRO-INFLAMMATORY AND PRO-FIBROGENIC ACTIONS OF THE HIV-ENVELOPE PROTEIN GP120 ARE MEDIATED BY INFLAMMASOME ACTIVATION AND MIR29B
1
University of Florence, Florence, Italy IRCCS, University of Pavia, Pavia, Italy 3 Istituto Superiore di Sanità, Rome, Italy 4 Ospedale Pediatrico Bambino Gesù, Rome, Italy 2
Introduction/aim: Patients with HCV/HIV co-infection show faster progression of liver fibrosis, in part associated with miRNA dysregulation and more severe inflammation. The HIV protein gp120 modulates directional migration and expression of profibrogenic cytokines in hepatic stellate cells (HSC), through engagement of the chemokine receptor CCR5. The NALP3 inflammasome is a critical pathway in the generation of proinflammatory signals during liver injury. Aim of this study was to evaluate the role miRNAs and inflammasome activation in mediating the effect HSC. Methods: HSC were isolated from normal human liver tissue. Inflammasome complex gene expression was measured by qRT-PCR. Levels of mature IL-1 were assayed by ELISA. miRNA expression was evaluated via RT-PCR. miR-29b mimic was transfected using Amaxa. Blood mononuclear cells (MNc) were isolated from healthy volunteers. Results: HSCs exposed to M-tropic gp120 (CN54) showed a time-dependent up-regulation of Pycard, NALP3, Caspase-1 and IL-1. ELISA showed increased levels of mature IL-1 in the supernatants of gp120-stimulated cell. Pre-incubation of HSC with neutralizing anti-CCR5 antibody reduced gp120-mediated IL-1 production, showing that this receptor is required for activation
Contents lists available at ScienceDirect
Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Abstracts of the 47th A.I.S.F. – Italian Association for the Study of the Liver – Annual Meeting 2014. Oral Communications
OC-01 MICROPARTICLES RELEASED AS A CONSEQUENCE OF LIPID-INDUCED TOXICITY PROMOTE NLRP3 INFLAMMASOME ACTIVATION IN HEPG2 CELLS C. Paternostro 1 , E. Benetti 2 , S. Cannito 1 , E. Novo 1 , F. Chiazza 2 , M. Rogazzo 2 , C. Bocca 1 , R. Fantozzi 2 , D. Povero 3 , A. Feldstein 3 , M. Collino 2 , M. Parola 1 1
Conclusions: Fat-laden cells, by releasing MPs in a paracrine way, can efficiently trigger inflammasome activation in surrounding hepatic cells, thus identifying an additional new molecular mechanism of inflammation in NASH pathogenesis. Reference [1] Povero D, et al. Science Signaling 2013;6(296):ra88.
http://dx.doi.org/10.1016/j.dld.2014.01.006
Department Clinical and Biological Sciences, University of Torino, Turin, Italy 2 Department Drug Science and Technology, University of Torino, Turin, Italy 3 Department of Pediatrics, University of California San Diego (UCSD), La Jolla, CA, USA
OC-02
Introduction: Hepatocytes or HepG2 cells overloaded with saturated lipo-toxic free fatty acids, a condition that mimick lipid accumulation occurring in the liver in some forms of steatohepatitis, have been recently reported to release proangiogenic microparticles (MPs) in a caspase 3-dependent manner, an event which occurs also in vivo and may have a role in the pathogenesis of NAFLD/NASH [1]. Aims: In the present study we investigated whether MPs released from fat-laden cells may affect in a paracrine way NLRP3 inflammasome, which is known to be activated in vivo in NAFLD/NASH conditions. Methods: MPs were collected and purified as released by fatladen HepG2 (i.e., HepG2 exposed for 24 h to 0.25 mM palmitic acid or PA), as recently described [1]. HepG2 resting cells were then incubated (15 min–24 h) with MPs, LPS (100 ng/mL–1 g/mL) or PA (150–500 M), the latter known to induce NLRP3 inflammasome in hepatocytes. Expression of NLRP-3, pro-caspase and cleaved caspase 1, pro-IL-1 and cleaved IL-1 was evaluated by Western blot analysis in cell lysates, whereas ELISA assays were used to measure IL-1 and IL-18 levels released by resting HepG2. Results: MPs induced a time-dependent increase in NLRP3 expression in resting HepG2 cells starting from 6 h and then reaching a plateau at 16–24 h, with a kinetics that overlapped the one exerted by PA and was delayed as compared to LPS (1–3 h). Interestingly, both MPs and PA, but not LPS, significantly induced caspase-1 activation and consequent release of IL-1 and IL-18 in a timedependent manner.
A. Cappon 1 , R. Bruno 2 , S. Gessani 3 , A. Masotti 4 , F. Marra 1
1590-8658/$36.00
PRO-INFLAMMATORY AND PRO-FIBROGENIC ACTIONS OF THE HIV-ENVELOPE PROTEIN GP120 ARE MEDIATED BY INFLAMMASOME ACTIVATION AND MIR29B
1
University of Florence, Florence, Italy IRCCS, University of Pavia, Pavia, Italy 3 Istituto Superiore di Sanità, Rome, Italy 4 Ospedale Pediatrico Bambino Gesù, Rome, Italy 2
Introduction/aim: Patients with HCV/HIV co-infection show faster progression of liver fibrosis, in part associated with miRNA dysregulation and more severe inflammation. The HIV protein gp120 modulates directional migration and expression of profibrogenic cytokines in hepatic stellate cells (HSC), through engagement of the chemokine receptor CCR5. The NALP3 inflammasome is a critical pathway in the generation of proinflammatory signals during liver injury. Aim of this study was to evaluate the role miRNAs and inflammasome activation in mediating the effect HSC. Methods: HSC were isolated from normal human liver tissue. Inflammasome complex gene expression was measured by qRT-PCR. Levels of mature IL-1 were assayed by ELISA. miRNA expression was evaluated via RT-PCR. miR-29b mimic was transfected using Amaxa. Blood mononuclear cells (MNc) were isolated from healthy volunteers. Results: HSCs exposed to M-tropic gp120 (CN54) showed a time-dependent up-regulation of Pycard, NALP3, Caspase-1 and IL-1. ELISA showed increased levels of mature IL-1 in the supernatants of gp120-stimulated cell. Pre-incubation of HSC with neutralizing anti-CCR5 antibody reduced gp120-mediated IL-1 production, showing that this receptor is required for activation