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 proangiogenicmicroparticles (MPs) in a caspase 3-dependent manner, an event which occurs also in vivo and may havea 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 fat-laden HepG2 (i.e., HepG2 exposed for 24 hr to 0.25 mMpalmitic acid or PA), as recently described (1). HepG2 resting cells were then incubated (15 min-24hrs) with MPs, LPS (100 ng/mL-1 µg/mL) or PA (150 – 500 µM), 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-1β was evaluated by Western blot analysis in cell lysates, whereas ELISA assays were used to measure IL-1β and IL-18 levels released by resting HepG2.Results.MPs induced a time-dependent increase in NLRP3 expression in resting HepG2 cells starting from 6hr and then reaching a plateau at 16-24 hrs, with a kinetics that overlapped the one exerted by PA and was delayed as compared to LPS (1-3 hrs). Interestingly, both MPs and PA, but not LPS, significantly induced caspase-1 activation and consequent release of IL-1β and IL-18 in a time-dependent manner. Conclusions.Fat-laden cells, by releasing MPs in a paracrine way, canefficiently trigger inflammasome activation in surrounding hepatic cells, thus identifying an additional new molecular mechanism of inflammation in NASH pathogenesis. References.1.Povero D et al., Science Signaling(2013),6(296),ra88.

Microparticles released as a consequence of lipid-induced toxicity promote NLRP3 inflammasome activation in HepG2 cells.

PATERNOSTRO, CLAUDIA;BENETTI, ELISA;CANNITO, STEFANIA;NOVO, ERICA;CHIAZZA, FAUSTO;BOCCA, Claudia;FANTOZZI, Roberto;COLLINO, Massimo;PAROLA, Maurizio
2014-01-01

Abstract

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 proangiogenicmicroparticles (MPs) in a caspase 3-dependent manner, an event which occurs also in vivo and may havea 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 fat-laden HepG2 (i.e., HepG2 exposed for 24 hr to 0.25 mMpalmitic acid or PA), as recently described (1). HepG2 resting cells were then incubated (15 min-24hrs) with MPs, LPS (100 ng/mL-1 µg/mL) or PA (150 – 500 µM), 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-1β was evaluated by Western blot analysis in cell lysates, whereas ELISA assays were used to measure IL-1β and IL-18 levels released by resting HepG2.Results.MPs induced a time-dependent increase in NLRP3 expression in resting HepG2 cells starting from 6hr and then reaching a plateau at 16-24 hrs, with a kinetics that overlapped the one exerted by PA and was delayed as compared to LPS (1-3 hrs). Interestingly, both MPs and PA, but not LPS, significantly induced caspase-1 activation and consequent release of IL-1β and IL-18 in a time-dependent manner. Conclusions.Fat-laden cells, by releasing MPs in a paracrine way, canefficiently trigger inflammasome activation in surrounding hepatic cells, thus identifying an additional new molecular mechanism of inflammation in NASH pathogenesis. References.1.Povero D et al., Science Signaling(2013),6(296),ra88.
The International Liver CongressTM 2014. 49th Annual Meeting of The European Association for the Study of Liver (EASL).
Londra
8-13 Aprile 2014
60
suppl 1
S153
S153
Paternostro C; Benetti E; Cannito S; Novo E; Chiazza F; Rogazzo M; Bocca C; Fantozzi R; Povero D; Feldstein A; Collino M; Parola M.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/155783
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