Background and aims. Hepatocytes or HepG2 cells overloaded with saturated lipotoxic free fatty acids, a condition that mimick lipid accumulation occurring in the liver in some forms of steato-hepatitis, have been recently reported to release pro-angiogenic micro-particles (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). Here we investigated whether MPs released from fat-laden cells may affect in a paracrine way NLRP3 inflammasome, which is known to be progressively 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 mM palmitic 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. In other experiments activated human THP1 macrophages (48 hrs activation by PMA 25 nM plus 24 hrs in fresh medium) were exposed up to 24 hrs to MPs released by fat-laden HepG2 cells. 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 were very early (i.e., 1-6 hrs) efficiently internalized by both HepG2 cells and THP1 macrophages, as revealed by means of confocal microscopy. MPs induced a time-dependent increase in the expression of NLRP3 inflammasome components 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 in particular of IL-1β in a time-dependent manner. Moreover, MPs also up-regulated NLRP3 inflammasome expression in THP1 human macrophages within 3-6 hrs, resulting in a significantly increased release of IL-1β. Conclusions. Fat-laden cells, by releasing MPs in a paracrine way, can efficiently trigger inflammasome activation in surrounding hepatic cells as well as macrophages, 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 by fat-laden cells activate in a paracrine way NLRP3 inflammasome in both HepG2 cells and macrophages.
MORELLO, ELISABETTA;PATERNOSTRO, CLAUDIA;BENETTI, ELISA;CANNITO, STEFANIA;NOVO, ERICA;CHIAZZA, FAUSTO;BOCCA, Claudia;FANTOZZI, Roberto;COLLINO, Massimo;PAROLA, Maurizio
2014-01-01
Abstract
Background and aims. Hepatocytes or HepG2 cells overloaded with saturated lipotoxic free fatty acids, a condition that mimick lipid accumulation occurring in the liver in some forms of steato-hepatitis, have been recently reported to release pro-angiogenic micro-particles (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). Here we investigated whether MPs released from fat-laden cells may affect in a paracrine way NLRP3 inflammasome, which is known to be progressively 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 mM palmitic 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. In other experiments activated human THP1 macrophages (48 hrs activation by PMA 25 nM plus 24 hrs in fresh medium) were exposed up to 24 hrs to MPs released by fat-laden HepG2 cells. 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 were very early (i.e., 1-6 hrs) efficiently internalized by both HepG2 cells and THP1 macrophages, as revealed by means of confocal microscopy. MPs induced a time-dependent increase in the expression of NLRP3 inflammasome components 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 in particular of IL-1β in a time-dependent manner. Moreover, MPs also up-regulated NLRP3 inflammasome expression in THP1 human macrophages within 3-6 hrs, resulting in a significantly increased release of IL-1β. Conclusions. Fat-laden cells, by releasing MPs in a paracrine way, can efficiently trigger inflammasome activation in surrounding hepatic cells as well as macrophages, thus identifying an additional new molecular mechanism of inflammation in NASH pathogenesis. References. 1.Povero D et al., Science Signaling(2013),6(296),ra88.
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