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

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.