Inflammasome activation in human and mouse macrophages engulfing autophagic dying cells

Abstract

Phagocytosis of PAMPs, DAMPs and certain dying cells can activate the inflammasome pathway in macrophages. In our study, we show that both human and mouse macrophages display a pro-inflammatory response to autophagic dying MCF-7 and Ba/F3 cells, but not to living, apoptotic, necrotic or necrostatin-1 treated ones. When we investigated this phenomenon, further it was found that caspase-1 was activated and IL-1β was processed and then secreted in a MyD88-independent manner. Neither caspase-1 inhibited nor caspase-1 deficient macrophages could trigger IL-1β release due to the lack of key component for pro-IL-1β cleavage and maturation before its secretion. Next we clarified which inflammasome is activated by autophagic dying cells and found that NALP-3 deficient macrophages displayed reduced IL-1β secretion, which was also observed in macrophages in which the NALP-3 gene was knocked down. Next, we investigated the upstream mechanism of NALP-3 inflammasome activation triggered by autophagic dying cells. Our results show that during phagocytosis of autophagic dying MCF-7 and Ba/F3 cells exogenous ATP is acting through P2X7 receptor, initiates K+ efflux, inflammasome activation and secretion of IL-1β from human and mouse macrophages. Calreticulin exposure on autophagic dying MCF-7 cells do not play role in inflammasome activation. ATP was secreted from human macrophages during co-incubation with autophagic dying MCF-7 cells which did not release ATP. However, autophagic dying Ba/F3 cells were the source the ATP which activated the P2X7 receptor and lead to inflammasome activation in mouse macrophages. We further showed that pannexin-1 channel is responsible for ATP secretion from autophagic dying Ba/F3 cells. MCF-7 and Ba/F3 cells dying with involvement of autophagy were capable of preventing crude LPS-induced pro-inflammatory cytokine release but pro-inflammatory cytokines were produced and secreted from human macrophages triggered by autophagic dying cells as a result of the secreted IL-1β. Finally, it was observed that injection of autophagic dying cells intraperitoneally induced an acute inflammatory reaction by recruiting neutrophils and monocytes/macrophages.