Inflammation driven by the NLRP3 inflammasome is coordinated through multiple signaling pathways and is regulated by subcellular organelles. Here, we tested the hypothesis that NLRP3 senses disrupted endosome trafficking to trigger inflammasome formation and inflammatory cytokine secretion. NLRP3-activating stimuli disrupted endosome trafficking and triggered localization of NLRP3 to vesicles positive for endolysosomal markers and for the inositol lipid PI4P. Chemical disruption of endosome trafficking sensitized macrophages to the NLRP3 activator imiquimod, driving enhanced inflammasome activation and cytokine secretion. Together, these data suggest that NLRP3 can sense disruptions in the trafficking of endosomal cargoes, which may explain in part the spatial activation of the NLRP3 inflammasome. These data highlight mechanisms that could be exploited in the therapeutic targeting of NLRP3.

The release of interleukin (IL)-1b from primary human monocytes in response to extracellular LPS occurs through the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome. In primary monocytes, in response to LPS, NLRP3 inflammasome activation is characterized by an independence of K + efflux and ASC speck formation and has been termed the 'alternative' pathway. Here, we report that pharmacological inhibition of V-ATPase with bafilomycin A1 exacerbated LPS-induced NLRP3 inflammasome activation in primary human monocytes. Inhibition of V-ATPase in the presence of extracellular LPS led to NLRP3-dependent, K + efflux-independent, ASC oligomerization and caspase-1 activation. Although V-ATPases are required for lysosomal acidification, we found that acidic lysosomal pH and protease activity were dispensable for this altered response, suggesting that V-ATPase inhibition triggered alternative signalling events. Therefore, V-ATPases may serve additional roles during NLRP3 inflammasome activation in primary human monocytes.

Inflammation driven by the NLRP3 inflammasome is coordinated through multiple signaling pathways and with a poorly defined regulation by sub-cellular organelles. Here, we tested the hypothesis that NLRP3 senses disrupted endosome trafficking to trigger inflammasome formation and inflammatory cytokine secretion. NLRP3-activating stimuli disrupted endosome trafficking and triggered localization of NLRP3 to vesicles positive for endosome markers and the inositol lipid PtdIns4P. Chemical disruption of endosome trafficking sensitized macrophages to the NLRP3 activator imiquimod driving enhanced inflammasome activation and cytokine secretion. Together these data suggest that NLRP3 is capable of sensing disruptions in the trafficking of endosomal cargoes, and that this may explain in part the spatial activation of the NLRP3 inflammasome complex. These data highlight new mechanisms amenable for the therapeutic targeting of NLRP3.