The NLRP6 inflammasome is activated by sterile or pathogen-induced endolysosomal damage.

The cytosolic innate immune sensor NLRP6 controls host defense against bacteria and viruses in the gastrointestinal tract, but the underlying mechanism is poorly understood. Here, we report that NLRP6 forms an inflammasome following endolysosomal damage caused by sterile triggers or bacterial pathogens such as Listeria monocytogenes in human intestinal epithelial cells (IECs). NLRP6 activation requires Listeriolysin O-dependent cytosolic invasion of L. monocytogenes and triggers IEC pyroptosis and IL-1β release via ASC/caspase-1-mediated GSDMD cleavage. NLRP6 activation requires its NACHT domain and ATP binding, whereas inflammasome formation is independent of bacterial pathogen-associated molecular patterns (PAMPs), such as lipoteichoic acid or dsRNA, which were previously reported to activate NLRP6. L. monocytogenes mutants deficient in cell-to-cell spread or escape from secondary vacuoles induce lower levels of cell death, linking bacteria-induced endolysosomal damage to NLRP6 activation. Finally, sterile endolysosomal damage recapitulates pathogen-induced NLRP6 activation and induces IEC pyroptosis. In summary, our study reveals that NLRP6 enables intestinal epithelial cells to detect endolysosomal damage, thereby mediating their response not only to pathogens but more generally to wide-ranging sources of pathological endolysosomal damage.