So, NLRP6 can initiate a canonical inflammasome and induces pyroptosis, establishing NLRP6 as a sensor for damage to the endolysosomal system.

Finally, sterile endolysosomal damage alone, using the lysosome rupturing compound LLOME, is sufficient to activate NLRP6.

On the bacterial side, bacterial PAMPs aren’t sufficient to activate NLRP6. However, cell to cell spread is important for activation as a mutant deficient for making actin tails is a worse activator of NLRP, as is the plcB mutant deficient in escape from a secondary vacuole.

By comparing NLRP6 and 3, they map the upstream region of the NACHT domain - the FISNA and its conserved poly basic motifs- as being important for receptor activation, but despite sequence similarities they show that each receptor is activated by distinct ligands.

Then they show that listeria based activation of NLRP6 in HIEC intestinal epithelial cells has all the hallmarks of canonical inflammasome activation including GSDMD cleavage.

First they show that WT listeria - but not the dhly mutant which doesn’t express the pore forming toxin Listeriolysin O that allows the bacteria to escape from endolysosomes - are sensed in a HEK reconstitution assay.