Mechanistic basis for nigericin-induced NLRP1 inflammasome activation in human epithelial cells

ABSTRACT Nigericin, an ionophore derived from Streptomyces hygroscopicus , is arguably the most commonly used tool compound to study the NLRP3 inflammasome. Recent findings, however, showed that nigericin also activates the NLRP1 inflammasome in human keratinocytes. In this study, we resolve the mechanistic basis of nigericin-driven NLRP1 inflammasome activation. In multiple non-hematopoietic cell types, nigericin rapidly and specifically inhibits the elongation stage of the ribosome cycle by depleting cytosolic potassium ions. This activates the ribotoxic stress response (RSR) sensor kinase ZAKɑ, p38 and JNK, as well as the hyperphosphorylation of the NLRP1 linker domain. As a result, nigericin-induced pyroptosis in human keratinocytes is blocked by extracellular potassium supplementation, ZAKɑ knockout or pharmacologic inhibitors of ZAKɑ and p38 kinase activities. By surveying a diverse panel of ionophores, we show that the electroneutrality of potassium efflux is essential to activate ZAKɑ-driven RSR, likely because a greater extent of K+ depletion is necessary to activate ZAKɑ-NLRP1 than NLRP3. These findings resolve the mechanism by which nigericin activates NLRP1 in nonhematopoietic cell types and demonstrate an unexpected connection between RSR, perturbations of potassium ion flux and innate immunity. GRAPHICAL ABSTRACT SIGNIFICANCE Nigericin is familiar to the inflammasome field as the most robust and commonly used NLRP3 inducer. It has enabled numerous breakthroughs in the field linking NLRP3 activation to potassium efflux. In this manuscript, we report that nigericin activates an alternate inflammasome sensor, NLRP1 in primary human skin, nasal and corneal epithelial cells. NLRP1 activation by nigericin requires K+ efflux-driven ribosome stalling and the ribotoxic stress response (RSR) sensor MAP3K, ZAKɑ. We further identify the key biophysical principles that explain why only a subset of K+ ionophores, exemplified by nigericin, function as ‘super’ inflammasome agonists that can activate either NLRP1 or NLRP3, depending on cell type. These results reveal an unexpected connection between RSR, potassium ion flux and innate immunity.