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NLRP1 is an innate immune sensor protein that activates inflammasome-driven pyroptotic cell death. Recent work demonstrates that human NLRP1 has evolved to sense viral infections. Whether and how human NLRP1 responds to other infectious agents is unclear. Here, and in a companion manuscript, we report that human NLRP1, as an integral component of the ribotoxic stress response (RSR), is activated by bacterial exotoxins that target human ribosome elongation factors EEF1 and EEF2, including Diphtheria Toxin (DT) fromCorynebacterium diphtheriae, exotoxin A fromPseudomonas aeruginosaand sidI fromLegionella pneumophila. In human keratinocytes, DT activates RSR kinases ZAKα, p38 and JNKs, upregulates a set of signature RSR transcripts and triggers rapid NLRP1-dependent pyroptosis. Mechanistically, these processes require 1) DtxR-mediated de-repression of DT production in the bacteria, as well as 2) diphthamide synthesis and 3) ZAKα/p38-driven NLRP1 phosphorylation in the host. In 3D human skin cultures,Corynebacterium diphtheriae infection disrupts barrier function and induces IL-1 driven inflammation. Pharmacologic inhibition of p38 and ZAKα suppresses keratinocyte pyroptosis and rescues barrier integrity ofCorynebacterium diphtheriae-treated organotypic skin. In summary, these findings implicate RSR and the NLRP1 inflammasome in antibacterial innate immunity and might explain certain aspects of diphtheria pathogenesis.KEY POINTSEEF1/EEF2-targeting bacterial exotoxins activate the human NLRP1 inflammasome.DT+ve toxigenicCorynebacterium diphtheriaeinduces ZAKα-driven RSR and NLRP1-driven pyroptosis in human keratinocytes.Identification of transcripts that are induced by multiple RSR agents across multiple cell types.p38 and ZAKα inhibition rescues epidermal integrity by limiting pyroptosis in 3D skin mode of cutaneous diphtheria.

Inflammasomes integrate cytosolic evidence of infection or damage to mount inflammatory responses. The inflammasome sensor NLRP1 is expressed in human keratinocytes and coordinates inflammation in the skin. We found that diverse stress signals induce human NLRP1 inflammasome assembly by activating MAP kinase p38: While the ribotoxic stress response to UV and microbial molecules exclusively activates p38 through MAP3K ZAKα, infection with arthropod-borne alphaviruses, including Semliki Forest and Chikungunya virus, activates p38 through ZAKα and potentially other MAP3K. We demonstrate that p38 directly phosphorylates NLRP1 and that serine 107 in the linker region is critical for activation. NLRP1 phosphorylation is followed by ubiquitination of NLRP1PYD, N-terminal degradation of NLRP1, and nucleation of inflammasomes by NLRP1UPA-CARD. In contrast, activation of NLRP1 by nanobody-mediated ubiquitination, viral proteases, or inhibition of DPP9 was independent of p38 activity. Taken together, we define p38 activation as a unifying signaling hub that controls NLRP1 inflammasome activation by integrating a variety of cellular stress signals relevant to the skin.

SummaryInflammasomes integrate cytosolic evidence of infection or damage to mount inflammatory responses. The inflammasome sensor NLRP1 is expressed in human keratinocytes and coordinates inflammation in the skin. We found that diverse stress signals converge on the activation of p38 kinases to initiate human NLRP1 inflammasome assembly: UV irradiation and microbial molecules that initiate the ribotoxic stress response critically relied on the MAP3 kinase ZAKα to activate p38 and ultimately human NLRP1. Infection with insect-transmitted alphaviruses, including Semliki Forest, Ross River, and Chikungunya virus, also activated NLRP1 in a p38-dependent manner. In the absence on ZAKα, inflammasome assembly was maintained, although at reduced levels, indicating contribution of other upstream kinases. NLRP1 activation by direct nanobody-mediated ubiquitination was independent of p38 activity. Stimulation of p38 by overexpression of MAP2 kinases MKK3 or MKK6 is sufficient for NLRP1 activation, and NLRP1 is directly phosphorylated by p38. Taken together, we define p38 activation as a unifying signaling hub that controls NLRP1 inflammasome activation by integrating a variety of cellular stress signals relevant to the skin.