Enteroviral 3C protease activates the human NLRP1 inflammasome in airway epithelia

A ligand is located, at long last! Members of the Nod-like receptor (NLR) family act as intracellular sensors of infection. Once they recognize pathogen-associated molecular patterns, they assemble into signaling complexes called inflammasomes, which induce proinflammatory cytokines and pyroptotic cell death. Although rodent NLR family pyrin domain containing 1 (NLRP1) can recognize bacterial toxins and protozoan pathogens, the ligands for human NLRP1 have remained elusive. Robinson et al. found that human NLRP1 senses and is activated by enteroviruses. During human rhinovirus (HRV) infection, the HRV 3C protease cleaves an autoinhibitory N-terminal fragment from NLRP1, which is subsequently degraded. The NLRP1 C-terminal fragment that is released then initiates inflammasome formation. This work offers insights into immune sensing of respiratory viral infections and provides an example of the N-terminal glycine degron pathway in human innate immunity. Science, this issue p. eaay2002 A rhinovirus protease provides a pathogen-derived trigger for the inflammasome, which may contribute to inflammatory respiratory disease. INTRODUCTION The innate immune system uses a variety of sensor proteins to detect infections and injuries. The nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domains-containing proteins (NLRPs) are a family of cytosolic sensors involved in the initiation of the host innate immune response. Upon activation, NLRPs form a multiprotein immune effector complex known as the inflammasome. Assembly of the inflammasome leads to caspase-1 activation, the secretion of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-18, and gasdermin D–dependent pyroptotic cell death. These processes orchestrate the downstream immune response to microbial infections and injuries. Genetic data support an important role for human NLRPs in antimicrobial defense. Dysregulated NLRP-driven inflammasome signaling also contributes to the pathogenesis of several autoimmune and autoinflammatory diseases. RATIONALE Understanding the molecular basis by which individual human NLRPs respond to specific stimuli is key to the study of innate immunity and inflammatory diseases. NLRP1 was one of the first human NLRPs to be described. Yet the pathogen-derived triggers for human NLRP1 have not been identified, and its mechanism of activation is much less understood than that of other NLRPs such as NLRP3. Although compelling evidence shows that rodent NLRP1 can be activated by specific bacterial toxins and protozoan infections, human NLRP1 is not triggered by these stimuli. Germline-activating NLRP1 mutations in humans cause a variety of inherited diseases characterized by sustained inflammation and hyperplasia in the skin and the airway. Thus, we hypothesized that some unknown molecule(s) from common skin or airway pathogens activate human NLRP1. RESULTS Here, we report that human NLRP1 senses and is activated by enteroviral infections. Enteroviruses cause a range of human diseases, including hand-foot-and-mouth disease, polio, and the common cold. In our model system, we show that NLRP1 is highly expressed in airway epithelia and is activated by the human rhinovirus (HRV), a type of enterovirus that causes the common cold. During infection, a HRV component known as 3C protease (HRV-3Cpro) cleaves NLRP1 specifically between amino acids Q130 and G131. This cleavage causes the autoinhibitory N-terminal fragment (amino acids 131 to 1212) to become recognized by the N-terminal glycine degron pathway and subsequently cleared by the proteasome. This sequence of events culminates in inflammasome activation mediated by the liberated NLRP1 C-terminal fragment (amino acids 1213 to 1474), resulting in caspase-1 activation, inflammatory cell death, and the secretion of inflammatory cytokines, such as IL-18, from infected airway epithelial cells. We also show that pharmacologic inhibition of the N-terminal glycine degron pathway by the NEDD8–cullin inhibitor MLN4924 effectively blocks the activation of NLRP1 inflammasome in HRV-infected primary human bronchial epithelial cells. CONCLUSION We establish that enteroviral 3Cpros, which have long been regarded as a key mediator of viral immune evasion, in fact act as a trigger for the NLRP1 inflammasome in primary human airway epithelial cells. These results provide a unified mechanism for proteolysis-mediated activation of NLRP1 in humans and rodents. Our results also reveal a role for the recently described N-terminal glycine degron pathway in human innate immunity. Thus, NLRP1 is a versatile sensor protein, which has evolved to respond to bacterial, viral, and protozoan infections in different species. As both HRV infection and inflammasome-dependent IL-1 cytokines are associated with acute exacerbations of asthma and chronic obstructive pulmonary disease, the NLRP1 inflammasome pathway holds potential as a therapeutic target in the treatment of inflammatory diseases involving the airway epithelia. Proposed mechanism of NLRP1 activation by 3Cpro in HRV-infected human airway epithelium. TLR, Toll-like receptor; RLR, retinoic acid-inducible gene I–like receptors; Pyd, pyrin domain; LRR, leucine-rich repeat; FIIND, function to find domain; CARD, caspase recruitment domain; ASC, apoptosis-associated speck-like protein containing a CARD A, Ala; C, Cys; E, Glu; G, Gly; Q, Gln; R, Arg; S, Ser; T, Thr. Immune sensor proteins are critical to the function of the human innate immune system. The full repertoire of cognate triggers for human immune sensors is not fully understood. Here, we report that human NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) is activated by 3C proteases (3Cpros) of enteroviruses, such as human rhinovirus (HRV). 3Cpros directly cleave human NLRP1 at a single site between Glu130 and Gly131. This cleavage triggers N-glycine–mediated degradation of the autoinhibitory NLRP1 N-terminal fragment via the cullinZER1/ZYG11B complex, which liberates the activating C-terminal fragment. Infection of primary human airway epithelial cells by live human HRV triggers NLRP1-dependent inflammasome activation and interleukin-18 secretion. Our findings establish 3Cpros as a pathogen-derived trigger for the human NLRP1 inflammasome and suggest that NLRP1 may contribute to inflammatory diseases of the airway.