Flagellin-induced NLRC4 phosphorylation primes the inflammasome for activation by NAIP5

The Nlrc4 inflammasome contributes to immunity against intracellular pathogens that express flagellin and type III secretion systems, and activating mutations in NLRC4 cause autoinflammation in patients. Both Naip5 and phosphorylation of Nlrc4 at Ser533 are required for flagellin-induced inflammasome activation, but how these events converge upon inflammasome activation is not known. Here, we showed that Nlrc4 phosphorylation occurs independently of Naip5 detection of flagellin because Naip5 deletion in macrophages abolished caspase-1 activation, interleukin (IL)-1β secretion, and pyroptosis, but not Nlrc4 phosphorylation by cytosolic flagellin of Salmonella Typhimurium and Yersinia enterocolitica. ASC speck formation and caspase-1 expression also were dispensable for Nlrc4 phosphorylation. Interestingly, Helicobacter pylori flagellin triggered robust Nlrc4 phosphorylation, but failed to elicit caspase-1 maturation, IL-1β secretion, and pyroptosis, suggesting that it retained Nlrc4 Ser533 phosphorylating-activity despite escaping Naip5 detection. In agreement, the flagellin D0 domain was required and sufficient for Nlrc4 phosphorylation, whereas deletion of the S. Typhimurium flagellin carboxy-terminus prevented caspase-1 maturation only. Collectively, this work suggests a biphasic activation mechanism for the Nlrc4 inflammasome in which Ser533 phosphorylation prepares Nlrc4 for subsequent activation by the flagellin sensor Naip5.Inflammasomes contribute critically to immunity and antimicrobial host defense of mammalian hosts. Their activation is tightly controlled because aberrant inflammasome signaling is harmful to the host, and results in inflammatory diseases (1, 2). Inflammasomes are a set of cytosolic multiprotein complexes that recruit and activate caspase-1, a key protease that triggers secretion of the inflammatory cytokines interleukin (IL)-1β and IL-18. In addition, caspase-1 induces pyroptosis, a proinflammatory and lytic cell death mode that contributes to pathogen clearance (3, 4). Several inflammasomes respond to a distinctive set of microbial pathogens (5). Activating mutations in the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) member Nlrc4 were recently shown to induce autoinflammation in patients (6–8). Moreover, the inflammasome assembled by Nlrc4 is critically important for clearing a variety of bacterial infections, including Salmonella enterica serovar Typhimurium (S. Typhimurium), Shigella flexneri, Pseudomonas aeruginosa, Burkholderia thailandensis, and Legionella pneumophila (3, 9–17). These intracellularly-replicating bacteria have in common that they propel themselves with flagella (18) and/or express bacterial type III secretion systems (T3SS) to translocate effector proteins into infected host cells (19). Members of the NLR apoptosis-inhibitory protein (Naip) subfamily recognize the cytosolic presence of the building blocks of these evolutionary conserved bacterial structures, and trigger Nlrc4 to assemble an inflammasome (20–25). C57BL/6J mice express four Naip proteins, Naip1, -2, -5, and -6, which are expressed from a multigene cluster located on chromosome 13qD1 (26). Mouse Naip1 and human NAIP bind T3SS needle proteins, Naip2 interacts with the T3SS basal rod component PrgJ, and Naip5 and Naip6 recognize flagellin (20, 22–25).In addition to these Naip sensors, recent work showed that phosphorylation of Nlrc4 at Ser533 is critical for activation of the Nlrc4 inflammasome following infection with S. Typhimurium and L. pneumophila, or transfection of purified S. Typhimurium flagellin (27). Reconstitution of immortalized Nlrc4^−/− macrophages with wild-type Nlrc4 restored S. Typhimurium- and L. pneumophila-induced inflammasome activation, whereas cells reconstituted with Nlrc4 S533A mutant were specifically defective in maturation of caspase-1, secretion of IL-1β, assembly of ASC (apoptosis-associated speck-like protein containing a CARD) specks and induction of pyroptosis by these pathogens (27). However, a central outstanding question is how these upstream events (i.e., bacterial recognition by Naip members and Nlrc4 phosphorylation) relate to each other. Naip binding of bacterial components may trigger Nlrc4 phosphorylation to induce inflammasome activation. Alternatively, Nlrc4 phosphorylation and Naip sensing of flagellin and T3SS may converge independently onto Nlrc4 inflammasome activation.Here, we approached this question by breeding Nlrc4^Flag/Flag mice that express Nlrc4 fused to a carboxy-terminal 3× Flag tag from both Nlrc4 alleles (27) with Naip5-deficient mice (22, 28). We found S. Typhimurium infection and cytosolic delivery of S. Typhimurium flagellin, S. Typhimurium PrgJ and Yersinia enterocolitica flagellin to induce Nlrc4 phosphorylation at Ser533 independently of Naip5. Interestingly, Helicobacter pylori (H. pylori) flagellin induced robust Nlrc4 Ser533 phosphorylation without caspase-1 activation, suggesting that Nlrc4 Ser533 phosphorylation and caspase-1 activation are molecularly decoupled. In agreement, the S. Typhimurium flagellin D0 domain was required and sufficient for Nlrc4 phosphorylation, whereas caspase-1 activation required the flagellin carboxy-terminus. Collectively, this work suggests a biphasic activation mechanism for the Nlrc4 inflammasome in which Ser533 phosphorylation primes Nlrc4 for subsequent activation by the flagellin sensor Naip5.