Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex Si Ming Mana, Lee J. Hopkinsa, Eileen Nugentb, Susan Coxc, Ivo M. Glücka, Panagiotis Tourlomousisa, John A. Wrighta, Pietro Cicutab, Tom P. Monied, and Clare E. Bryanta,1 aDepartment of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, United Kingdom; bSector of Biological and Soft Systems, The Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, United Kingdom; cRandall Division of Cell and Molecular Biophysics, Kings College, London WC2R 2LS, United Kingdom; and dDepartment of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom Edited* by Vishva M. Dixit, Genentech, San Francisco, CA, and approved April 15, 2014 (received for review February 15, 2014) Pathogen recognition by nucleotide-binding oligomerization do- Escherichia coli (5), Listeria monocytogenes (6, 7), Pseudomonas main-like receptor (NLR) results in the formation of a macromo- aeruginosa (5), Salmonella enterica serovar Typhimurium (S. Typhi- lecular protein complex (inflammasome) that drives protective murium) (5, 8–10), and Yersinia species (11). In mouse macro- inflammatory responses in the host. It is thought that the number phages, the NLRC4 inflammasome responds to flagellin and type III of inflammasome complexes forming in a cell is determined by the secretion system-associated needle or rod proteins (5, 8, 9) after number of NLRs being activated, with each NLR initiating its own their detection by NLR family, apoptosis inhibitory protein (NAIP) inflammasome assembly independent of one another; however, 5 or NAIP6 and NAIP1 or NAIP2, respectively (12–15). Phos- we show here that the important foodborne pathogen Salmonella phorylation of NLRC4 at a single, evolutionarily conserved residue, enterica serovar Typhimurium (S. Typhimurium) simultaneously Ser 533, by PKCδ kinase is required for NLRC4 inflammasome activates at least two NLRs, whereas only a single inflamma- assembly (16). The NLRP3 inflammasome is activated by a large some complex is formed in a macrophage. Both nucleotide-binding repertoire of DAMPs, including ATP, nigericin, maitotoxin, domain and leucine-rich repeat caspase recruitment domain 4 and uric acid crystals, silica, aluminum hydroxide, and muramyl di- nucleotide-binding domain and leucine-rich repeat pyrin domain 3 peptide (17–20). NLRP3 is also activated by bacterial PAMPs from are simultaneously present in the same inflammasome, where many species, including Aeromonas species (4, 21), L. monocytogenes both NLRs are required to drive IL-1β processing within the Salmo- (6, 7, 22), Neisseria gonorrhoeae (23), S. Typhimurium (10), Strep- nella-infected cell and to regulate the bacterial burden in mice. tococcus pneumoniae (24), and Yersinia species (11). The mecha- Superresolution imaging of Salmonella-infected macrophages nisms by which NLRC4 and NLRP3 inflammasomes contribute to revealed a macromolecular complex with an outer ring of apopto- host defense against bacterial pathogens are emerging; however, sis-associated speck-like protein containing a caspase activation little is known about the dynamics governing inflammasome as- and recruitment domain and an inner ring of NLRs, with active sembly in infections caused by bacteria that activate multiple NLRs, caspase effectors containing the pro–IL-1β substrate localized in- such as S. Typhimurium (10), A. veronii (4), and Yersinia (11). ternal to the ring structure. Our data reveal the spatial localization NLRP3 does not have a CARD and requires ASC to interact of different components of the inflammasome and how different with the CARD of procaspase-1. This interaction requires a members of the NLR family cooperate to drive robust IL-1β pro- charged interface around Asp27 of the procaspase-1 CARD (25). cessing during Salmonella infection. Significance innate immunity | bacteria | caspase-1 | caspase-8 | ASC The nucleotide-binding oligomerization domain-like receptor nflammasomes are cytosolic multimeric protein complexes (NLR) family members, NLRC4 and NLRP3, activate the inflam- Iformed in the host cell in response to the detection of patho- masome to provide host defenses against infection. The precise gen-associated molecular patterns (PAMPs) or danger-associated molecular constituents of an inflammasome are unknown; molecular patterns (DAMPs). Formation of the inflammasome in however, it is believed that receptor-specific complexes con- response to PAMPs is critical for host defense because it facilitates taining apoptosis-associated speck-like protein containing processing of the proinflammatory cytokines pro–IL-1β and pro– a caspase activation and recruitment domain (ASC) and cas- IL-18 into their mature forms (1). The inflammasome also initiates pase-1 are formed. Here, we used confocal and superresolution microscopy to show that in macrophages infected with Sal- host cell death in the form of pyroptosis, releasing macrophage- monella resident microbes to be killed by other immune mechanisms (2). Typhimurium, a pathogen that activates two distinct NLRs, ASC forms an outer ring-like structure that comprises The current paradigm is that there are individual, receptor-specific NLRC4, NLRP3, caspase-1, caspase-8, and pro–IL-1β within the inflammasomes consisting of one nucleotide-binding oligomeriza- same macromolecular complex. These results suggest that the tion domain-like receptor (NLR; leucine-rich repeat–containing) inflammasome is a highly dynamic macromolecular protein or PYHIN [pyrin domain and hematopoietic expression, interferon- complex capable of recruiting different NLRs and effectors to inducible nature, and nuclear localization (HIN) domain-containing] coordinate inflammasome responses to infection. receptor, the adaptor protein apoptosis-associated speck-like protein IMMUNOLOGY containing a caspase activation and recruitment domain (CARD; Author contributions: S.M.M., L.J.H., E.N., P.C., and C.E.B. designed research; S.M.M., L.J.H., ASC), and caspase-1 (3). How the protein constituents of the inflam- E.N., I.M.G., P.T., and J.A.W. performed research; E.N., S.C., and P.C. contributed new masome are spatially orientated is unclear. reagents/analytic tools; S.M.M., L.J.H., E.N., S.C., P.T., T.P.M., and C.E.B. analyzed data; Nucleotide-binding domain and leucine-rich repeat caspase and S.M.M., T.P.M., and C.E.B. wrote the paper. recruitment domain 4 (NLRC4) and nucleotide-binding domain The authors declare no conflict of interest. and leucine-rich repeat pyrin domain 3 (NLRP3) are the best- *This Direct Submission article had a prearranged editor. characterized inflammasomes, especially with respect to their 1To whom correspondence should be addressed. E-mail: [email protected]. responses to pathogenic bacteria. The NLRC4 inflammasome is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. activated primarily by bacteria, including Aeromonas veronii (4), 1073/pnas.1402911111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1402911111 PNAS | May 20, 2014 | vol. 111 | no. 20 | 7403–7408 Downloaded by guest on September 25, 2021 Whether ASC is also required for the assembly of the NLRC4 the cytosol of unstimulated BMMs (Fig. S1A). Each infected cell inflammasome is less clear. NLRC4 contains a CARD that can contained one ASC speck with a diameter of 0.6–0.7 μm after interact directly with the CARD of procaspase-1 (26); however, infection with S. Typhimurium (Fig. 1A). Protein oligomeriza- ASC is required for some of the responses driven by NLRC4 (27). tion assays confirmed that ASC formed oligomers following Macrophages infected with S. Typhimurium or other pathogens Salmonella infection (Fig. 1B). Specks that appeared in a cross- exhibit formation of a distinct cytoplasmic ASC focus or speck, section orientation had an apparent hole in the middle (Fig. 1A). which can be visualized under the microscope and is indicative of A similar ASC “ring” structure was observed in human THP-1 inflammasome activation (10, 28, 29). Our laboratory and others macrophages (Fig. 1C and Fig. S1B). We used Bayesian locali- have shown that only one ASC speck is formed per cell irre- zation superresolution microscopy to obtain higher magnifica- spective of the stimulus used (29–32). However, many bacteria tion images of the specks in cross-section (33). After infection of activate two or more NLRs, and it is unclear whether a singular THP-1 cells or BMMs with S. Typhimurium (MOI of 10) for 30 inflammasome is formed at a time or if multiple inflammasomes min, immunolabeled ASC specks from different cells were vi- are formed independent of each other, with each inflammasome sualized and the images were compared with each other. Anal- containing one member of the NLR family. ysis of multiple cross-sections of an ASC speck revealed that it In this study, we describe the endogenous molecular con- forms a ring-like structure (Fig. 1D). Immunolabeling of ASC stituents of the Salmonella-induced inflammasome and their spa- using two different antibodies revealed the same ring-like tial orientation. In cross-section, ASC forms a large external ring structure (Fig. S1C), suggesting that this structure is not due with the NLRs and caspases located internally. Critically, NLRC4, to the effect of a particular antibody. Further analysis of the NLRP3, caspase-1, and caspase-8 coexist in the same ASC speck to ASC specks showed the presence of side filaments frequently coordinate pro–IL-1β processing. All ASC specks