Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity

Abstract

Inflammasomes are a family of cytosolic multiprotein complexes that initiate innate immune responses to pathogenic microbes by activating the caspase 1 protease1,2. Although genetic data support a critical role for inflammasomes in immune defence and inflammatory diseases3, the molecular basis by which individual inflammasomes respond to specific stimuli remains poorly understood. The inflammasome that contains the NLRC4 (NLR family, CARD domain containing 4) protein was previously shown to be activated in response to two distinct bacterial proteins, flagellin4,5 and PrgJ6, a conserved component of pathogen-associated type III secretion systems. However, direct binding between NLRC4 and flagellin or PrgJ has never been demonstrated. A homologue of NLRC4, NAIP5 (NLR family, apoptosis inhibitory protein 5), has been implicated in activation of NLRC4 (refs 7–11), but is widely assumed to have only an auxiliary role1,2, as NAIP5 is often dispensable for NLRC4 activation7,8. However, Naip5 is a member of a small multigene family12, raising the possibility of redundancy and functional specialization among Naip genes. Here we show in mice that different NAIP paralogues determine the specificity of the NLRC4 inflammasome for distinct bacterial ligands. In particular, we found that activation of endogenous NLRC4 by bacterial PrgJ requires NAIP2, a previously uncharacterized member of the NAIP gene family, whereas NAIP5 and NAIP6 activate NLRC4 specifically in response to bacterial flagellin. We dissected the biochemical mechanism underlying the requirement for NAIP proteins by use of a reconstituted NLRC4 inflammasome system. We found that NAIP proteins control ligand-dependent oligomerization of NLRC4 and that the NAIP2–NLRC4 complex physically associates with PrgJ but not flagellin, whereas NAIP5–NLRC4 associates with flagellin but not PrgJ. Our results identify NAIPs as immune sensor proteins and provide biochemical evidence for a simple receptor–ligand model for activation of the NAIP–NLRC4 inflammasomes.

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Figure 1: NAIP2 is required in macrophages for inflammasome activation in response to PrgJ.
Figure 2: Reconstitution of the NAIP5–NLRC4 inflammasome in 293T cells.
Figure 3: NAIP5 is required for formation of a hetero-oligomeric complex that contains NLRC4, NAIP5 and flagellin.
Figure 4: NAIP paralogues confer specificity to the NLRC4 inflammasome.

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Acknowledgements

Work in R.E.V.’s laboratory is supported by Investigator Awards from the Burroughs Wellcome Fund and the Cancer Research Institute and by NIH grants AI075039, AI080749 and AI063302. We thank J. von Moltke, A. Kintzer and B. Krantz for provision of reagents; S. Mariathasan and V. Dixit for the gift of anti-NLRC4 antibodies and Nlrc4−/− mice; J. D. Sauer and D. Portnoy for development of Listeria strains to deliver PrgJ and FlaA; and E. Michelle Long and W. Dietrich for pCDNA3-NAIP constructs and anti-NAIP antibodies. We thank A. Roberts for her initial efforts to knock down NAIP2, M. Fontana for validating NAIP knockdowns, and J. von Moltke and members of the Barton and Vance laboratories for discussions.

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E.M.K. and R.E.V. conceived the experiments and wrote the paper. E.M.K. performed the experiments.

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Correspondence to Russell E. Vance.

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The authors declare no competing financial interests.

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Kofoed, E., Vance, R. Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature 477, 592–595 (2011). https://doi.org/10.1038/nature10394

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