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 protease. Although genetic data support a critical role for inflammasomes in immune defence and inflammatory diseases, 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, flagellin and PrgJ, 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 role, as NAIP5 is often dispensable for NLRC4 activation. However, Naip5 is a member of a small multigene family, 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.

Figure 1. NAIP2 is required in macrophages for inflammasome activation in response to PrgJ

(a–c) Primary bone-marrow derived macrophages expressing shRNAs targeting NAIP2 (or controls) were infected with flagellin-deficient Listeria monocytogenes (MOI = 5) expressing a secreted ActA100-PrgJ (pPrgJ) or ActA100-FlaA (pFlaA) fusion protein under IPTG-inducible control. (a, b) Cell death (± s.d.) was measured in triplicate by LDH release 6 hours after infection, or (c) Active CASP1 (p10) was measured by western blotting of cell supernatants. (d, e) NAIP2 knockdown cells were infected with wildtype or flagellin-deficient (FliC/FljB⁻) Salmonella Typhimurium and inflammasome activation was measured by (d) LDH release (± s.d.) at 3h after infection or (e) CASP1 processing. Data shown are representative of two (c, e) or three (a, b, d) independent experiments. *, p<0.02 as compared to scramble (Student’s t-test, two-tailed).

Figure 2. Reconstitution of the NAIP5/NLRC4 inflammasome in 293T cells

(a) GFP-marked expression vectors encoding NLRC4, NAIP5, CASP1 and/or flagellin (FlaA) were transiently transfected into 293T cells. Cells were imaged for differential interference contrast (DIC) and GFP fluorescence 48 hours later. Dead cells were stained with 7AAD. (b) GFP-high cells and (c) 7AAD positive cells were quantified (± s.d.) as in a, but with specific expression vectors omitted from the transfection as indicated. CASP1(C284A) is a catalytically dead mutant. (d, e) 293T cells were transfected as indicated and analyzed as above. Data shown (± s.d.) are representative of at least three independent experiments. *, p<0.02 (Student’s t test, two tailed); ns, not significant.

Figure 3. NAIP5 is required for formation of a hetero-oligomeric complex that contains NLRC4, NAIP5 and flagellin

(a) 293T cells were transfected as indicated, followed by analysis by Blue Native-PAGE or SDS-PAGE, and western blotting. *NS, non-specific band. (b) 293T cells were transfected as indicated and lysates were separated by a first dimension of Blue Native-PAGE followed by a second dimension of SDS-PAGE. (c, d) 293T cells were transfected as indicated and samples were processed and analyzed as in a. Data shown are representative of at least three independent experiments.

Figure 4. NAIP Paralogs Confer Specificity to the NLRC4 Inflammasome

(a) 293T cells were co-transfected with wild-type NAIP5 and NLRC4, alone or in combination with 6x-Myc-FlaA or 6x-Myc-PrgJ followed by Blue Native PAGE 48 hours later. *NS, non-specific band. Whole cell lysates were also separated by conventional 4–12% SDS-PAGE to control for expression of each transfected gene construct (left panel). (b) 293T cells were transfected with wild-type NAIP2 and NLRC4 and analyzed as in a. (c) 293T cells were transfected with wild-type NAIP6 and NLRC4, and analyzed as in a. Data shown are representative of at least three independent experiments.