Critical function for Naip5 in inflammasome activation by a conserved carboxy-terminal domain of flagellin

Abstract

Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes and what they sense remain poorly defined. Here we demonstrate that 35 amino acids of the carboxyl terminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in the intracellular sensor Naip5. These mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Our data clarify the molecular basis for the cytosolic response to flagellin.

Figure 1. The C-terminus of flagellin is necessary and sufficient to trigger IPAF- and caspase-1-dependent macrophage death

(a) Flow cytometry of wild-type, caspase-1-deficient and IPAF-deficient macrophages transduced with retroviruses expressing flagellin from L. pneumophila (FlaA) or a control gene (Irgb10) followed by an internal ribosomal entry site (IRES) and GFP. (b,c) Graphs of flow cytometry data on wild-type or IPAF-deficient macrophages transduced with retroviruses expressing a control protein, full length FlaA, or flagellin lacking the C-terminal 2, 4 or 48 amino acids (FlaACΔ2, FlaACΔ4, FlaACΔ48) (b) or flagellin lacking the N-terminal 309 amino acids (FlaANΔ309) (c). (d) Flow cytometry of wild-type or IPAF-deficient macrophages transduced with retroviruses expressing FlaANΔ309-IRES-GFP (left) or fusions of GFP and the following truncations of the C-terminal end of flagellin: C166 (GFP-C166), C35 (GFP-C35) and C20 (GFP-C20). Results are representative of 2–5 independent experiments.

Figure 2. The cytotoxic C-terminus of flagellin is highly conserved and is distinct from the region sensed by TLR5

(a) Model of the salmonella flagellin monomer as it appears within the assembled flagellar filament. Isoleucine 411 is critical for ‘sensing’ of flagellin by TLR5, and corresponds to I391 in L. pneumophila flagellin. (b) Cell death assayed by release of lactate dehydrogenase (LDH) from B6 macrophages infected with indicated L. pneumophila strains (MOI = 1). Error bars represent s.d. of triplicate samples. (c) Alignment of the C-terminal 35 amino acids of flagellin; bolded leucines in Lp and St flagellin were mutated to alanine in the experiments in Fig. 3. (d,e) Graphs of flow cytometry data on wild-type (WT) or IPAF-deficient (IPAF-KO) macrophages transduced with retroviruses expressing either full-length flagellin from L. pneumophila (Lp-FlaA), S. typhimurium (St-FliC), P. aeruginosa (Pa-FliC) or S. flexneri (Sf-FliC) upstream of IRES-GFP (d) or the C-terminal 35 amino acids (C35) of L. pneumophila (Lp) or S. typhimurium (St) flagellin fused to the C-terminus of GFP (e). The percentage of macrophages that were GFP+ is indicated. Results are representative of 2–5 independent experiments.

Figure 3. Leucines in the C-terminus of L. pneumophila flagellin are critical for activation of the inflammasome

(a) Flow cytometry of wild-type and IPAF-deficient macrophages transduced with GFP-C35 harboring the mutations L470A (GFP-C35A), L472A and L473A (GFP-C35AA), or L470A, L472A and L473A (GFP-C35AAA). (b) Immunoblot of IPAF-deficient macrophages expressing the constructs in a with anti-GFP. (c) Cell death indicated by release of lactate dehydrogenase (LDH) ±s.d. from wild-type macrophages infected with wild-type (WT), flagellin-deficient (ΔflaA) or ΔflaA L. pneumophila complemented with wild-type flagellin (ΔflaA ::flaA) or the leucine to alanine mutants L470A (ΔflaA::flaA-A), L472A and L473A (ΔflaA::flaA-AA), all three mutations (ΔflaA::flaA-AAA), salmonella flagellin ((ΔflaA ::fliC), or the fliI mutant (fliI::Cm), which is non-flagellated but still expresses flaA, . Macrophages were assayed for release of LDH after 4 hours of infection. (d) ELISA assay for release of IL-1β from Pam3CSK4-primed bone-marrow-derived macrophages infected at an MOI of 1 with the indicated strains. (e) Replication of the indicated L. pneumophila strains, in wild-type and IPAF-deficient macrophages, measured by colony forming units. Results are representative of 2–3 independent experiments.

Figure 4. Naip5 is required for activation of caspase-1 and release of IL-1β induced by L. pneumophila

(a) Cell death assayed by release of lactate dehydrogenase (LDH) from peritoneal macrophages infected by wild-type (WT) or flagellin-deficient (ΔflaA) L. pneumophila (MOI = 1). Chr13-AJ mice carry chromosome 13 and the Naip5 locus from A/J mice on the B6 background. (b) Immunoblot of processed p10 of active caspase-1 detected in supernatants of bone-marrow-derived macrophages infected with wild-type (WT) or flagellin-deficient (ΔflaA) L. pneumophila at an MOI of 1. (c) Sandwich ELISA assay for release of IL-1β ±s.d. from Pam3CSK4-primed wild-type (B6), Naip5-deficient (Naip5-KO) or Naip5-heterozygous (Naip5-het) bone-marrow-derived macrophages infected with wild-type (WT) or flagellin-deficient (ΔflaA) L. pneumophila at an MOI of 1. Results are representative of 2–5 independent experiments.

Figure 5. Naip5 is required for restriction of L. pneumophila replication in macrophages

(a) Luminescence assay to measure replication of wild-type (WT), ΔflaA or type IV secretion-deficient ΔdotA L. pneumophila in bone-marrow-derived macrophages (MOI = 0.01). Values represent mean ± s.d. of triplicate samples. (b) Replication measured by colony forming units of ΔflaA, or ΔflaA complemented with either wild-type flaA (ΔflaA::flaA) or with the triple leucineto-alanine mutant (ΔflaA::flaA-AAA, see Fig. 3); colony forming units were evaluated at daily intervals after infection. Results are representative of 2–3 independent experiments.

Figure 6. Role of Naip5 in recognition of L. pneumophila, S. typhimurium and P. aeruginosa

(a) Graph of flow cytometry data on macrophages from the indicated strains transduced with retroviruses expressing GFP fused to the C-terminal 35 amino acids of flagellin from L. pneumophila (Lp-C35) or S. typhimurium (St-C35). (b,c) Cell death indicated by release of lactate dehydrogenase (LDH) ±s.d. from bone marrow-derived macrophages infected with wild-type (WT), SPI-1 type III secretion system-deficient (ΔinvA) or flagellin-deficient (ΔfliCfljB) S. typhimurium (MOI = 2) (b) or in bone marrow-derived macrophages by wild-type P. aeruginosa (MOI = 10) (c). (d) Flow cytometry of macrophages transduced with retroviruses expressing a control protein or full-length L. pneumophila flagellin (FlaA). Results are representative of at least 3 independent experiments.