Listeria monocytogenes engineered to activate the Nlrc4 inflammasome are severely attenuated and are poor inducers of protective immunity

Abstract

Inflammasomes are intracellular multiprotein signaling complexes that activate Caspase-1, leading to the cleavage and secretion of IL-1β and IL-18, and ultimately host cell death. Inflammasome activation is a common cellular response to infection; however, the consequences of inflammasome activation during acute infection and in the development of long-term protective immunity is not well understood. To investigate the role of the inflammasome in vivo, we engineered a strain of Listeria monocytogenes that ectopically expresses Legionella pneumophila flagellin, a potent activator of the Nlrc4 inflammasome. Compared with wild-type L. monocytogenes, strains that ectopically secreted flagellin induced robust host cell death and IL-1β secretion. These strains were highly attenuated both in bone marrow-derived macrophages and in vivo compared with wild-type L. monocytogenes. Attenuation in vivo was dependent on Nlrc4, but independent of IL-1β/IL-18 or neutrophil activity. L. monocytogenes strains that activated the inflammasome generated significantly less protective immunity, a phenotype that correlated with decreased induction of antigen-specific T cells. Our data suggest that avoidance of inflammasome activation is a critical virulence strategy for intracellular pathogens, and that activation of the inflammasome leads to decreased long-term protective immunity and diminished T-cell responses.

Fig. 1.

L. monocytogenes minimally activates the inflammasome. Wild-type (closed symbols) or Caspase-1^−/− (open symbols) mice were infected with 1 × 10⁵ wild-type L. monocytogenes and CFU per organ were determined 48 h postinfection. Data are representative of at least two independent experiments.

Fig. 2.

L. monocytogenes strains that ectopically secrete flagellin hyperinduce host cell death and IL-1β secretion. Cell death (A) and IL-1β (B) secretion were measured following 6 h of infection at an MOI of 5 in wild-type, Caspase-1^−/−, ASC^−/−, or Nlrc4^−/− bone marrow-derived macrophages. Data are representative of at least three independent experiments. *P < 0.05 by Student's t-test.

Fig. 3.

Inflammasome activation attenuates L. monocytogenes virulence. Wild-type (A) or Naip5^−/−/Nlrc4^−/− (B) mice were infected with 1 × 10⁴ wild type (closed symbols) or L. monocytogenes-L.p.FlaA (open symbols) and CFU was determined 48 h postinfection in the spleen (circles) and liver (triangles). Data are representative of at least two independent experiments. *P < 0.05 by Mann-Whitney test.

Fig. 4.

Neither neutrophil depletion or ASC deficiency rescue the L. monocytogenes-L.p.FlaA virulence defect. (A) Wild-type mice were mock treated (closed symbols) or treated with 10 μg/mL of RB6-8C5 anti-GR1 antibody (open symbols) and infected with either 5 × 10³ wild-type + 5 × 10³ LLO_S44A mutants (squares) or 5 × 10³ wild-type + 5 × 10³ L. monocytogenes-L.p.FlaA (triangles) and competitive index was determined 48 h postinfection. Data are representative of at least two experiments. (B) Wild-type (circles) or ASC^−/− (triangles) mice were infected with 1 × 10⁴ wild-type (closed symbols) or L. monocytogenes-L.p.FlaA (open symbols) and CFU were determined 48 h postinfection. Data are representative of at least two independent experiments. *P < 0.05 by Mann-Whitney test.

Fig. 5.

Immunization with inflammasome activating L. monocytogenes results in a loss of protective immunity. Wild-type mice were immunized with1 × 10⁷ (triangles) or 1 × 10³ (squares) ΔactA/inlB (closed symbols) or ΔactA/inlB L. monocytogenes-L.p.FlaA (open symbols) bacteria. Thirty days postimmunization, mice were challenged with 2 × 10⁵ wild-type bacteria and CFU per organ were analyzed 68 to 72 h postchallenge. Data are representative of at least two independent experiments. *P < 0.05 by Mann-Whitney test.

Fig. 6.

L. monocytogenes that activates the Nlrc4 inflammasome impairs the primary specific CD8⁺ T-cell response. C57BL/6 (A) or Caspase1^−/− (B) mice were injected with 1 × 10⁷ CFU of ΔactA/inlB or ΔactA/inlB L. monocytogenes-L.p.FlaA–expressing OVA and B8R epitope. Seven days postimmunization, the percentage of antigen-specific IFN-γ⁺ CD8⁺ T cells was determined using intracellular cytokine staining after in vitro restimulation with the indicated peptide. Values in each plot represent the mean ± SD of antigen-specific cells within a CD8⁺ cell gate among splenocytes from four to five animals per groups. One representative experiment of two to four is shown.