The Nlrc4 Inflammasome Contributes to Restriction of Pulmonary Infection by Flagellated Legionella spp. that Trigger Pyroptosis

Abstract

The Nlrc4 inflammasome is triggered in response to contamination of the host cell cytoplasm with bacterial flagellin, which induces pyroptosis, a form of cell death that accounts for restriction of bacterial infections. Although induction of pyroptosis has been extensively investigated in response to Salmonella typhimurium and Legionella pneumophila, little is known regarding the role of the inflammasome for restriction of non-pneumophila Legionella species. Here, we used five species of the Legionella genus to investigate the importance of the inflammasome for restriction of bacterial infection in vivo. By infecting mice deficient for inflammasome components, we demonstrated that caspase-1 and Nlrc4, but not Asc, contribute to restriction of pulmonary infection with L. micdadei, L. bozemanii, L. gratiana, and L. rubrilucens. L. longbeachae, a non-flagellated bacterium that fails to trigger pyroptosis, was not restricted by the inflammasome and induced death in the infected mice. In contrast to L. longbeachae, flagellin mutants of L. pneumophila did not induce mice death; therefore, besides bypassing the Nlrc4 inflammasome, L. longbeachae may employ additional virulence strategies to replicate in mammalian hosts. Collectively, our data indicate that the Nlrc4 inflammasome plays an important role in host protection against opportunistic pathogenic bacteria that express flagellin.

Keywords: Legionella; Nlrc4; caspase-1; inflammasome; pulmonary infection.

Figure 1

Caspase-1 contributes for restriction of in vivo infection with L. micdadei, L. bozemanii, L. gratiana, and L. rubrilucens. C57BL/6 (closed circles) and caspase-1^−/− (open circles) mice were infected intranasally with 1.0 × 10⁶ Legionella spp. per mouse. (A) L. micdadei. (B) L. bozemanii. (C) L. gratiana. (D) L. rubrilucens. Mice were sacrificed 4, 48, and 96 h after infection and dilutions of the lung homogenates were added to CYE agar plates for CFU determination. Each dot represents a single animal and the horizontal lines represent the average. Six to 10 mice were used per group. Data are representative of those found in four independent experiments. An asterisk indicates a P value of <0.05.

Figure 2

Nlrc4, but not Asc, contributes for restriction of in vivo infection with L. micdadei, L. bozemanii, L. gratiana, and L. rubrilucens. C57BL/6 (closed circles), Nlrc4^−/− (open circles), and Asc^−/− (closed squares) mice were infected intranasally with 1.0 × 10⁶ Legionella spp. per mouse. (A) L. micdadei. (B) L. bozemanii. (C) L. gratiana. (D) L. rubrilucens. Mice were sacrificed 4 or 48 h after infection and dilutions of the lung homogenates were added to CYE agar plates for CFU determination. Each dot represents a single animal and the horizontal lines represent the average. Three to 10 mice were used per group. Data are representative of those found in three independent experiments. An asterisk indicates a P value of <0.05.

Figure 3

Caspase-1 does not influence pulmonary infection by L. longbeachae. C57BL/6 (closed circles) and caspase-1^−/− (opened circles) mice were infected intranasally with Legionella longbeachae for determination of CFU in the lungs (A) and mice survival (B–D). (A) Mice were sacrificed after 4 or 48 h after infection and dilutions of the lung homogenates were added to CYE agar plates for CFU determination. Each dot represents a single animal and the horizontal lines represent the average. Six to 10 mice were used per group. (B–D) Survival of mice inoculated with 10⁵ (B); 10⁶ (C); or 10⁷ (D) L. longbeachae per mice over a period of 10 days. Ten mice were used per group. Data are representative of those found in four independent experiments. An asterisk indicates a P value of <0.05.

Figure 4

Legionella longbeachae, but not flaA mutants of L. pneumophila, is lethal for mice. (A) C57BL/6 mice were inoculated with 10⁵ (diamonds); 10⁶ (triangles); or 10⁷ (squares) L. longbeachae (L. long.) per mouse and the survival was monitored over a period of 10 days. (B) C57BL/6 mice were inoculated 10⁵, 10⁶, or 10⁷ CFUs of wild-type Legionella pneumophila (WT L.p.) or with flagellin mutants L. pneumophila (flaA). The survival was monitored over a period of 10 days. Ten mice were used per each group. Data are representative of those found in four independent experiments.