Asc and Ipaf Inflammasomes direct distinct pathways for caspase-1 activation in response to Legionella pneumophila

Abstract

Caspase-1 activation is a key feature of the innate immune response of macrophages elicited by pathogens and a variety of toxins. Here, we determined the requirement for different adapter proteins involved in regulating host processes mediated by caspase-1 after macrophage infection by Legionella pneumophila. The adapter protein Asc was found to be important for caspase-1 activation during L. pneumophila infection. Activation of caspase-1 through Asc did not require the flagellin-sensing pathway involving the host nucleotide-binding domain and leucine-rich repeat-containing protein Ipaf (NLRC4). Asc-dependent caspase-1 activation was inhibited by high extracellular potassium levels, whereas Ipaf-dependent activation was unaffected by potassium treatment. Activation of caspase-1 in macrophages occurred independently of Nalp3 and proteasome activity, suggesting that a previously uncharacterized mechanism for caspase-1 activation through Asc may be triggered by L. pneumophila. Rapid pore formation and pyroptosis induced by L. pneumophila required caspase-1, Ipaf, and bacterial flagellin but occurred independently of Asc. Equivalent levels of active interleukin-18 (IL-18) were detected in the lungs of mice infected with a flagellin-deficient strain of L. pneumophila and Asc-deficient mice infected with wild-type L. pneumophila. Active IL-18 was undetectable in the lungs of Asc-deficient mice infected with an L. pneumophila flagellin mutant, indicating independent roles for Ipaf and Asc in caspase-1-mediated processing and release of IL-18 in vivo. Ipaf-dependent activation of caspase-1 restricted bacterial replication in vivo, whereas Asc was dispensable for restriction of L. pneumophila replication in mice. Thus, L. pneumophila-mediated caspase-1 activation involves the coordinate activities of inflammasomes differentially regulated by Ipaf and Asc.

FIG. 1.

Caspase-1 activation by macrophages in response to L. pneumophila involves both Ipaf and Asc. BMMs from wild-type (WT), Asc^−/−, Ipaf^−/−, or Casp1^−/− mice were either noninfected or infected with wild-type or dotA L. pneumophila at an MOI of 10. (A and B) IL-1β (A) and IL-18 (B) levels in culture supernatants at 8 h postinfection were quantified by ELISA. Data are represented as averages ± standard deviations. (C) Immunoblot of the p10 fragment of caspase-1 from combined supernatants and pellets of BMMs obtained at 4 h postinfection. Data are representative of four (A) or three (B and C) experiments. ND, measurable quantities not detected.

FIG. 2.

L. pneumophila activates caspase-1 through Asc, independent of the Ipaf/flagellin pathway. BMMs from wild-type (WT), Asc^−/−, Ipaf^−/−, or Casp1^−/− mice were infected with the indicated L. pneumophila variants at an MOI of 10. (A and B) IL-1β (A) and IL-18 (B) levels in culture supernatants at 8 h postinfection were quantified by ELISA. Data are represented as averages ± standard deviations. (C) Immunoblot of the p10 fragment of caspase-1 from combined supernatants and pellets of BMMs obtained at 4 h postinfection. Data are representative of four (A) or three (B and C) experiments. ND, measurable quantities not detected.

FIG. 3.

Asc-dependent IL-1β and IL-18 release by macrophages in response to L. pneumophila is inhibited by high extracellular potassium. BMMs from wild-type, Asc^−/−, Ipaf^−/−, or Casp1^−/− mice were infected with wild-type or flaA L. pneumophila at an MOI of 10. During infection, cells were cultured in the presence of an additional 50 mM KCl; control cells were incubated with an additional 50 mM NaCl. IL-1β (A), IL-18 (B), and TNF-α (C) levels in culture supernatants at 8 h postinfection were quantified by ELISA. Data are represented as averages ± standard deviations. Data are representative of four (A) or three (B and C) experiments.

FIG. 4.

High extracellular potassium levels inhibit Asc-dependent caspase-1 activation. An immunoblot of the p10 fragment of caspase-1 from combined supernatants and pellets of BMMs from wild-type (WT), Asc^−/−, or Ipaf^−/− mice is shown. Cells were infected for 4 h with wild-type, dotA, or flaA L. pneumophila at an MOI of 10 in the presence of an additional 50 mM NaCl or 50 mM KCl. Data are representative of three experiments.

FIG. 5.

Macrophages deficient in both Ipaf and Asc fail to activate caspase-1 in response to L. pneumophila. BMMs from wild-type (WT), Asc^−/−, Ipaf^−/−, Ipaf^−/− Asc^−/−, or Casp1^−/− mice were infected with the indicated L. pneumophila variants at an MOI of 10. (A and B) IL-1β (A) and IL-18 (B) levels in culture supernatants at 8 h postinfection were quantified by ELISA. Data are represented as averages ± standard deviations. (C) Immunoblot of the p10 fragment of caspase-1 from combined supernatants and pellets of BMMs obtained at 4 h postinfection. Data are representative of four (A and C) or two (B) experiments. ND, measurable quantities not detected.

FIG. 6.

L. pneumophila pathways for caspase-1 activation do not involve Nalp3 or the proteasome. (A) BMMs from wild-type (WT), Asc^−/−, Nalp3^−/−, or Casp1^−/− mice were infected with wild-type or flaA L. pneumophila at an MOI of 10 and assayed for IL-1β in supernatants at 8 h postinfection. (B and C) Wild-type or Asc-deficient (Asc^−/−) BMMs were pretreated with LPS (1 μg/ml) for 3 hours, followed by infection with wild-type or dotA L. pneumophila in the presence of dimethyl sulfoxide (DMSO) (mock treated) or MG132 and assayed for IL-1β or IL-18 in culture supernatants at 6 h postinfection. Data are represented as averages ± standard deviations. (D) LDH release assays were performed at 6 h postinfection for wild-type macrophages infected with wild-type or dotA L. pneumophila in the presence or absence of MG132. Values represent the percentage of LDH released compared to that for cells lysed with Triton X-100. Data are represented as averages ± standard deviations. Data are representative of two experiments.

FIG. 7.

Pore formation and cell lysis induced by L. pneumophila requires caspase-1, Ipaf, and bacterial flagellin but not Asc. BMMs from wild-type (WT), Asc^−/−, Ipaf^−/−, or Casp1^−/− mice were either noninfected or infected with the indicated L. pneumophila variants at an MOI of 50. (A and D) Macrophages were assayed for PI uptake at 2 h postinfection by flow cytometry. Noninfected controls are represented in gray with a dashed outline, and the overlaid samples are represented by a solid black line. (B, C, and E) LDH release assays were performed at the indicated times for wild-type BMMs infected with indicated L. pneumophila variants (B and E) or wild-type, Asc^−/−, Ipaf^−/−, or Casp1^−/− BMMs infected with wild-type L. pneumophila (C). Values represent the percentage of LDH released compared to that for cells lysed with Triton X-100. Data points are averages ± standard deviations. Data for wild-type macrophages infected with wild-type L. pneumophila are the same in panels B and C. Macrophages were incubated in the presence of an additional 50 mM KCl or 50 mM NaCl where indicated (D and E). Data are representative of five (A), four (B and C), or three (D and E) experiments.

FIG. 8.

L. pneumophila induces IL-18 release through Ipaf/flagellin- and Asc-dependent pathways in the mouse lung. Wild-type (WT), Ipaf^−/−, Asc^−/−, and Casp1^−/− mice were infected with wild-type, dotA, or flaA L. pneumophila through intranasal inoculation. (A) At 24 h postinfection, bronchoalveolar lavage fluid was assayed for IL-18 by ELISA as described above. IL-18 levels indicated are the averages ± standard errors for five mice in all groups except that of C57BL/6 mice infected with PBS or dotA L. pneumophila, which contained four mice. (B and C) For assaying L. pneumophila growth, wild-type mice infected with the indicated L. pneumophila variants (B) and wild-type, Ipaf^−/−, Asc^−/−, or Casp1^−/− mice infected with wild-type L. pneumophila (C) were sacrificed at various time points, and the lungs were harvested for quantifying CFU. Each point is displayed as the average ± standard error for four mice. Data for C57BL/6 mice infected with wild-type L. pneumophila are the same in panels B and C. An asterisk indicates a significant difference (P < 0.05) when comparing the indicated sample to a wild-type mouse infected with wild-type L. pneumophila. Data are representative of three experiments.