Salmonella Infection Induces Recruitment of Caspase-8 to the Inflammasome To Modulate IL-1β Production

Si Ming Man, Panagiotis Tourlomousis, Lee Hopkins, Tom P. Monie, Katherine A. Fitzgerald and Clare E. Bryant
J Immunol November 15, 2013, 191 (10) 5239-5246; DOI: https://doi.org/10.4049/jimmunol.1301581

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Salmonella infection induces recruitment of caspase-8 to the ASC inflammasome. (A) Wild type (Caspase-8 +/+ ripk3 +/+), Caspase-8 +/− ripk3 −/−, and Caspase-8 −/− ripk3 −/− primary BMMs were infected with S. Typhimurium (STm; MOI 10) for 30 min and stained for ASC (magenta), active caspase-1 (green), caspase-8 (red), and DNA (blue). Scale bar, 7.5 μm. (B) Percentage of ASC foci that harbored a caspase-1 focus, caspase-8 focus, or both caspase-1 and caspase-8 foci in (A). (C) Wild type, caspase-1 −/− (caspase-11 −/−), and Asc −/− BMMs were infected with S. Typhimurium for 30 min and stained for ASC (red), active caspase-8 (green), and DNA (blue). Scale bar, 10 μm. (D) Percentage of ASC foci containing a caspase-8 focus in (C). (E) Wild type BMMs were stimulated with flagellin from S. Typhimurium for 1 h and stained for ASC (magenta), active caspase-1 (green), caspase-8 (red), and DNA (blue). Scale bar, 7.5 μm. Original magnification ×100 for (A), (C), and (E). (F) Percentage of ASC foci that harbored a caspase-1 focus, caspase-8 focus, or both caspase-1 and caspase-8 foci in (E). Cells were fixed and stained with a rabbit anti-ASC Ab and a rat anti-caspase-8 Ab (A, E) or caspase-8 FLICA stain (C). At least 100 (B, F) or 200 (D) ASC-focus–containing BMMs were counted in each independent experiment. Data are from three independent experiments.

  • FIGURE 2.
    FIGURE 2.

    Salmonella infection induces recruitment of caspase-8 into the inflammasome where it undergoes proteolysis. (A) Unprimed or LPS-primed wild type primary BMMs were infected with S. Typhimurium (STm; MOI 10) for 30 min. Endogenous caspase-8 was immunoprecipitated using an anti–caspase-8 Ab. Western blotting was used to detect caspase-8, ASC, and NLRP3. (B) Western blot analysis of caspase-8 proteolysis in wild type, Nlrc4 −/−, Nlrp3 −/−, Asc −/−, and caspase-1−/− (caspase-11−/−) BMMs infected with STm for 30 min. Data are representative of two (B) or three (A) independent experiments. *H chain of the IP Ab.

  • FIGURE 3.
    FIGURE 3.

    Salmonella infection drives caspase-8–dependent IL-1β production. (A) Unprimed wild type (Caspase-8 +/+ ripk3 +/+), caspase-8 +/− ripk3 −/−, caspase-8 −/− ripk3 −/−, and caspase-8 +/− ripk3 +/− primary BMMs were infected with S. Typhimurium (STm; MOI 1) for 1, 2, 6, and 24 h and levels of IL-1β were measured. (B) Western blot analysis of the cleaved IL-1β p17 subunit (processed IL-1β), cleaved caspase-1 p10 subunit (active caspase-1), cleaved caspase-8 p18 subunit (active caspase-8), procaspase-1, NLRP3, pro–IL-1β, and β-actin in the supernatant or cell lysate of LPS-primed BMMs. (C) BMMs were infected with S. Typhimurium (MOI 1) for 2, 6, and 24 h and levels of TNF-α were measured. (D) LPS-primed wild type and caspase-1 −/− (caspase-11 −/−) BMMs were infected with S. Typhimurium in the presence or absence of a caspase-8 inhibitor (30 μM) for 2 or 6 h. Western blot analysis of the cleaved IL-1β p17 subunit (processed IL-1β), pro–IL-1β, and β-actin in the supernatant or cell lysate. Data are representative of two (C, D) or three (A, B) independent experiments and error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

  • FIGURE 4.
    FIGURE 4.

    Recruitment of pro–IL-1β to the ASC focus is dependent on caspase-1 and unaffected by inhibition of caspase-8. (A and B) LPS-primed wild type or caspase-1 −/− (caspase-11 −/−) BMMs were infected with S. Typhimurium for 30 min in the presence of DMSO (vehicle control) or a caspase-8 inhibitor (50 μM) and were immunostained for active caspase-1 (green), ASC (magenta), pro–IL-1β/IL-1β (red), and DNA (blue). Scale bar, 10 μm. Original magnification ×100. (B) Percentage of ASC foci that harbored caspase-1, pro–IL-1β/IL-1β, or both. At least 100 ASC-focus–containing BMMs were counted for each treatment in each independent experiment. Data are representative of three independent experiments, and error bars represent SEM.

  • FIGURE 5.
    FIGURE 5.

    Salmonella infection does not induce early cell death via Caspase-8. (A) Wild type (Caspase-8 +/− ripk3 +/−), Caspase-8 +/− ripk3 −/−, and Caspase-8 −/− ripk3 −/− primary BMMs were infected with S. Typhimurium (STm) for 2, 6, and 24 h, and levels of lactate dehydrogenase were measured. (B) LPS-primed wild type (caspase-8 +/+ ripk3+/+), caspase-8+/− ripk3 −/−, caspase-8 −/− ripk3 −/−, and caspase-8 +/− ripk3 −/− BMMs were infected with S. Typhimurium (MOI 10) for 30 min or 1 h, and levels of lactate dehydrogenase were measured in the lysate. Data are representative of three independent experiments, and error bars represent SEM.

Additional Files

  • Data Supplement

    Files in this Data Supplement:

    • Supplemental Figures 1-4 (PDF, 877 Kb) - Description:
      Supplemental Figure 1. NLRC4 is required for ASC focus formation in response to infection by S. Typhimurium (S.Tm).
      Supplemental Figure 2. Caspase-8 is required for the induction of pro-IL-1β mRNA following S. Typhimurium (S. Tm) infection.
      Supplemental Figure 3. Absence of Caspase-8 does not impair early IL-18 production.
      Supplemental Figure 4. NLRC4 and caspase-1, but not ASC, induce cell death in response to infection by S. Typhimurium (S.Tm).
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section