Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages

Abstract

Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1beta processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial-host interactions.

Figure 1. Flagellin-Independent Caspase-1 Activation and Pyroptosis in Shigella-Infected Macrophages

(A) Immunoblot for FliC expression. Bacterial whole cell lysates were loaded from S. typhimurium strains: wild-type (WT), flagellar mutant (fliA::Tn10); or S. flexneri strains: WT, flagellin mutant (ΔfliC), flagellin overexpressor (ΔfliC/fliC). (B) RT-PCR for fliC mRNA expression in Shigella. As the controls, phoA or ipaB expression was examined. (C) LDH release from BMMs infected with Shigella WT, ΔfliC, or ΔfliC/fliC. Error bars represent mean ± SD. (D) Activation of caspase-1, assessed by immunoblot for the processed p10 fragment after infection with Shigella WT, ΔfliC, or TTSS mutant (S325). (E) Processing of IL-1β, assessed by immunoblot. Results are representative of three experiments.

Figure 2. Ipaf and ASC Are Required for Shigella-Induced Caspase-1 and IL-1β Processing

Wild-type, caspase-1-deficient, Ipaf-deficient, or ASC-deficient BMMs were infected with Shigella WT. Immunoblot for caspase-1 p10 (A, C, and E) and for IL-1β (B, D, and F). Blots are representative of three experiments.

Figure 3. The Early Phase of Pyroptosis Induced by Shigella Is Dependent on Ipaf but Not ASC

Caspase-1-deficient (A and D), Ipaf-deficient (B and E), or ASC-deficient BMMs (C and F) were infected with Shigella WT (A–C) or TTSS mutant S325 (D–F). LDH released from infected BMMs were quantified at the indicated time. Error bars represent mean ± SD.

Figure 4. Autophagy Induced by Amino Acid Starvation or Rapamycin Treatment Is Independent of Caspase-1, Ipaf, and ASC

(A) GFP-LC3-expressing wild-type BMMs and caspase-1-deficient, Ipaf-deficient, or ASC-deficient BMMs were incubated in amino acid–starved conditions for 2 h. GFP fluorescence and differential interference contrast (DIC) were shown separately. Scale bar = 10 μm. (B) BMMs with GFP-LC3-associated autophagosomal particles were quantified. Error bars represent mean ± SD. (C) Endogenous LC3-I to LC3-II conversion upon rapamycin treatment was analyzed by western blotting using anti-LC3 antibody. The immunoblot for β-actin was indicated as an internal control.

Figure 5. Caspase-1 Deficiency Promotes Autophagosome Maturation in Macrophages Infected with Shigella Independently of VirG Protein

GFP-LC3-expressing wild-type or caspase-1-deficient BMMs were infected with Shigella WT, ΔvirG, or TTSS mutant S325. As a control, GFP alone expressing wild-type or caspase-1-deficient BMMs were infected with Shigella WT. (A) At 30 min after infection, the infected cells were immunostainted with Cy5-labeled anti- Shigella LPS antibody (colored red) and examined using a confocal microscope. The merged image with Cy5 bacteria and GFP fluorescence, and differential interference contrast (DIC) were also shown. Scale bars = 10 μm. (B) GFP-LC3-associated intracellular bacteria were quantified. Error bars represent mean ± SD.

Figure 6. Endogenous LC3-I to LC3-II Conversion in BMMs Infected by Shigella

Wild-type, caspase-1-deficient, Ipaf-deficient, or ASC-deficient BMMs were infected with Shigella WT, ΔvirG, or TTSS mutant S325. At 30 min after infection, the total lysates of BMMs were prepared and analyzed by western blotting using anti-LC3 antibody. The immunoblot for β-actin was indicated as an internal control.

Figure 7. Differential Regulation of Shigella-Induced Autophagy by Ipaf and ASC

GFP-LC3-expressing Ipaf-deficient or ASC-deficient BMMs were infected with Shigella WT or TTSS mutant. (A) At 30 min after infection, the infected cells were immunostainted with Cy5-labeled anti-Shigella LPS antibody (colored red) and examined using a confocal microscope. The merged image with Cy5 bacteria and GFP fluorescence, and differential interference contrast (DIC) were also shown. Scale bars = 10 μm. (B) GFP-LC3-associated intracellular bacteria were quantified. Error bars represent mean ± SD.

Figure 8. Autophagy Inhibitor 3-MA Enhances Caspase-1-Independent or Ipaf-Independent Cell Death of Macrophages Infected with Shigella

Wild-type (A and D), caspase-1-deficient (B and E), or Ipaf-deficient BMMs (C and F) were infected with Shigella WT (A–C) or TTSS mutant (S325) (D–F) in the presence or absence of 3-MA (10 mM). Error bars represent mean ± SD. *, p < 0.05 (Mann–Whitney U test).