Immune Netw.  2012 Dec;12(6):284-290. 10.4110/in.2012.12.6.284.

Salmonella Promotes ASC Oligomerization-dependent Caspase-1 Activation

• Hwang I
• Park S
• Hong S
• Kim EH
• Yu JW

• KMID: 2150761
• DOI: http://doi.org/10.4110/in.2012.12.6.284

Abstract

Innate immune cells sense and respond to the cytoplasmic infection of bacterial pathogens through NLRP3, NLRC4 or AIM2 inflammasome depending on the unique molecular pattern of invading pathogens. The infection of flagellin- or type III secretion system (T3SS)-containing Gram-negative bacteria such as Salmonella enterica serovar Typhimurium (S. typhimurium) or Pseudomonas aeruginosa (P. aeruginosa) triggers NLRC4-dependent caspase-1 activation leading to the secretion of proinflammatory cytokines such as interleukin-1-beta (IL-1beta) and IL-18. Previous studies have shown that apoptosis-associated speck-like protein containing a CARD (ASC) is also required for Salmonella-induced caspase-1 activation, but it is still unclear how ASC contributes to the activation of NLRC4 inflammasome in response to S. typhimurium infection. In this study, we demonstrate that S. typhimurium triggers the formation of ASC oligomer in a potassium depletion-independent manner as determined by in vitro crosslinking and in situ fluorescence imaging. Remarkably, inhibition of potassium efflux failed to block Salmonella-promoted caspase-1 activation and macrophage cell death. These results collectively suggest that ASC is substantially oligomerized to facilitate the activation of caspase-1 in response to S. typhimurium infection. Contrary to NLRP3 inflammasome, intracellular potassium depletion is not critical for NLRC4 inflammasome signaling by S. typhimurium.

Keyword

Inflammasome; Caspase-1; Salmonella; ASC oligomerization

MeSH Terms

Cell Death
Cytokines
Cytoplasm
Gram-Negative Bacteria
Interleukin-18
Macrophages
Optical Imaging
Potassium
Pseudomonas aeruginosa
Salmonella
Salmonella enterica
Cytokines
Interleukin-18
Potassium

Figure

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  Figure 1 Prevention of potassium efflux blocks the caspase-1 activation by nigericin, but not by Salmonella. (A) LPS-primed BMDMs were untreated or pretreated with zVAD (10 µM), KCl or glybenclamide (glyben, 50 µM) for 30 min as indicated, and then infected with S. typhimurium (MOI 30) as described in Materials and methods. Cultural supernatants (Sup) or soluble lysates (Lys) were immunoblotted with anti-caspase-1 antibody. (B) LPS-primed BMDMs were pretreated with the indicated inhibitors as in (A) and stimulated with nigericin (Niger, 5 µM) for 45 min. Supernatants or lysates were immunoblotted with the appropriate antibodies as indicated.

• 

  Figure 2 Prevention of potassium efflux blocks the ASC oligomerization by nigericin, but not by Salmonella. (A) LPS-primed BMDMs were pretreated with the indicated inhibitors and stimulated with nigericin (5 µM) for 45 min. The cross-linked pellets (Pell), soluble lysates (Lys) and supernatants (Sup) were immunoblotted with anti-caspase-1 or anti-ASC antibodies. (B) LPS-primed BMDMs were infected with S. typhimurium (Sal, MOI 30) or treated with nigericin (5 µM) in the presence or absence of glybenclamide, and assayed as described in (A).

• 

  Figure 3 Potassium efflux-independent ASC oligomerization by Salmonella infection. (A) PMA-primed THP-1-ASC-GFP cells were infected with S. typhimurium (Sal) at a MOI of 10 or 30 or stimulated with nigericin (10 µM). The number of ASC pyroptosome was counted using a fluorescent microscope and the ASC speck-containing cells were represented as a relative percentage compared to the total cell number. Cultural supernatants or lysates were immunoblotted with anti-caspase-1 or anti-IL-1β antibodies. (B) PMA-primed THP-1-ASC-GFP cells were infected with S. typhimurium (MOI 30) or treated with nigericin (5µM), and assayed as described in (A). The asterisk indicates a significant difference compared to only nigericin-treated cells (n=3, p<0.05).

• 

  Figure 4 Differential effect of potassium efflux on inflammasome-mediated macrophage cell death. LPS-primed mouse BMDMs were infected with S. typhimurium (MOI 30, A and C) or stimulated with nigericin (5 µM, B and C) together with the pretreatment of the indicated inhibitors. Cell death was determined by LDH release into extracellular medium as described in Materials and methods. Asterisks indicate significant differences compared to only nigericin-treated cells (B, n=3, p<0.005; C, n=3, p<0.001).

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