Involvement of the AIM2, NLRC4, and NLRP3 inflammasomes in caspase-1 activation by Listeria monocytogenes

Abstract

Infection with Listeria monocytogenes can cause meningitis and septicemia in newborn, elderly, or immunocompromised individuals. Pregnant women are particularly susceptible to Listeria, leading to a potentially fatal infection. Cytosolic Listeria activates the proinflammatory caspase-1 and induces the processing and secretion of interleukins IL-1beta and IL-18 as well as caspase-1-dependent pyroptosis. This study elucidates the role of various inflammasome components of host macrophages in the proinflammatory response to infection with Listeria. Here, we have used macrophages from AIM2-, NLRC4-, NLRP3-, and ASC-deficient mice to demonstrate that AIM2, NLRC4, and NLRP3 inflammasomes as well as the adaptor protein ASC all contribute to activation of caspase-1 in Listeria-infected macrophages. We show that Listeria DNA, which escapes into the cytosol of infected macrophages, triggers AIM2 oligomerization, caspase-1 activation, and pyroptosis. Interestingly, we found that flagellin-deficient Listeria, like Francisella tularensis, is recognized primarily by the AIM2 inflammasome, as no caspase-1 activation or cell death was observed in AIM2-deficient macrophages infected with this Listeria mutant. We further show that prior priming of NLRC4-deficient macrophages with LPS is sufficient for Listeria-induced caspase-1 activation in these macrophages, suggesting that TLR4 signaling is important for activation of the AIM2 and NLRP3 inflammasomes by Listeria in the absence of NLRC4. Taken together, our results indicate that Listeria infection is sensed by multiple inflammasomes that collectively orchestrate a robust caspase-1 activation and proinflammatory response.

Fig. 1

Listeria flagellin is important for activation of the AIM2 inflammasome. a–b WT and AIM2-KO macrophages were left uninfected (Control) or infected for 5 h with WT (wild type), flaA (flagellin-deficient) or Δhly (LLO-deficient) Listeria monocytogenes (LM) at MOI 50. c–d WT and AIM2-KO macrophages were left uninfected or infected for 5 h with different MOIs of flaA Listeria, or primed with 200 ng/ml of LPS for 4 h, then pulsed with 5 μM Nigericin for 45 min as indicated. Culture supernatants (Sup) and cell lysates (Lysates) were harvested and analyzed by immunoblotting with the indicated antibodies (a and c). Culture supernatants were also analyzed by LDH release assay (b and d). Data are representative of at least three experiments

Fig. 2

NLRC4 and NLRP3 are required for activation of caspase-1 in Listeria-infected LPS-nonprimed macrophages. a WT, AIM2-KO, NLRC4-KO, and NLRP3-KO macrophages were left untreated or infected for 5 h with LM-WT or flaA Listeria at MOI 50. Culture supernatants and cell lysates were collected after infection and analyzed by immunoblotting with the indicated antibodies. b Release of LDH into culture supernatants of the macrophages in a was assayed with the CytoTox 96 LDH-release kit. Data are representative of at least three experiments

Fig. 3

LPS priming restores Listeria-induced inflammasome activation in NLRC4-KO macrophages. a WT, AIM2-KO, NLRC4-KO, and NLRP3-KO macrophages were primed with 200 ng/ml of LPS for 4 h before infection for 5 h with WT or flaA deletion mutant Listeria at MOI 50. Culture supernatants and cell lysates were collected after infection and analyzed by immunoblotting with the indicated antibodies. b Release of LDH into culture supernatants of the macrophages in a was assayed with the CytoTox 96 LDH-release kit. Data are representative of at least three experiments

Fig. 4

ASC is indispensable for caspase-1 activation in Listeria-infected macrophages. WT (a) or ASC-KO (c) macrophages were left untreated or infected for 5 h with WT or flaA Listeria at the indicated MOI. Culture supernatants and cell lysates were collected after infection and analyzed by immunoblotting with the indicated antibodies. Data are representative of at least three experiments. b and d Release of LDH into culture supernatants of the macrophages in a and c, respectively

Fig. 5

Cytoplasmic DNA derived from Listeria triggers AIM2 oligomerization. NLRP3-KO-AIM2-GFP-N1 macrophages were left uninfected (a) or infected for 5 h with WT Listeria at MOI 50 (b) and then stained with the DNA-specific blue Hoechst dye. c NLRP3-KO-AIM2-GFP-N1 macrophages were infected for 5 h with Hoechst-labeled WT Listeria at MOI 50. Live cell images were then collected by confocal microscopy. The white arrow in the blue, green, and merged channels indicate the Hoechst-labeled Listeria cytoplasmic DNA. Data are representative of at least three experiments

Fig. 6

A schematic diagram showing that NLRC4 signals upstream of the NLRP3 and AIM2 inflammasomes in LPS-nonprimed macrophages infected with Listeria. Asterisks indicate activated inflammasomes. See discussion for details