Multiple Nod-like receptors activate caspase 1 during Listeria monocytogenes infection - PubMed (original) (raw)

Multiple Nod-like receptors activate caspase 1 during Listeria monocytogenes infection

Sarah E Warren et al. J Immunol. 2008.

Abstract

Listeria monocytogenes escapes from the phagosome of macrophages and replicates within the cytosolic compartment. The macrophage responds to L. monocytogenes through detection pathways located on the cell surface (TLRs) and within the cytosol (Nod-like receptors) to promote inflammatory processes aimed at clearing the pathogen. Cytosolic L. monocytogenes activates caspase 1, resulting in post-translational processing of the cytokines IL-1beta and IL-18 as well as caspase 1-dependent cell death (pyroptosis). We demonstrate that the presence of L. monocytogenes within the cytosolic compartment induces caspase 1 activation through multiple Nod-like receptors, including Ipaf and Nalp3. Flagellin expression by cytosolic L. monocytogenes was detected through Ipaf in a dose-dependent manner. Concordantly, detection of flagellin promoted bacterial clearance in a murine infection model. Finally, we provide evidence that suggests cytosolic L. monocytogenes activates caspase 1 through a third pathway, which signals through the adaptor protein ASC. Thus, L. monocytogenes activates caspase 1 in macrophages via multiple pathways, all of which detect the presence of bacteria within the cytosol.

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1

FIGURE 1

Cytosolic Listeria monocytogenes in macrophages induces IL-1_β_ secretion, which is partially dependent upon the NLRs Nalp3 and Ipaf. WT, Nalp3−/−, or Ipaf−/− BMM were stimulated with LPS to induce proIL-1_β_ expression before infection with WT or LLO-deficient L. monocytogenes (MOI 6). IL-1_β_ secretion was assessed by ELISA as a measure of caspase 1 activation at (A) 1 h or (B) 4 h post infection. Error bars, SD. Representative of three independent experiments.

FIGURE 2

FIGURE 2

Ipaf is activated in response to cytosolic L. monocytogenes flagellin. A, LPS stimulated macrophages were transfected with purified flagellin from S. typhimurium (FljB) or L. monocytogenes (FlaA) or hook protein (FlgE) (negative control). IL-1_β_ secretion was assessed after 1 h by ELISA. B, LPS stimulated WT or Ipaf_−/− BMM were transfected with 31 ng of either FlaA or FlgE and IL-1_β secretion was assessed after 2 h. Representative of three independent experiments.*, p < 0.05.

FIGURE 3

FIGURE 3

flgK L. monocytogenes are amotile and secrete increased amounts of flagellin into the environment. A, Schematic of the structure of L. monocytogenes flagella. CM, Cell membrane; and PGN, peptidoglycan. B, Motility plate demonstrating that WT and LLO L. monocytogenes are motile but flaA and flgK L. monocytogenes are not. C, Proteins were precipitated from bacterial pellets and supernatants and run on western blot to assess flagellin expression and secretion. P, Pellet (bacteria associated); and S, supernatant (secreted). **, Nonspecific band.

FIGURE 4

FIGURE 4

Flagellin-dependent variation in caspase 1 processing in L. monocytogenes_-infected macrophages is dependent on Ipaf. A, LPS-primed WT macrophages were infected with WT, flaA, flgK, or ΔLLO L. monocytogenes at MOI 6. IL-1_β secretion from supernatants was assessed 4 h post infection. *, p < 0.05. B, WT macrophages were infected at MOI 6 for 4 h then proteins precipitated from supernatants and combined with cell lysates. Samples were Western blotted then probed for the p10 fragment of caspase 1. U, Uninfected; and **, nonspecific band. L. monocytogenes infected Ipaf_−/− macrophages were analyzed for (C) IL-1_β secretion and (D) caspase 1 processing as in A and B above. Error bars, SD. Representative of three experiments. E, LPS-primed WT macrophages were infected with WT, flaA, flgK, or ΔLLO L. monocytogenes at MOI 1, 3, 6, or 12. IL-1_β_ secretion from supernatants was assessed 4 h post infection.

FIGURE 5

FIGURE 5

Detection of flagellin enhances bacterial clearance. C57BL/6 mice were injected with 104 L. monocytogenes i.v. Five days later, bacterial clearance was assessed by plating organ homogenates and counting CFUs. Geometric mean of 10 mice per strain is shown (−).*, p < 0.05 and **, p < 0.01. NS, Not significant. Ten mice per cohort.

FIGURE 6

FIGURE 6

Ipaf signaling and flagellin detection is independent of ASC. Macrophages were infected with L. monocytogenes strains (MOI 6) for 4 h before IL-1_β_ secretion was determined by ELISA. A) WT, _ASC_−/−, or Ipaf_−/−/ASC_−/− macrophages infected with WT or ΔLLO mutant L. monocytogenes B) _ASC_−/− macrophages infected with WT, flaA, flgK, or ΔLLO L. monocytogenes. Error bars, s.d. Representative of three experiments. *p = 0.05; ***p < 0.001.

FIGURE 7

FIGURE 7

A third receptor activates inflammasome following L. monocytogenes infection. Macrophages were infected with L. monocytogenes strains (MOI 6) and IL-1_β_ secretion determined after four hours. A) WT, _Nalp3_−/−, and _ASC_−/− macrophages infected with WT or ΔLLO mutant L. monocytogenes. B) WT, _Nalp3_−/−, and _ASC_−/− macrophages were infected with either flaA or ΔLLO mutant L. monocytogenes. Error bars, s.d. Representative of three experiments. *p = 0.05.

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