Listeriolysin O (LLO), an hly-encoded cytolysin from Listeria monocytogenes, plays an essential role in the entry of this pathogen into the macrophage cytoplasm and is also a key factor in inducing the production of IFN-γ during the innate immune stage of infection. In this study, we examined the involvement of LLO in macrophage production of the IFN-γ-inducing cytokines IL-12 and IL-18. Significant levels of IL-12 and IL-18 were produced by macrophages upon infection with wild-type L. monocytogenes, whereas an LLO-deficient mutant (the L. monocytogenes Δhly) lacked the ability to induce IL-18 production. Complementation of Δhly with hly completely restored the ability. However, when Δhly was complemented with ilo encoding ivanolysin O (ILO), a cytolysin highly homologous with LLO, such a restoration was not observed, although ILO-expressing L. monocytogenes invaded and multiplied in the macrophage cytoplasm similarly as LLO-expressing L. monocytogenes. Induction of IL-18 was diminished when pretreated with a caspase-1 inhibitor or in macrophages from caspase-1-deficient mice, suggesting the activation of caspase-1 as a key event resulting in IL-18 production. Activation of caspase-1 was induced in macrophages infected with LLO-expressing L. monocytogenes but not in those with Δhly. A complete restoration of such an activity could not be observed even after complementation with the ILO gene. These results show that the LLO molecule is involved in the activation of caspase-1, which is essential for IL-18 production in infected macrophages, and suggest that some sequence unique to LLO is indispensable for some signaling event resulting in the caspase-1 activation induced by L. monocytogenes.

Listeria monocytogenes (LM)3 is a Gram-positive facultative intracellular bacterium that often causes life-threatening infections in immunocompromised hosts, including newborns and elderly people (1, 2, 3, 4). The pathogenicity of LM can be attributed to the invasion and subsequent intracellular parasitism in a variety of host cells such as hepatocytes, fibroblasts, and epithelial cells. Professional phagocytes, such as macrophages, are also the major target cells of LM because the pathogen can survive and grow inside macrophages, even once being trapped in phagosomes after phagocytosis. Virulence factors encoded in Listeria pathogenicity island 1 are required for the evasion of intracellular bactericidal mechanisms by LM. Among the group of virulence factors, listeriolysin O (LLO), a 56-kDa cytolysin encoded by hly, is the most important virulence determinant and plays an essential role in bacterial escape from the phagosome into the cytoplasm where the pathogen multiplies efficiently (5, 6, 7).

In mice infected with LM, innate immune cells such as macrophages and dendritic cells are activated to release proinflammatory cytokines, including TNF-α, IL-1, and IL-6. In addition to these proinflammatory cytokines, IL-12 and IL-18, which are IFN-γ-inducing cytokines, are also released from innate immune cells and subsequently induce the production of IFN-γ from NK cells and NK dendritic cells (8, 9). Such an initial IFN-γ response is not only essential for the host defense against primary LM infection but is also important for the establishment of T cell-mediated acquired immunity, which is required for the protection of the host against secondary challenge with LM (10, 11).

In contrast to the established role of IFN-γ for the host defense, the mechanism of IFN-γ induction in the initial stage of infection with LM has been elucidated only partially. On the basis of the fact that an infection with the LM strain lacking LLO, which is incapable of escape into the cytosol and intracellular multiplication, never induces a significant level of IFN-γ response (12, 13), LLO seems to play a critical role in the induction of IFN-γ. Regarding the contribution of LLO to the induction of IFN-γ response, there may be several possibilities. One possibility is that LLO serves just as cytolytic protein and simply enables the bacteria to escape from the phagosomal compartment. Then the recognition of the bacterial ligand(s) by some cytoplasmic pattern recognition receptor in the macrophage cytoplasm, for example, the Nod-like receptor (NLR), may result in the activation of the signaling pathway required for the induction of IFN-γ-inducing cytokines. Another possibility is the direct stimulation of the signaling cascade by LLO itself as an essential ligand after serving as the protein toxin necessary for evasion into the cytoplasm. As LLO is known to modulate various cellular responses (14), it is likely that the LLO molecule itself may induce or enhance the production of IFN-γ by activating macrophages as a bacterial modulin.

Listeria ivanovii (LI) is an animal pathogen and carries a gene cluster that is highly analogous to the Listeria pathogenicity island 1 of LM (15). LI produces ivanolysin O (ILO) encoded by ilo, a cytolysin that shows ∼80% homology with LLO in amino acid sequence (16). Although LI is capable of evasion into and multiplication inside the macrophage cytoplasm like LM, IFN-γ responses after infection with LI in vitro and in vivo were very low as compared with those induced by LM (17). It is therefore unlikely that only the bacterial entry into the macrophage cytoplasm is sufficient for the induction of IFN-γ production. The comparison between LM and LI may not be the best tool to test the second possibility mentioned above as these two species are not isogenic although they belong to the same genus Listeria, and there may be some critical difference in the ligands other than the difference between LLO and ILO. To overcome this problem, isogenic LM mutants producing LLO or ILO were constructed by gene complementation of an LLO-deficient LM mutant with hly and ilo. In our previous study using these isogenic mutants, we examined whether the initial IFN-γ response is due simply to the entry of LM into the macrophage cytoplasm or whether the presence of LLO itself is required. It was found that LLO-producing LM, but not ILO-producing LM, strongly induced the production of IFN-γ on LM infection in vitro and in vivo (11). The results clearly indicated that the LLO molecule is involved by itself in the induction of host IFN-γ response and not by enabling bacterial cells to be delivered into the macrophage cytoplasm.

IFN-γ production in the host as an innate immune response to LM is highly dependent on the release of two major IFN-γ-inducing cytokines, IL-12 and IL-18 (18). In contrast to IL-12 production, which never requires further processing, the release of IL-18 as an active form definitely requires a cleavage of pro-IL-18 by caspase-1 (19). Recent reports have shown that caspase-1 activation is induced efficiently only by LM, which is capable of escaping from the phagosome, but not by the LLO mutant incapable of evasion into the cytosol (20, 21). In a study that emphasized the importance of intracellular parasitism for caspase-1 activation, the cytosolic flagellin of Salmonella appeared to be responsible for caspase-1 activation, but no particular component of Listeria was identified (22). These findings raised a possibility that LLO itself is one of the candidates for the bacterial molecule responsible for caspase-1 activation.

In this study, we have examined the molecular basis for the induction of IL-12 and IL-18 in macrophages stimulated with LM by using isogenic mutants that differ only in the cytolytic protein, with special reference to the induction of cytokine gene expression and caspase-1 activation.

Female mice of C57BL/6 (normal, TLR4 knockout) strains were purchased from Japan SLC. Caspase-1 knockout C57BL/6 mice were provided by H. Tsutsui (Hyogo Medical College, Hyogo, Japan). Mice were maintained in specific-pathogen-free conditions and used at 7–9 wk of age. All the experimental procedures performed on mice were approved by the Animal Ethics and Research Committee of Kyoto University Graduate School of Medicine, Kyoto, Japan.

The parental wild-type LM strain used in this study was L. monocytogenes EGD (serovar 1/2a). Three isogenic mutants, Δhly, Δhly::hly, and Δhly::ilo, were constructed from wild-type LM using the homologous recombination method and the similarity in the expression level of each cytolysin was shown in a previous study (11). Bacteria were grown overnight in brain-heart infusion broth (EIKEN Chemical) at 37°C with shaking. One volume of the overnight culture was added to 100 volumes of fresh brain-heart infusion medium and cultured further for 5 h. Bacterial cells were washed, suspended in PBS supplemented with 10% glycerol, and stored in aliquots at −80°C. The concentration of bacteria was determined by plating 10-fold serially diluted suspensions on a tryptic soy agar (EIKEN Chemical) plate and counting the number of colonies after cultivation for 24 h.

Peritoneal exudate cells (PECs) of mice were obtained 3 days after an i.p. injection of 2 ml of thioglycolate medium (EIKEN Chemical). After washing with RPMI 1640, PECs were incubated on culture plates at 37°C for 3 h in culture medium that consisted of RPMI 1640 supplemented with 10% FCS. After incubation, the cells were washed with RPMI 1640 and adherent PECs were used for infection study. Bone marrow cells were obtained from tibiae of mice and then cultured in RPMI 1640 supplemented with 10% FCS, gentamicin (10 μg/ml; Wako Pure Chemical Industries), and recombinant mouse M-CSF (100 ng/ml; R&D Systems) for 5 days. After washing with RPMI 1640, adherent bone marrow-derived macrophages were collected. The cells were plated at 1.5 × 105 cells/well in 96-well microplates or at 2 × 106 cells/well in 6-well microplates for detection of cytokines or active caspase-1, respectively. The cells were infected with bacteria at a multiplicity of infection (MOI) of 1 for 30 min at 37°C.

Adherent PECs were seeded into a 24-well plate at 5 × 105 cells/well and then infected with bacteria at a MOI of 1 for 30 min at 37°C. Cells were washed three times and cultured for 3 h at 37°C in the presence of 10 μg/ml gentamicin. After several washings, the cells were fixed by 3% paraformaldehyde and incubated overnight at 4°C with a blocking solution that is PBS containing 10% Blocking One (Nacalai) and 0.1% saponin (Nacalai). F-actin formation was visualized by the staining of infected cells with Alexa Fluor 488-phalloidin (Invitrogen), and the bacterial cell was stained by treatment with rabbit anti-Listeria polyclonal Ab (ViroStat) in blocking solution at room temperature for 1 h in a dark room and then with Alexa Fluor 594-anti-rabbit IgG Ab (Invitrogen) at room temperature for 1 h in a dark room. Cells were examined under fluorescent microscope for actin cloud or actin tail formation.

Neutralization of cytokines in culture was done as reported previously (23, 24). The neutralizing Ab specific for IL-12 (goat, polyclonal) and that for IL-18 (rat, clone 93-10C) were purchased from R&D Systems and Medical & Biological Laboratories, respectively. As control Abs, normal rat IgG (ICN Biomedicals) and normal goat IgG (R&D Systems) were used. After infection with bacteria for 0.5 h, Abs were added to cell culture medium at 5 μg/ml.

Levels of cytokines in culture supernatants were determined by two-site sandwich ELISA as reported previously (11, 25). Briefly, bacteria were added to the cell cultures and incubated at 37°C for 30 min. The infected cells were cultured for an additional 24 h in the presence of 10 μg/ml gentamicin. Culture supernatants were then collected and stored at −80°C until the cytokine measurement. The ELISA kit for TNF-α was purchased from eBiosciences. For the titration of other cytokines, pairs of biotin-labeled and unlabeled mAbs specific to IL-18 (Medical & Biological Laboratories), IL-12p70 (Endogen), and IFN-γ (Endogen) were used.

Total cellular RNA was extracted using NucleoSpin RNA II (Macherey-Nagel), according to the manufacturer’s instructions. The collected RNA (0.2 μg) was treated with RNase-free DNase (Promega) to eliminate contaminating DNA before being subjected to reverse transcription using random primers (Invitrogen) and ReverTra Ace (TOYOBO). Quantitative real-time RT-PCR was performed on ABI PRISM 7000 (Applied Biosystems) using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). Results were analyzed with ABI PRISM 7000 SDS software. The following mouse primer sequences were designed using Applied Biosystems Primer Express software: tnfα, 5′-ATGCTGGGACAGTGACCTGG-3′ (forward) and 5′-CCTTGATGGTGGTGCATGAG-3′ (reverse); il-12p40, 5′-GGATGGAAGAGTCCCCCAAA-3′ (forward) and 5′-CTGGAAAAAGCCAACCAAGC-3′ (reverse); il-18, 5′-GAAAGCCGCCTCAAACCTTC-3′ (forward) and 5′-CATTGTTCCTGGGCCAAGAG-3′ (reverse); caspase-1, 5′-GCCCACTGCTGATAGGGTGA-3′ (forward) and 5′-CCCGGGAAGAGGTAGAAACG-3′ (reverse); and β-actin, 5′-GCCCTGAGGCTCTTTTCCAG-3′ (forward) and 5′-TGCCACAGGATTCCATACCC-3′ (reverse). Gene-specific transcript levels were normalized to the amount of β-actin mRNA.

A caspase-1-specific inhibitor, N-benzyloxycarbonyl-Tyr-Val-Ala-Asp-fluoromethyl ketone (z-YVAD-fmk), was purchased from R&D Systems. After infection with bacteria for 30 min, this inhibitor dissolved in DMSO was added to cell culture medium at several concentrations. For the control wells, DMSO without inhibitor was added.

The cells were cultured for several hours at 37°C with gentamicin after infection, with each LM strain at a MOI of 1 for 30 min. After cultivation, supernatants were collected, and cells were lysed in the buffer containing 1% Nonidet P-40 supplemented with 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1.5 μg/ml aprotinin, and 2 mM DTT. Six milliliters of culture supernatants were precipitated with 7 μg of rabbit polyclonal Ab for mouse caspase-1 p10 (Santa Cruz Biotechnology) in the presence of protein G-Sepharose (GE Healthcare). The cell lysates and precipitates were subjected to SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes by electroblotting. The membranes were immunoblotted with anti-caspase-1 Ab or anti-β-actin Ab (Sigma-Aldrich).

Culture supernatants were collected, centrifuged, and transferred to new tubes. LDH activity was measured using an LDH cytotoxicity detection kit (TaKaRa BIO). The percentage of LDH release was calculated by using the following formula: percentage of release = 100 × (experimental LDH release − spontaneous LDH release)/(maximal LDH release − spontaneous LDH release). To determine the maximal LDH release, cells were treated with 1% Triton X-100.

Adherent PECs seeded in 24-well microplates at 5 × 105 cells/well were infected with bacteria at a MOI of 1. After cultivation for 21 h, the cells were washed three times with PBS and then fixed in 4% paraformaldehyde. Fragmented DNA was labeled by the TUNEL method using MEBSTAIN Apoptosis Kit Direct (Medical & Biological Laboratories) according to the manufacturer’s instructions. Total nucleus was visualized by 4′,6-diamidino-2-phenylindole staining (Dojindo). Bacterial cells were stained by using rabbit anti-Listeria polyclonal Ab (VivoStat) and Alexa Fluor 594-labeled anti-rabbit Ig G Ab (Invitrogen). The cells were examined under a fluorescent microscope, and cells positive for each fluorescence were enumerated.

An expression vector was constructed by ligation of the prfA and hly promoter region into the multiple cloning sites of pAT28, which contains a spectinomycin resistance gene, with Ligation High (Toyobo), and then gene fragments of hly full length, hly Trp492→Ala492 (W492A), hly domain 1–3, and domain 4 were ligated downstream of hly promoter. The following primer sequences were used: prfA, 5′-cGATGAGCTCTTAATTTAATTTTCCCAAGTAGCAG-3′ (forward) and 5′-ACGCCCCGGGATGAACGCTCAAGCAGAAG-3′ (reverse); hly promoter, 5′-CGATCCCGGGAATGGCCCCCTCCTTTGAT-3′ (forward) and 5′-CGCGGTACCGATATCCTTTGCTTCAGTTTG-3′ (reverse); hly, 5′-CGATTGCGCATCTGCATTCAATAAAG-3′ (forward); hly d4, 5′-CGATCCCGGGAAAATTAACATCGATCACTC-3′ (forward); hly W492A, 5′-TTGGGAATGGGCGAGAACGGTAA-3′ (forward); hly, 5′-GCTCTAGATTATTCGATTGGATTATCTAC-3′ (reverse); hly d1–3, 5′-CGTCTAGATTATGTATAAGCTTTTGAAG-3′ (reverse). The primers for prfA were designed to generate restriction sites for SacI and SmaI. The primers for hly promoter were designed to generate restriction sites for SmaI and KpnI/EcoRV. The F-primers for hly and hly d4 were designed to generate restriction sites for FspI and SmaI, respectively. The reverse primers for hly and hly d1–3 were designed to generate restriction sites for XbaI. The resulting plasmid was introduced into the competent cells of Δhly::ilo by electroporation. Transformants were selected on brain-heart infusion agar plates supplemented with spectinomycin (250 μg/ml; Nacalai Tesque). The expression of each LLO molecule produced by transformed Δhly::ilo strains was confirmed by Western blotting using anti-LLO Ab, although the Ab showed a weak level of cross-reactivity to ILO. The similarity in the levels of ILO production by these ILO-producing strains carrying the plasmid harboring the hly fragment was confirmed by Western blotting using anti-ILO Ab. These polyclonal Abs for LLO or ILO were prepared by hyperimmunization of a normal white rabbit with recombinant LLO or recombinant ILO emulsified in Freund’s complete adjuvant.

For comparisons between two groups, Student’s t test was used when the variances of the groups were judged to be equal by F test. Multigroup comparisons of mean values were made according to the ANOVA and the Fisher’s protected least significant difference post hoc test after the confirmation of homogeneity of variances among the groups by using Bartlett’s test. Statistical significance was determined as p < 0.05.

In the in vitro infection of macrophages prepared from C57BL/6 mice, two Δhly strains complemented with hly or ilo exhibited a similar level of ability to escape from the phagosome of macrophages (Fig. 1,A). An essential requirement for LLO in the induction of IFN-γ by LM was confirmed by using whole PECs from C57BL/6 mice, the cell population containing IFN-γ-producing cells such as NK cells (Fig. 1,B). The IFN-γ-inducing ability of wild-type LM that was abolished by the deletion of hly could be restored when Δhly was complemented with hly but not ilo. These findings were consistent with our previous report in which PECs from C3H/HeN mice were used (11). In the culture system used in this study, both IL-12 and IL-18 were shown to be important for IFN-γ production induced by LLO-expressing LM, as neutralizing Abs against these two cytokines significantly inhibited the IFN-γ response (Fig. 1 C).

FIGURE 1.

LLO- and ILO-expressing LM similarly escape from phagosomes but differently induce IFN-γ and IL-18. Whole PECs (B and C), adherent PECs (A and D–F), or bone marrow-derived macrophages (G) were infected with each LM strain. A, Cells were cultured for an additional 3 h in the presence of gentamicin, then bacteria and F-actin were stained and 300 bacteria were counted. The percentage of bacteria positive for associating F-actin was calculated for each strain. The filled bars represent the mean of three independent wells and the error bars indicate the SD. B–G, Cells were cultured for an additional 24 h in the presence (C) or absence (B, DG) of each neutralizing Ab, and the amounts of each cytokine were then determined by ELISA. Data represent the mean of triplicate assays and SD. Similar results were obtained in three independent experiments. ∗, p < 0.01. WT, Wild type.

FIGURE 1.

LLO- and ILO-expressing LM similarly escape from phagosomes but differently induce IFN-γ and IL-18. Whole PECs (B and C), adherent PECs (A and D–F), or bone marrow-derived macrophages (G) were infected with each LM strain. A, Cells were cultured for an additional 3 h in the presence of gentamicin, then bacteria and F-actin were stained and 300 bacteria were counted. The percentage of bacteria positive for associating F-actin was calculated for each strain. The filled bars represent the mean of three independent wells and the error bars indicate the SD. B–G, Cells were cultured for an additional 24 h in the presence (C) or absence (B, DG) of each neutralizing Ab, and the amounts of each cytokine were then determined by ELISA. Data represent the mean of triplicate assays and SD. Similar results were obtained in three independent experiments. ∗, p < 0.01. WT, Wild type.

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For the assessment of IL-12 and IL-18 production in response to each LM strain, we used culture supernatants of adherent peritoneal macrophages to rule out the possible influences of the products from other nonadherent cells like IFN-γ. A similar level of production of IL-12p70 was observed in all groups of macrophages infected with any of the LM strains used, indicating that induction of IL-12 is not dependent on the escape of bacteria from phagosome and a sort of cytolysin. Another caspase-independent cytokine, TNF-α, was also produced even by macrophages stimulated with Δhly and incapable of evasion into the cytosol (Fig. 1, D and E). By contrast, the pattern of IL-18 induction by LM strains was quite similar to that of IFN-γ (11). The IL-18-inducing ability of wild-type LM was lost completely by deletion of hlyhly) but was successfully restored by complementation with hlyhly::hly). Interestingly, such a significant level of restoration was not observed when Δhly was complemented with ilohly::ilo) (Fig. 1,F). The critical difference in the ability to induce IL-18 production between Δhly::hly and Δhly::ilo was observed also in bone marrow-derived macrophages (Fig. 1 G). These data indicate that LLO plays an important role in the induction of IL-18 in infection with LM and that the IL-18 response is dependent not on extraphagosomal evasion mediated by either LLO or ILO, but on the LLO molecule itself.

IL-18 is first synthesized as pro-IL-18, then processed to mature form and secreted from cells in response to appropriate stimuli. To clarify which stage of IL-18 production is stimulated by LLO, we next analyzed the levels of IL-18 mRNA in macrophages infected with each mutant strain. In contrast to the significant difference in the production of mature IL-18 shown above, there was no significant difference in the ability to induce the expression of IL-18 among all of the LM strains used (Fig. 2). Therefore the difference appeared to be dependent on the process that follows IL-18 gene expression.

FIGURE 2.

Expression of mRNA for various cytokines after infection with LM strains. Adherent PECs were infected with each LM strain. The cells were cultured for an additional 5 h in the presence of gentamicin. Total RNA was extracted and subjected to quantitative real-time RT-PCR for detection of mRNA for TNF-α, IL-12p40, and IL-18. Data represent the mean of triplicate assays and SD. N. D., Not detected; WT, wild type.

FIGURE 2.

Expression of mRNA for various cytokines after infection with LM strains. Adherent PECs were infected with each LM strain. The cells were cultured for an additional 5 h in the presence of gentamicin. Total RNA was extracted and subjected to quantitative real-time RT-PCR for detection of mRNA for TNF-α, IL-12p40, and IL-18. Data represent the mean of triplicate assays and SD. N. D., Not detected; WT, wild type.

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Caspase-1, known also as IL-1β-converting enzyme, cleaves pro-IL-18 into mature form (20, 26, 27, 28). When macrophages were pretreated with z-YVAD-fmk, a caspase-1-specific inhibitor, IL-18 production induced by wild-type LM was decreased in a dose-dependent manner (Fig. 3,A). A nonspecific inhibitory effect of z-YVAD-fmk was ruled out, because the same concentration of this inhibitor did not affect the production of TNF-α (Fig. 3,B). To further confirm the involvement of caspase-1 in LLO-dependent IL-18 maturation, macrophages from caspase-1-deficient mice were infected with wild-type LM and the levels of IL-18 in culture supernatants were then compared with those in macrophages from normal mice. As expected, there was a significant reduction of IL-18 production in caspase-1-deficient macrophages, whereas the same cells produced TNF-α at a level comparable to that produced by the cells from normal mice (Fig. 3, C and D).

FIGURE 3.

Caspase-1-dependent IL-18 production on LM infection. A and B, Adherent PECs were infected with wild-type LM. The cells were cultured for an additional 24 h in the presence of gentamicin with or without z-YVAD-fmk. C and D, Adherent PECs from normal and caspase-1 knockout mice were infected with wild-type LM. Cells were cultured for 24 h in the presence of gentamicin and then the culture supernatants were collected. The amounts of cytokines were determined by ELISA. Data represent the mean of triplicate assays and SD. Similar results were obtained in three independent experiments. ∗, p < 0.01.

FIGURE 3.

Caspase-1-dependent IL-18 production on LM infection. A and B, Adherent PECs were infected with wild-type LM. The cells were cultured for an additional 24 h in the presence of gentamicin with or without z-YVAD-fmk. C and D, Adherent PECs from normal and caspase-1 knockout mice were infected with wild-type LM. Cells were cultured for 24 h in the presence of gentamicin and then the culture supernatants were collected. The amounts of cytokines were determined by ELISA. Data represent the mean of triplicate assays and SD. Similar results were obtained in three independent experiments. ∗, p < 0.01.

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On the basis of the above result, we have compared the expression and processing of caspase-1 between recombinant LM mutants producing either LLO or ILO. Quantitative real-time RT-PCR detection of caspase-1 mRNA in the peritoneal macrophages showed a constitutive expression even in the absence of infection, and there was no significant level of further induction by infection with any strain (Fig. 4,A). Caspase-1 synthesized as an immature form is converted into active caspase-1 composed of p10 and p20 fragments by proteolytic cleavage and is released from the cells soon after the conversion (29, 30). To detect the active form of caspase-1 efficiently, we enriched caspase-1 in the culture supernatants by immunoprecipitation using anti-caspase-1 Ab, according to our own modification of the method reported recently (29). LLO-producing Δhly::hly strongly induced the processing of immature caspase-1 as determined by the detection of a p10 fragment (Fig. 4,B). A faint band could be observed also in lysate of macrophages infected with Δhly::ilo; however, it never reached to the level observed by Δhly::hly during 20 h of cultivation. Following the detection of the p10 fragment in the cell lysate, the activated form of caspase-1 became detectable only in the supernatant from the culture of macrophages infected with Δhly::hly (Fig. 4,B, bottom). The very faint level of p10 fragment induced by Δhly::ilo infection never increased even if the culture time was extended to 30 h or of the MOI was increased to 10 (data not shown). The active form of caspase-1 was also detected in the supernatant from wild-type LM-infected macrophages, but not from Δhly-infected cells (Fig. 4 C). These results clearly indicated that caspase-1 activation induced upon infection with LM is dependent on not only the entry of bacteria into the macrophage cytoplasm but also the LLO molecule itself.

FIGURE 4.

Expression and activation of caspase-1 in macrophages infected with LM strains. A, Adherent PECs were infected with each LM strain. The cells were cultured for an additional 5 h in the presence of gentamicin. Total RNA was extracted and subjected to quantitative real-time RT-PCR for detection of mRNA for caspase-1. Data represent the mean of triplicate assays and SD. B, The cells were cultured for an indicated time, and the culture supernatants were then collected. Adherent cells were lysed with 1% Nonidet P-40 lysis buffer. Active caspase-1 in the supernatants was immunoprecipitated and immunoblotted using a rabbit anti-caspase-1 Ab. C, After cultivation for 20 h, the active form of caspase-1 in the supernatants of the cells infected with each LM strain was detected. WT, Wild type.

FIGURE 4.

Expression and activation of caspase-1 in macrophages infected with LM strains. A, Adherent PECs were infected with each LM strain. The cells were cultured for an additional 5 h in the presence of gentamicin. Total RNA was extracted and subjected to quantitative real-time RT-PCR for detection of mRNA for caspase-1. Data represent the mean of triplicate assays and SD. B, The cells were cultured for an indicated time, and the culture supernatants were then collected. Adherent cells were lysed with 1% Nonidet P-40 lysis buffer. Active caspase-1 in the supernatants was immunoprecipitated and immunoblotted using a rabbit anti-caspase-1 Ab. C, After cultivation for 20 h, the active form of caspase-1 in the supernatants of the cells infected with each LM strain was detected. WT, Wild type.

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It has been reported that LM causes an unique type of cell death of the infected host macrophages, which is distinguished as pyroptosis from other forms of programmed cell death by its requirement of caspase-1 activation and the loss of plasma membrane integrity (21, 31). Other intracellular bacteria, Salmonella and Shigella, also induce pyroptosis accompanied by the release of LDH in a caspase-1-dependent manner only at an early phase of cell death, but not in late phases (32, 33). To know whether caspase-1-dependent events other than IL-18 maturation occur during infection with LLO-producing LM, we next determined LDH released from infected macrophages. The amount of LDH released from normal macrophages was higher than that from caspase-1-deficient macrophages when infection was done with Δhly::hly (Fig. 5, A and B). In contrast, Δhly::ilo induced a significantly lower level of LDH release compared with Δhly::hly, and there was no significant difference in the amount of LDH released after infection with Δhly::ilo between normal and caspase-1-deficient macrophages. Upon pyroptotic cell death, DNA cleavage occurred and could be detected by the TUNEL method (31, 34). Therefore we also tried to determine the level of pyroptosis by the visualization of fragmented DNA in Listeria-infected cells. As shown in Fig. 5,C and Table I, TUNEL-positive cells were observed frequently in normal macrophages infected with Δhly::hly but not in those infected with Δhly::ilo. The DNA fragmentation induced by Δhly::hly was caspase-1 dependent, because the number of TUNEL-positive cells was fewer and similar to the control level in the absence of caspase-1. These data indicated that LLO-producing LM, but not ILO-producing LM, induces a caspase-1-dependent pyroptosis in addition to the processing of IL-18 and supported our finding that the delivery of bacteria into the cytoplasm is not sufficient but that the presence of LLO is required for the induction of caspase-1 activation upon LM infection.

FIGURE 5.

Detection of caspase-1-dependent cell death in macrophages infected with Δhly::hly or Δhly::ilo. A and B, Adherent PECs were infected with Δhly::hly or Δhly::ilo. The cells were cultured for an indicated time in the presence of gentamicin. The culture supernatants were collected and the LDH activity was assayed. Data represent the mean of triplicate assays and SD. Similar results were obtained in three independent experiments. ∗, p < 0.01. C, The cells were cultured for 21 h and visualized by staining for Listeria (red), fragmented DNA (green), and total nucleus (blue). DAPI, 4′,6-Diamidino-2-phenylindole.

FIGURE 5.

Detection of caspase-1-dependent cell death in macrophages infected with Δhly::hly or Δhly::ilo. A and B, Adherent PECs were infected with Δhly::hly or Δhly::ilo. The cells were cultured for an indicated time in the presence of gentamicin. The culture supernatants were collected and the LDH activity was assayed. Data represent the mean of triplicate assays and SD. Similar results were obtained in three independent experiments. ∗, p < 0.01. C, The cells were cultured for 21 h and visualized by staining for Listeria (red), fragmented DNA (green), and total nucleus (blue). DAPI, 4′,6-Diamidino-2-phenylindole.

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Table I.

Percentage of TUNEL-positive (TUNEL+) cells in Listeria-infected cells or total cells

MacrophageBacteriaTUNEL+/Listeria-Infected Cells (%) (Green/Red+ Cell × 100)aTUNEL+/Total Cells (%) (Green/Blue × 100)a
Normal None  1.95 ± 0.40 
 Δhly::hly 28.80 ± 1.83 15.83 ± 0.76 
 Δhly::ilo 4.60 ± 0.72 2.06 ± 0.20 
    
Caspase-1 None  1.95 ± 0.20 
 Δhly::hly 5.73 ± 0.55 3.30 ± 0.36 
 Δhly::ilo 7.10 ± 1.41 3.06 ± 0.65 
MacrophageBacteriaTUNEL+/Listeria-Infected Cells (%) (Green/Red+ Cell × 100)aTUNEL+/Total Cells (%) (Green/Blue × 100)a
Normal None  1.95 ± 0.40 
 Δhly::hly 28.80 ± 1.83 15.83 ± 0.76 
 Δhly::ilo 4.60 ± 0.72 2.06 ± 0.20 
    
Caspase-1 None  1.95 ± 0.20 
 Δhly::hly 5.73 ± 0.55 3.30 ± 0.36 
 Δhly::ilo 7.10 ± 1.41 3.06 ± 0.65 
a

Value represents means and SDs of three independent wells. Three hundred cells were examined in each well.

Because LLO has been reported as a ligand for TLR4 when added from outside of the cells (35), we examined the involvement of TLR4 in the production of IL-18 and the activation of caspase-1 in response to wild-type LM. Macrophages obtained from normal or TLR4-deficient mice were infected with LLO-producing wild-type LM and the culture supernatants were then subjected to cytokine assays. As shown in Fig. 6, A–D, the levels of cytokines, including IL-18, in supernatants of LM-infected TLR4-deficient macrophages were comparable to those of infected macrophages from normal mice, indicating that TLR4 is not involved in the production of IL-18 and other cytokines in response to the infection with LM. Furthermore, wild-type LM induced similar levels of caspase-1 activation in normal and TLR4-deficient macrophages (Fig. 6 E). From these results, it is indicated that the LLO-dependent caspase-1 activation and the subsequent production of mature IL-18 are not due to the recognition of LLO by TLR4.

FIGURE 6.

Cytokine production and caspase-1 activation in TLR4 knockout macrophages. A–D, Whole PECs (A) and adherent PECs (B–D) from normal and TLR4 knockout mice were infected with wild-type LM. Cells were cultured for 24 h in the presence of gentamicin and then the culture supernatant was collected. The amount of each cytokine was determined by using ELISA specific for each cytokine. Data represent the mean of triplicate assays and SD. Similar results were obtained in three independent experiments. ∗, p < 0.01. E, Adherent PECs from normal and TLR4 knockout mice were infected with wild-type LM. Cells were cultured for 20 h in the presence of gentamicin and the culture supernatants were collected. Active caspase-1 in the supernatants was immunoprecipitated and immunoblotted using a rabbit anti-caspase-1 Ab.

FIGURE 6.

Cytokine production and caspase-1 activation in TLR4 knockout macrophages. A–D, Whole PECs (A) and adherent PECs (B–D) from normal and TLR4 knockout mice were infected with wild-type LM. Cells were cultured for 24 h in the presence of gentamicin and then the culture supernatant was collected. The amount of each cytokine was determined by using ELISA specific for each cytokine. Data represent the mean of triplicate assays and SD. Similar results were obtained in three independent experiments. ∗, p < 0.01. E, Adherent PECs from normal and TLR4 knockout mice were infected with wild-type LM. Cells were cultured for 20 h in the presence of gentamicin and the culture supernatants were collected. Active caspase-1 in the supernatants was immunoprecipitated and immunoblotted using a rabbit anti-caspase-1 Ab.

Close modal

To further confirm the involvement of LLO in caspase-1 activation induced upon LM infection, we constructed a Δhly::ilo strain additionally expressing the full-length LLO by using the pAT28 expression vector. The activation of caspase-1 was observed when macrophages were infected with the Δhly::ilo carrying full-length LLO expression vector, whereas Δhly::ilo transformed with empty vector could not induce caspase-1 activation (Fig. 7,B). Among the four domains comprising the whole LLO molecule, domain 4 is known as a cholesterol-binding domain and contains Trp-rich undecapeptide, which is highly conserved among cholesterol-dependent cytolysins and essential for the binding to membrane cholesterol (36). It has been previously reported that the domain 1–3 molecule of truncated LLO completely loses its cytolytic activity and the substitution of the third Trp of the undecapeptide (amino acid residue 492 of LLO holotoxin) with Ala severely attenuates the cytolytic activity of LLO (36, 37). Therefore, we next constructed Δhly::ilo strains carrying each vector for the expression of LLO W492A or domain 1–3 of LLO to know whether cholesterol-binding and subsequent cytolysis by this cytolysin is required for the activation of caspase-1 in macrophages infected with LM. The production of different LLO molecules encoded by each plasmid was confirmed by Western blotting using an anti-LLO polyclonal Ab (Fig. 7,A). The impaired ability of Δhly::ilo in caspase-1 activation was restored by the introduction of a plasmid harboring the gene for full-length LLO, and such an effect was not affected even by the elimination of cytolytic activity as clearly shown by LLO W492A or LLO domain 1–3 (Fig. 7,B). By contrast, complementation with a plasmid harboring the domain 4 of LLO, a molecule capable of cholesterol binding without cytolytic activity (38), never resulted in the acquisition of the ability for caspase-1 activation (Fig. 7,B). As there was no significant difference in the level of F-actin-positive bacteria inside macrophages among these plasmid-carrying Δhly::ilo strains (Fig. 7 C), it was clearly indicated that domain 1–3 is the region responsible for the activation of caspase-1 in infected macrophages.

FIGURE 7.

Detection of the domain (d) of LLO responsible for caspase-1 activation. A, Culture supernatants of Δhly::ilo strains transformed with each LLO-expressing vector were applied to SDS-PAGE and subsequent Western blotting using anti-LLO Ab (upper panel) or anti-ILO Ab (lower panel). B, Adherent PECs were infected with each Δhly::ilo strain, cultured for 20 h in the presence of gentamicin and spectinomycin (250 μg/ml), and culture supernatants were then collected. Active caspase-1 in the supernatants was immunoprecipitated and immunoblotted using a rabbit anti-caspase-1 Ab. C, Adherent PECs were infected with each Δhly::ilo strain, cultured for 3 h in the presence of gentamicin and spectinomycin, and then bacteria and F-actin were stained and 300 bacteria were counted. The percentage of bacteria positive for associating F-actin was calculated for each strain. The filled bars represent the mean of three independent wells, and the error bars indicate the SD.

FIGURE 7.

Detection of the domain (d) of LLO responsible for caspase-1 activation. A, Culture supernatants of Δhly::ilo strains transformed with each LLO-expressing vector were applied to SDS-PAGE and subsequent Western blotting using anti-LLO Ab (upper panel) or anti-ILO Ab (lower panel). B, Adherent PECs were infected with each Δhly::ilo strain, cultured for 20 h in the presence of gentamicin and spectinomycin (250 μg/ml), and culture supernatants were then collected. Active caspase-1 in the supernatants was immunoprecipitated and immunoblotted using a rabbit anti-caspase-1 Ab. C, Adherent PECs were infected with each Δhly::ilo strain, cultured for 3 h in the presence of gentamicin and spectinomycin, and then bacteria and F-actin were stained and 300 bacteria were counted. The percentage of bacteria positive for associating F-actin was calculated for each strain. The filled bars represent the mean of three independent wells, and the error bars indicate the SD.

Close modal

We previously showed that an entry of bacteria into the cytoplasm was not sufficient for the IFN-γ response of mice to LLO-producing LM by both in vitro and in vivo experiments using two isogenic strains that differed only in LLO and ILO (11). In this study, we examined the difference between Δhly::hly and Δhly::ilo in the induction of IL-12 and IL-18, the two major IFN-γ-inducing cytokines, to elucidate the mechanism of LLO-dependent IFN-γ response to LM. It was noteworthy that LLO-producing LM, but not ILO-producing LM, strongly induced the production of IL-18. Because mRNA expression did not depend exclusively on LLO, we next examined the activation of caspase-1, which is essential for the maturation and secretion of biologically active IL-18. It was strongly suggested that the dependence of the IFN-γ response on LLO is due to the LLO-dependent induction of caspase-1 activation and subsequent IL-18 production and that the IL-18 response is dependent on not only the entry of bacteria into the macrophage cytoplasm but probably also on the distinct activity of LLO as a signaling ligand.

On the basis of a previous report (18) and our own similar observation (not shown) that IL-12 is not induced in mice deficient for MyD88, an adaptor molecule of almost all TLRs, upon infection with LM, it is clear that TLR-dependent recognition of bacterial ligands is essentially required for the induction of IL-12 production. By contrast, production of IL-18 is reported to be induced regardless of the absence of MyD88 (39). Our present finding that TLR4, a recognition receptor for LLO, was not involved in the activation of caspase-1 and production of IL-18 in macrophages infected with LM is consistent with these previous observations. Generally, IL-18 is constitutively expressed, and the produced pro-IL-18 remains inside the cells until being cleaved by activated caspase-1. Therefore both MyD88-dependent production of IL-12 and TLR4- and MyD88-independent cleavage of pro-IL-18 are likely the key processes for the induction of IFN-γ upon LM infection. Some distinct activity of LLO appeared to be involved in the latter process that cannot be induced by ILO.

Although the involvement of LLO in caspase-1-mediated IL-18 induction upon infection with LM was revealed clearly in this study, the mechanism remains to be elucidated. One possibility to be considered is the difference in the cytotoxic effect between LLO and ILO. These two cytolysins are highly homologous and exhibited a quite similar function regarding the contribution to the escape of Listeria from phagosome into the cytoplasm. After the escape of bacteria from the hazardous phagosome by means of cytolysin into the cytosol, a nutrient-rich niche for multiplication, it is important for the cytosolic bacteria to minimize further cytolytic activity to prevent host cell damage. For that, two mechanisms have been proposed: the dependency of cytolytic activity on acidic pH (40) and the presence of an N-terminal Pro-Glu-Ser-Thr (PEST)-like sequence, which is thought to target for phosphorylation and/or degradation in eukaryote cells (41). Although both LLO and ILO exhibited a similar optimal pH for cytolytic activity (42), the hemolytic activity of recombinant ILO was relatively higher than that of recombinant LLO (17). Besides, the sequence analysis of the ilo gene revealed the absence of the PEST-like sequence that is present in LLO (data not shown). In the present experimental results, the level of cytolysis induced by ILO-producing LM was marginal and rather lower than that induced by LLO-producing LM in caspase-1-deficient cells (Fig. 5). Moreover, these LM strains induced similar levels of TNF-α and IL-12 production by macrophages (Fig. 1). Therefore the above possibility could be ruled out, and the difference in the ability to induce IL-18 between LLO-producing LM and ILO-producing LM should be explained by other reasons. Construction of the Δhly strains complemented with genes encoding chimeric proteins between LLO and ILO and recombinant strains expressing cytolysins mutated for PEST-like sequence, are under way, and the molecular basis for the LLO-dependent activation of caspase-1 should be clarified in the near future.

From our results using LLO-producing LM and ILO-producing LM, there may be two major possibilities as follows: 1) LLO activates some signaling pathway that leads to caspase-1 activation without the participation of any other ligands; and 2) LLO induces the activation of caspase-1 in cooperation with other bacterial ligands or merely enhances that induced by other bacterial ligands. If either of these possibilities is the case, ILO itself must have no or less ability to induce caspase-1 activation compared with LLO. In our previous study using recombinant LLO and ILO, it was shown that ILO is far less capable of inducing cytokines than LLO when added from outside of the cells in vitro (17). Therefore our assumption is that there is some molecular structure in LLO that is exclusively important for caspase-1 activation in the cytosol of macrophages. Indeed, our results using Δhly::ilo strains additionally expressing the recombinant proteins of LLO molecule (Fig. 7) clearly indicated that domain 1–3 is the region responsible for such an activity after the escape of bacteria into the cytosolic space and also that the ability of the LLO molecule for membrane binding or membrane damage is not involved in the activation of caspase-1. In a further study, experiments on a transfection of macrophages with LLO- or ILO-expressing vector or an intracytosolic injection of recombinant cytolysin are to be conducted.

Compared with virulence gene expression in broth-cultured LM, the expression of LLO is believed to be up-regulated inside macrophages (43). It is therefore reasonable that some cytoplasmic sensor molecule, rather than a cell surface receptor, recognizes LLO, resulting in caspase-1 activation. It has been reported that LLO is recognized by TLR4 and activates a signaling pathway downstream of TLR4 (35). Moreover, recombinant LLO protein induced the production of various cytokines by splenocytes or PEC cultures in a TLR4-dependent manner, whereas recombinant ILO protein did not induce their production (17, 35). However, our present results indicated that TLR4 was not involved in the production of IL-18 and the activation of caspase-1 in macrophages infected with LM (Fig. 6). Several recent reports have shown that some bacterial ligands are recognized not only by TLRs but also by NLRs such as Ipaf/CARD12 and Nalp3/cryopyrin/Pypaf1, which are cytoplasmic proteins containing a leucine-rich repeat domain, a nucleotide-binding domain, and each signaling domain. Flagellin from Salmonella or Legionella, which is known as a TLR5 ligand (44), induces caspase-1 activation through an Ipaf-dependent pathway when it is in the cytoplasm (45, 46, 47). The small antiviral compounds imiquimod and R-848, which are TLR7 ligands (48), are also known to induce the activation of caspase-1 in a Nalp3-dependent manner (49). Moreover, Nalp1b, an NLR protein, is involved in the caspase-1-dependent cell death of mouse macrophages induced by lethal toxin, which is a protein toxin produced by Bacillus anthracis, suggesting that lethal toxin induces the activation of caspase-1 through Nalp1b directly or indirectly (50). In the case of infection with Listeria, caspase-1 activation in macrophages is reported to depend upon an apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain, ASC, an adaptor molecule that links upstream NLRs to caspase-1 (20). Furthermore, it is reported that potassium efflux and the P2X7 receptor are not required for caspase-1 activation induced by Listeria infection (22). On the basis of these recent findings, it is highly possible that LM-induced caspase-1 activation is triggered by the recognition of cytosolic LLO by some NLR protein. The experiments in this line will be conducted also by using recombinant strains under construction.

In conclusion, this study has clearly indicated that LM-induced IFN-γ production in mice is ascribed mainly to the presence of LLO due to a distinct activity of inducing the activation of caspase-1 after evasion into the cytosolic space of infected macrophages.

We thank Keisuke Kuida (Vertex Pharmaceuticals) and Hiroko Tsutsui (Hyogo Medical University) for providing caspase-1 knockout mice.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Culture and Sports of Japan, a Grant-in-Aid for Scientific Research (B) and (C), and a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science.

3

Abbreviations used in this paper: LM, Listeria monocytogenes; ILO, ivanolysin O; LI, Listeria ivanovii; LDH, lactate dehydrogenase; LLO, listeriolysin O; MOI, multiplicity of infection; NLR, Nod-like receptor; PEC, peritoneal exudate cell; PEST, Pro-Glu-Ser-Thr; z-YVAD-fmk, N-benzyloxycarbonyl-Tyr-Val-Ala-Asp-fluoromethyl ketone.

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