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(Journal of Leukocyte Biology. 2002;72:743-751.)
© 2002 by Society for Leukocyte Biology

Salmonella virulence factor SipB induces activation and release of IL-18 in human dendritic cells

Donatus Dreher*, Menno Kok{dagger}, Carolina Obregon*, Stephen G. Kiama{ddagger},§, Peter Gehr{ddagger} and Laurent P. Nicod*

* Division of Pneumology, University Hospital of Geneva, Switzerland;
{dagger} Department of Genetics and Microbiology and Department of Medical Biochemistry, University of Geneva, Switzerland;
{ddagger} Institute of Anatomy, University of Berne, Switzerland; and
§ Department of Veterinary Anatomy, University of Nairobi, Kenya

Correspondence: Dr. Donatus Dreher, Division of Pneumology, Centre Médical Universitaire, 1, rue Michel-Servet, 1211 Geneva-4, Switzerland. E-mail: dreher{at}dim.hcuge.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin-18 (IL-18) plays an important role in innate and acquired immunity, in particular against intracellular pathogens. However, little is known about the microbial factors that trigger IL-18 secretion by dendritic cells (DCs). To determine the influence of bacterial virulence factors on the activation and release of IL-18, we infected human monocyte-derived DCs with virulence mutants of the facultative intracellular pathogen Salmonella typhimurium. Our results show that infection by S. typhimurium causes caspase-1-dependent activation of IL-18 and triggers the release of IL-18 in human DCs. The secretion of IL-18 by the DCs was closely correlated with the ability of the S. typhimurium strains to induce apoptosis. We demonstrate that activation and release of IL-18 are blocked by mutations in the Salmonella sipB gene, which encodes a virulence factor that activates caspase-1 to induce apoptosis. These findings indicate that the activation and release of IL-18 induced by bacterial virulence factors may represent one component of innate immunity against the intracellular bacteria.

Key Words: antigen-presenting cells • cytokines • innate immunity • bacteria • host defense


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin-18 (IL-18), originally identified as interferon-{gamma} (IFN-{gamma})-inducing factor [1 , 2 ], plays an important role in the induction of innate immunity [3 ] and is involved in the regulation of T helper cell type 1 (Th1) and type 2 (Th2) immune responses [4 ]. IL-18 is a key factor in the host defense against various (facultative) intracellular microorganisms in the mouse, such as Salmonella typhimurium [5 ], Listeria monocytogenes [6 ], Cryptococcus neoformans [7 ], and Mycobacterium tuberculosis [8 ]. Increased, circulating IL-18 levels were also detected in the human in response to M. tuberculosis [9 ] and Mycobacterium leprae [10 ] infection. However, despite the critical role of IL-18 in clearing these intracellular pathogens, the factors that elicit IL-18 secretion during infection are not yet fully explained.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APC) and control the induction of Th1 and Th2 immune responses [11 ]. DCs take up bacteria at their site of entry and migrate to the local lymphatic organs to present processed antigens to naive or primed T cells [11 , 12 ]. Besides their crucial function in the induction of acquired immune responses, DCs have recently been found to play a prime role in innate immunity in that they can sense pathogens [13 ] and immediately generate a protective cytokine response [14 ]. The regulation of IL-18 secretion in human DCs by different immunomodulatory compounds [15 , 16 ] and by the interaction of DCs with T cells [17 ] has been investigated previously. Yet, data about the induction of IL-18 secretion in DCs by live microorganisms are sparse. It has been shown lately that M. tuberculosis can stimulate the secretion of IL-18 in human DCs [18 ], but it is not known how the pathogen triggers the secretion and whether it induces the processing of IL-18 into its biologically active form as well.

S. typhimurium, a Gram-negative, facultative intracellular bacterium that is a frequent cause of food-borne infections in humans, is widely used to study host-pathogen interactions [19 ]. APC are natural targets of infection by Salmonellae [12 ], and we recently showed that S. typhimurium infects human DCs as well [20 ]. In the present work, we investigated the response of DCs to infection by S. typhimurium to better understand the regulation of IL-18 secretion during microbial infection. We explored virulent and attenuated bacterial strains of different genetic background to identify the factors that would trigger the secretion of IL-18 in the infected cells. Our results show that infection by S. typhimurium provokes the release of IL-18 in the DCs and triggers the processing of IL-18 into its biologically active form. Our study clearly attributes the activation and the release of IL-18 to a bacterial virulence factor, SipB, which was hitherto known to induce apoptosis and caspase-1 activation in the infected cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human DCs
Human DCs were generated from peripheral blood monocytes (PBMC) as previously described [20 ], following the methods originally given by Sallusto and Lanzavecchia [21 ]. PBMC were isolated from buffy coats of healthy donors by Ficoll Paque (Research Grade, AP Biotech, Uppsala, Sweden) density gradient centrifugation. After 1 h of adherence in RPMI 1640 (Life Technologies, Rockville, MD), containing 10% fetal calf serum (FCS; Seromed, Berlin, Germany), glutamine (2 mM), and penicillin/streptomycin [complete culture medium (CCM)], dishes were rinsed with Hanks’ balanced salt solution (HBSS), and adherent cells were incubated overnight in CCM. Loosely adherent monocytes were then recovered by three rinses with HBSS. Immature DCs (iDCs) were obtained from the monocytes by culture during 6 days in CCM, supplemented with granulocyte macrophage-colony stimulating factor (10 ng/mL; Immugenex, Los Angeles, CA) and IL-4 (10 ng/mL; R&D Systems, Minneapolis, MN). If not otherwise stated, these iDCs were used for infection (see below).

Bacterial strains
Bacterial strains used in this study were virulent S. typhimurium strains (C53, SL1344, and ATCC14028), attenuated strains derived from these strains that are under consideration as vaccine vectors (PhoPc, PhoP-, AroA) [22 ], and mutants of the virulent strains that lack the sipB gene (SipB, SD11). The characteristics of these S. typhimurium strains are summarized in Table 1 . Prior to the infection of human DCs, bacteria were grown overnight in Luria broth (LB) and diluted 20x in LB with 300 mM KCl and 0.5% KNO3 at 37°C without agitation. Bacterial concentrations were followed by measurement of optical density (OD) of 450 nm. When the OD reached values between 3 and 5 x 108 colony-forming units (CFU)/mL, the cultures were diluted accordingly in prewarmed RPMI to obtain a suspension of 5.0 x 107 CFU/mL. For infection or exposure to different agents, iDCs were washed, and 2 x 105 cells were seeded in 1 mL CCM without antibiotics. Cells were infected by the addition of bacterial suspensions to obtain a multiplicity of infection (M.O.I.) of 25 bacteria/cell [20 ]. Infection was allowed for 30 min, after which extracellular bacteria were killed with gentamycin (60 mg/L). In parallel experiments, the iDCs were exposed to heat-killed S. typhimurium (PhoPc, 5 min at 95°C, 25 bacteria/cell), Salmonella-derived lipopolysaccharide (LPS; 1 µg/mL; Sigma Chemical Co., St. Louis, MO), or C2 ceramide (N-acetylsphingosine, 100 µM; Sigma Chemical Co.). In experiments involving the caspase-1 inhibitor acetyl-tyrosyl-valyl-alanyl-chloromethyl-ketone (acetyl-YVAD-CMK Calbiochem, San Diego, CA), the inhibitor was added 1 h before infection and kept in the culture medium until the cells were recovered. Infections and exposures were carried out in 0.1% FCS, and cells and supernatants were recovered at 24 h, unless otherwise stated.


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  		Table 1. Characteristics and Origins of S. typhimurium Strains Examined in this Study

 
Electron microscopy
The morphology of DCs after infection was assessed by transmission electron microscopy (TEM). DCs were washed three times in ice-cold phosphate-buffered saline (PBS; pH 6.8), fixed in 2.5% glutaraldehyde in PBS, postfixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer, and contrasted in 0.5% uranyl acetate in 0.05 M maleate buffer. This was followed by dehydration in a graded series of ethanol and gradual replacement of ethanol with propylene oxide, before infiltrating and embedding cells in epoxy resin. Ultrathin sections were cut using a Reichert ultramicrotome, picked on carbon-coated copper grids, stained with uranyl acetate, counterstained with lead citrate, and observed with a Philips 300 TE microscope under an accelerating voltage of 60 kV.

Flow cytometry
Apoptosis and necrosis were assessed by flow cytometry as previously described [20 ]. In short, apoptotic cells were stained with phycoerythrin (PE)-labeled annexin V (10 µg/mL during 10 min at room temperature; PharMingen, San Diego, CA), which detects the translocation of phosphatidylserine to the outer layer of the cell membrane in the early stages of apoptosis [28 ], whereas necrotic cells were stained with propidium iodide (PI; 1 µg/mL immediately before measure; PharMingen), which indicates a permeabilization of the cell membrane. Cells were gated on the basis of their light-scattering properties, and two-color flow cytometry of PI versus PE was performed on a FACScan (Becton Dickinson, Mountain View, CA). To quantify the uptake of the virulent strain SL1344 and the sipB-deficient strain SL1344 by the DCs, bacteria were transformed with plasmid coding for enhanced green fluorescent protein (eGFP) under Ptac promoter control [29 ] to express eGFP constitutively, with comparable fluorescence intensity per CFU. Confocal microscopy showed that the contribution of bacteria sticking from the outside of the cells to the eGFP signal was negligible. One-color flow cytometry was performed with the FACScan to determine the percentage of GF cells among the DCs that were gated on the basis of their light-scattering properties.

Enzyme-linked immunosorbent assay (ELISA)
To measure secretion of IL-18 into the cell culture supernatant, 100 µL supernatant was subjected to a commercial ELISA kit (Diaclone, France), which recognized pro-IL-18 and mature IL-18 with a minimal detection limit of less than 45 pg/ml. Secretion of IL-12 by DCs or of IFN-{gamma} by T cells [see below: autologous mixed leukocyte reaction (AMLR)] was determined with commercial ELISA kits (R&D Systems and Diaclone, respectively).

Western blotting
Cell culture supernatants (5x1 mL/2x105 DCs) were incubated for 2 h at 4°C with 0.5 µg/mL anti-IL-18 monoclonal antibody (mAb; R&D Systems), recognizing pro-IL-18 and mature IL-18, followed by precipitation with protein G-sepharose beads (Sigma Chemical Co.) for 2.5 h at 4°C. The beads were washed two times with PBS before proteins were detached by boiling-in during 5 min at 95°C in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (SB). Cells (5x2x105 DCs) were washed and directly dissolved in the SB (5 min at 95°C). After separation on 12% SDS-PAGE and electroblotting, IL-18 was revealed by chemiluminescence (ECL-Plus, Amersham, Little Chalfont, UK) using the anti-IL-18 mAb and a peroxidase-labeled antibody against mouse immunoglobulin G. Pro-IL-18 and mature IL-18 were distinguished by their different molecular weights (24 kDa and 18 kDa, respectively).

AMLR
To perform AMLRs, the nonadherent cells were recovered after the initial adherence of the PBMC, and T cells were enriched on nylon wool columns, as described previously [30 ]. Until exposure to the autologous DCs, the T cells were frozen in RPMI containing 40% FCS and 10% Dulbecco’s modified Eagle’s medium, and viable cells were recovered by Ficoll gradient centrifugation prior to use. At 24 h after infection of the DCs with S. typhimurium, 5 x 103 DCs were incubated with 1.5 x 105 T cells in triplicates during 5 days in 200 µL CCM. At the end of this period, IFN-{gamma} secreted by the T cells was measured in the supernatant by ELISA (see above). Means of the triplicates were calculated in each of the independent experiments.

Statistical analysis
Results are reported as means ± SE of independent experiments performed with cells from separate donors, unless otherwise stated. The nonparametric Kruskal-Wallis and Wilcoxon signed ranks tests were used for significance testing between groups and between paired data, respectively; two-sided P values are given.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The genetic background of S. typhimurium influences the secretion of IL-18 by infected DCs
We have previously shown that the genetic background of S. typhimurium vaccine strains profoundly influences the induction of cytokines in human DCs, such as tumor necrosis factor {alpha}, IL-12[p70], and IL-10 [20 ]. Therefore, we also sought to evaluate the effect of different S. typhimurium strains on the secretion of the immunostimulatory cytokine IL-18 by the DCs. Figure 1A shows that the secretion of IL-18 by a S. typhimurium wild-type strain and different attenuated strains varied significantly according to their genetic background. Strongest IL-18 secretion was obtained with the wild-type and the attenuated mutant PhoPc. No increase of IL-18 secretion was observed when cells were exposed to heat-killed bacteria. We had previously observed that the S. typhimurium wild-type strain and the PhoPc mutant induced apoptosis more efficiently than the PhoP- and AroA mutants, whereas heat-killed bacteria did not kill DCs at all [20 ]. Therefore, we compared the secretion of IL-18 with the induction of apoptosis by DCs exposed to the different S. typhimurium strains (Fig. 1B) . These results indicated a significant correlation between apoptosis and IL-18 secretion for the different strains.



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  		Figure 1. (A) Attenuated S. typhimurium strains stimulate to a different extent the secretion of IL-18 by the DCs. iDCs were infected by the S. typhimurium wild-type ATCC14028 or by the attenuated strains PhoPc, PhoP-, and AroA (M.O.I. 25 bacteria/cell for all strains; see Materials and Methods) or were exposed to the same number of heat-killed PhoPc. At 24 h after exposure, the concentration of IL-18 in the supernatants (1 mL/2x105 cells) was determined by ELISA (see Materials and Methods). Bars are means ± SE of 8–10 independent experiments. Differences between groups were assessed with the Kruskal Wallis test: *, P < 0.05 for comparisons with the controls (medium); ***, P < 0.001 as compared with the control; {Psi}, P < 0.05 for comparisons of ATCC14028 or PhoPc with PhoP- or AroA. (B) IL-18 secretion is closely correlated to the capacity of the S. typhimurium strains to induce apoptosis. iDCs were exposed as described in A. The IL-18 secretion data are the same as in A. At 24 h after exposure, apoptosis was determined by flow cytometry with annexin-V-PE, detecting early and late stages of apoptosis. Results are expressed as percentage of all gated cells (see Materials and Methods). Apoptosis data are means of 6–11 independent experiments. The line was obtained by linear regression of the IL-18 data against the apoptosis data (correlation coefficient r=0.95; P<0.05).

 
The virulence factor SipB is responsible for the induction of apoptosis in the DCs
The ability of S. typhimurium to induce apoptosis in murine macrophages depends at least partially on virulence genes encoded in the SPI-1 region, in particular the gene sipB, which codes for Salmonella invasion protein B [23 , 31 ]. This SipB protein was reported to bind to and activate caspase-1 (also known as IL-1ß-converting enzyme), which is the enzyme that processes IL-18 into its mature form [32 , 33 ]. To examine whether the virulence factor SipB was important for the induction of apoptosis in human DCs as well, we exposed the cells to the virulent S. typhimurium strain C53 or to the mutant strain SipB, which is identical to C53 except for an insertion inactivating the sipB gene [34 ]. At 24 h after infection, apoptosis was detected by binding PE-labeled annexin-V to the cell membrane, as described previously [20 ]. In the same experiments, the early and the late stages of apoptosis were distinguished by PI, which stains late-stage apoptotic cells (PE+/PI+) and necrotic cells (PE-/PI+) but not early-stage apoptotic cells (PE+/PI–) or the viable cells (PE-/PI-). Figure 2A shows that the strain C53 strongly induced the late stage of apoptosis and to a lesser extent, also the early stage of apoptosis. In contrast, the SipB-deficient strain did not increase the total number of apoptotic cells (early or late apoptosis) as compared with the control. Almost identical results were obtained for the virulent strain SL1344 and its mutant SD11, which completely lacks the SPI-1 region where sipB is encoded (not shown).



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  		Figure 2. (A) The virulent strain C53 but not its sipB- mutant induces apoptosis in the DCs. iDCs were infected by the virulent strain C53 or by the sipB- mutant SipB. At 24 h after exposure, DCs were gated-in by their light-scattering properties (panel 1), and apoptosis and necrosis were determined by flow cytometry with annexin-V-PE and PI in controls (panel 2) and in cells infected by the strains C53 (panel 3) or SipB (panel 4). PE-positive and PI-negative cells were counted as the early stage of apoptosis (lower right quadrant); PE-positive and PI-positive cells were counted as the late stage of apoptosis (upper right quadrant), and counts were expressed as percentage of all gated cells. (B) C53 and the sipB- mutant infect the DCs, but only C53 produces the morphological signs of apoptosis. iDCs were infected by the strain C53 or by its sipB- mutant SipB. At 24 h after infection, cells were processed for light microscopy (LM) or for TEM (EM). Cells in apoptosis are recognized in LM by the condensation of the nuclear chromatin in cells infected by C53 (arrows in panel 1) but not in those infected by SipB (panel 2) and in EM, by the fragmentation of the nucleus in a cell infected by C53 (panel 3) but not in that infected by SipB (panel 4). N, Nucleus; S, S. typhimurium; P, spacious phagosome where the virulent strain has multiplied. Original LM scale bar = 25 µm; original EM scale bar = 2 µm.

 
To verify whether DCs exposed to the strain C53 indeed underwent apoptotic cell death and to confirm in particular for the SipB-deficient strain the presence of the bacteria inside the DCs, we performed light and TEM at 24 h after infection. This analysis revealed the morphological signs of apoptosis in DCs exposed to C53 but not in cells exposed to the sipB- mutant, despite the fact that the cells were efficiently infected by both strains (Fig. 2B) . Very similar apoptosis and infection patterns were seen with the virulent strain SL1344 and its derivative SD11, respectively (not shown). To quantify the uptake of the virulent strain as compared with its sipB--deficient derivative, we also infected DCs with fluorescent SL1344 and SD11 strains (see Materials and Methods). Fluorescence-activated cell sorter analysis at 24 h after infection showed that the uptake of both strains by the iDCs was equally efficient (40.6±2% and 38.8±1.8% positive cells, respectively, as compared with 1.1±1% in the controls; n=3).

Release of IL-18 is correlated with apoptosis and is dependent on the virulence factor SipB
To test whether mutations that affected the capacity of S. typhimurium to induce apoptosis would also influence the secretion of IL-18, we compared both parameters at 24 h after infection. Figure 3A shows the percentage of apoptotic cells after exposure to LPS, heat-killed bacteria, the different live bacteria, or C2 ceramide, an agent that induces apoptosis in the DCs [35 ]. Our results show that the S. typhimurium parent strain C53 but not its sipB- mutant SipB triggered apoptosis to a similar extent as C2 ceramide. Again, the parent strain SL1344 and its SPI-1 mutant SD11 induced the same pattern as the strains C53 and SipB, respectively. It is interesting that the bacterial product LPS alone, which is known to induce DC maturation, appeared to confer some protection against spontaneous apoptosis in uninfected cells. Figure 3B shows that the virulent S. typhimurium strains C53 and SL1344 increased the secretion of IL-18 to more than five times the baseline level. In contrast, the induction of IL-18 secretion was largely blunted in the sipB- mutants SipB and SD11, neither of which expresses a functional SipB protein. C2 ceramide induced a very strong IL-18 secretion, and LPS had no significant effect.



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  		Figure 3. (A) Different mutations affecting the expression of sipB block the induction of apoptosis. iDCs were infected by the virulent S. typhimurium parent strains C53 or SL1344 or by their sipB- mutants SipB and SD11, respectively (M.O.I. 25 for all strains), or were exposed to S. typhimurium-derived LPS (1 µg/mL) or C2 ceramide (100 µM), an agent that induces apoptosis. At 24 h after exposure, apoptosis was determined by flow cytometry with annexin-V-PE, detecting early and late stages of apoptosis, and was expressed as percentage of all gated cells. Bars are means ± SE of 5–12 independent experiments. Differences between groups were assessed with the Kruskal Wallis test: *, P < 0.05 for comparisons with the controls (medium); **, P < 0.01 as compared with the control; ***, P < 0.001 as compared with the control; {Psi}, P < 0.05 for comparisons between the strains. (B) The same sipB mutations largely block IL-18 secretion. iDCs were exposed as described in A. At 24 h after exposure, the concentration of IL-18 in the supernatants (1 mL/2x105 cells) was determined by ELISA. Bars are means ± SE of 7–17 independent experiments. Differences between groups were assessed with the Kruskal Wallis test: *, P < 0.05 for comparisons with the controls (medium); ***, P < 0.001 as compared with the control; {Psi}, P < 0.05 for comparisons between the strains.

 
The virulence factor SipB induces the activation of IL-18
IL-18 is synthesized as a 24-kDa precursor protein, pro-IL-18, which is proteolytically processed by caspase-1 to the 18-kDa mature form, mature IL-18 [32 , 33 ]. Only the proteolytically cleaved form, mature IL-18, binds to the IL-18 receptor complex and is biologically active [4 , 33 ]. To determine whether S. typhimurium would induce the activation of IL-18 in the DCs, we investigated the processing of IL-18 by Western blotting (Fig. 4 ). Our results show that after infection with virulent strain C53, IL-18 released in the supernatant was predominantly the 18-kDa mature form, indicating that C53 not only induced the release but also the activation of this cytokine. In contrast, after infection with the sipB- strain SipB, IL-18 recovered from the supernatant was predominantly the 24-kDa precursor form. Also, when IL-18 release was induced by the apoptosis-inducing agent C2 ceramide, only the biologically inactive pro-IL-18 could be detected in the supernatants at 24 h after exposure (not shown).



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  		Figure 4. The virulent S. typhimurium strain C53 but not the sipB- mutant triggers the activation of IL-18. iDCs were infected by the strain C53 or by its sipB- mutant, SipB (M.O.I. 25 for both strains). At 24 h after infection, 1 x 106 DCs were dissolved in SB. The supernatant of these cells (5 mL) was immunoprecipitated with a mAb against IL-18, and the precipitate was dissolved in SB (see Materials and Methods, Western blotting). Recombinant, mature IL-18 (20 ng) was directly dissolved in SB (mat-IL-18) or diluted in culture medium (5 mL) and immunoprecipitated as described above [mat-IL-18 (IP)]. After SDS-PAGE and electroblotting, IL-18 was recognized by chemiluminescence using the mAb. Pro-IL-18 is revealed at the 24-kD position and activated, mature IL-18 at the 18-kD position, which were determined by molecular weight markers.

 
The activation of IL-18 by the virulence factor SipB is mediated by caspase-1
Caspase-1 activity, which is critical for the processing of IL-18, has been linked to the release of this cytokine from the cells [32 ]. To assess whether activation and release of IL-18 by the SipB virulence factor were mediated by caspase-1, we used the cell permeable, irreversible inhibitor acetyl-YVAD-CMK (50 µM), which blocks the proteolytic activity of caspase-1 [23 ]. Although the inhibitor completely blocked processing of pro-IL-18 into mature IL-18 in C53-infected cells (Fig. 5A ), the inhibition of caspase-1 did not influence the secretion of IL-18 into the supernatant (Fig. 5B) . This indicated that activation of IL-18 was not necessary for its release from the infected cells.



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  		Figure 5. (A) Inhibition of caspase-1 blocks the activation of IL-18 after infection by virulent S. typhimurium. iDCs were infected by the strain C53 (M.O.I. 25). YVAD: One hour before exposure and during exposure, cells were treated with the cell-permeable, irreversible caspase-1 inhibitor YVAD-CMK (50 µM). At 24 h after infection, IL-18 from the supernatant (5 mL/1x106 DCs) was immunoprecipitated, and Western blotting with the cells and with the precipitate from the supernatant was performed as described in Figure 4 . (B) The inhibition of caspase-1 does not prevent the release of IL-18 from the cells. iDCs were infected by the strain C53 (M.O.I. 25). YVAD: As described in A. At 24 h after exposure, the concentration of IL-18 in the supernatants (1 mL/2x105 cells) was determined by ELISA. Bars are means ± SE of three independent experiments.

 
The induction of apoptosis by SipB in the DCs is not caspase-1-dependent
In murine macrophages, the induction of apoptosis by Salmonella SipB has been shown to be mediated by caspase-1 [23 ]. To assess the role of caspase-1 activation in the apoptosis of human DCs, we compared the effects of the caspase-1 inhibitor acetyl-YVAD-CMK (50 µM) on the induction of apoptosis by the virulent Salmonella strain C53, its sipB-deficient derivative SipB, and the proapoptotic agent C2 ceramide (Fig. 6 ). Whereas the caspase-1 inhibitor had no effect on the Salmonella-induced apoptosis, it did significantly reduce the percentage of apoptotic cells in the DCs exposed to C2 ceramide. In conclusion, Salmonella SipB regulated apoptosis, IL-18 maturation, and IL-18 release in the DCs. However, only the maturation of IL-18 was dependent on caspase-1. In contrast, C2 ceramide induced apoptosis and IL-18 release but not the maturation of IL-18. The induction of apoptosis by C2 ceramide was caspase-1-dependent.



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  		Figure 6. Inhibition of caspase-1 does not prevent the induction of apoptosis by virulent S. typhimurium. iDCs were infected by the virulent S. typhimurium parent strain C53 or by its sipB- mutant SipB (M.O.I. 25 for both strains) or were exposed to C2 ceramide (100 µM). YVAD: One hour before exposure and during exposure, cells were treated with the cell-permeable caspase-1 inhibitor YVAD-CMK (50 µM). At 24 h after exposure, apoptosis was determined by flow cytometry with annexin-V-PE, as described in Figure 3 . Bars are means ± SE of three to eight independent experiments. Differences between groups were assessed with the Kruskal Wallis test: **, P < 0.01 as compared with the control; {Psi}, P < 0.05 for the comparison between exposures with and without the caspase-1 inhibitor.

 
S. typhimurium-infected DCs become potent inducers of IFN-{gamma}
The capacity of infected DCs to stimulate the secretion of IFN-{gamma} was tested with autologous T cells (see Materials and Methods, AMLR). These experiments revealed that DCs exposed to live PhoPc, an attenuated strain that expresses SipB and induces apoptosis in a substantial part of the DCs [20 ], very efficiently stimulate IFN-{gamma} in the T cells (710±172 ng/ml as compared with 114±63 ng/ml using heat-killed bacteria and 5±3 ng/ml with unexposed DCs; n=3). Experiments using DCs infected by the SipB strain showed that they also induced IFN-{gamma} in the T cells to some extent (184±18 ng/ml). However, it was not possible to compare the SipB-deficient mutants with their virulent parent strains, as the latter would successively reinfect and kill the DCs and T cells during the 5-day coincubation period. PhoPc and SipB efficiently induced in the DCs the secretion of IL-12 [p70] (124±77 pg/ml and 647±54 pg/ml, respectively, as compared with 7.7±3 pg/ml in the controls; n=5), which acts in synergy with IL-18 to stimulate the production of IFN-{gamma} by T cells [36 ]. Experiments using a blocking antibody against the IL-18 receptor (mAb 840, R&D Systems) inhibited the secretion of IFN-{gamma} in response to PhoPc by up to 32%, suggesting that IL-12 and/or other cytokines produced by the infected cells could not fully substitute for the effects of IL-18.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been suggested that DCs play a prime role in the innate response and provide a link between innate and acquired immunity [13 , 14 ]. If so, DCs need to be able to distinguish between dangerous pathogens and inoffensive particles. Members of the Toll-like receptor family mediate the recognition of pathogen-associated molecular patterns (PAMPs) by the DCs [37 ]. Our previous work indicated that besides the PAMPs, S. typhimurium mutations, which influenced the expression of the bacterial virulence factors, had a profound impact on DC activation and the secretion of Th1 versus Th2 cytokine patterns [20 ]. Therefore, we searched for alternative pathways that might allow the DCs to recognize and to signal the danger from virulent pathogens. In the present study, we identify a new link between a Salmonella virulence factor and the emission of the immunostimulatory cytokine IL-18 by the DCs.

Salmonellae may enter DCs by active invasion or by passive phagocytosis [12 ]. Immature DCs are efficient phagocytes and take up heat-killed S. typhimurium as readily as inert particles [38 ]. However, our results showed that only live S. typhimurium triggered the release of IL-18 from the DCs. Moreover, we found that IL-18 secretion varied strongly according to the genetic background of S. typhimurium. These findings suggested that the extent at which S. typhimurium triggers the secretion of IL-18 in the DCs depends on factors produced by live bacteria during infection. Because the pattern of IL-18 secretion induced by the different S. typhimurium vaccine strains resembled the pattern of apoptosis caused by the same strains in the DCs, we sought to identify the molecular mechanisms that might link IL-18 secretion and apoptosis. S. typhimurium is known to actively induce apoptosis in murine macrophages by translocation of the virulence factor SipB in the cytoplasma of its host cell through a type III protein-secretion system [23 , 39 ]. Our results showed that mutations that abolish the expression of the sipB gene not only blocked the ability of S. typhimurium to induce apoptosis but also controlled the secretion of IL-18 by the infected DCs. These results could not be explained by differences in infectivity of the strains, as the sipB- mutants were taken up by the DCs as efficiently as their virulent parent strains. Whereas the induction of apoptosis by the SipB protein has been described previously in other cell types, this is the first time that the role of this bacterial virulence factor has been demonstrated in the secretion of IL-18.

IL-18 is synthesized as a larger precursor that lacks a secretory leader signal. Yet, IL-18 can be secreted in the biologically active, mature form or in the precursor form [40 ]. Our results clearly showed that S. typhimurium not only triggered the release of IL-18 but also the processing of pro-IL-18 into mature IL-18. The secretion and the activation of IL-18 were dependent on the SipB virulence factor, as sipB- mutants were not able to release IL-18 from the DCs or to induce its conversion to mature IL-18. To test the possibility that the release of IL-18 from the DCs was a consequence of the conversion from pro-IL-18 into mature IL-18, we blocked the processing of IL-18 with a caspase-1 inhibitor. We found that the release of IL-18 by S. typhimurium was independent of the processing of pro-IL-18 to mature IL-18 by caspase-1. This finding was in accordance with previous reports documenting the release of pro-IL-18 from human DCs [40 , 41 ]. A probable explanation for the release of pro-IL-18 from the DCs after infection with S. typhimurium would be the progression of cells from early to late stages of apoptosis, leading to permeabilization or even disintegration of the cell membrane. The induction of apoptosis in the infected DCs was not blocked by the caspase-1 inhibitor, indicating that other apoptotic pathways might be triggered by S. typhimurium. In mouse macrophages, the Salmonella virulence factor SipB was recently found to simultaneously activate caspase-1 and caspase-2, which both induce apoptosis [42 ].

The secretion of the biologically active, mature form of IL-18 by the DCs appeared to be a specific, caspase-1-mediated effect of the Salmonella virulence factor SipB, rather than a consequence of Salmonella-induced cell death and subsequent leakage of the cytokine from the dying cells. No mature IL-18 was recovered from control cells or their supernatants, although apoptosis occurred spontaneously in a significant fraction of the DCs. Moreover, the proapoptotic agent C2 ceramide did not release the mature form of IL-18 from the cells, despite the role of caspase-1 in ceramide-mediated apoptosis. This fact might be explained by previous findings that the caspase-1-dependent activation of IL-18 in DCs is facilitated by bacterial factors, such as LPS [16 ]. An alternative explanation would be that ceramide-induced cell death progressed very rapidly, and pro-IL-18 was leaked from the dying cells before activation took place.

In caspase-1 -/- knockout mice, the spread of S. typhimurium from the Peyer’s patches is severely compromised [43 ]. This finding underlines the important role of caspase-I, a molecular target of the Salmonella virulence factor SipB, in the bacterial infection strategy. SipB plays a dual role in this strategy: First, as part of the bacterial type III protein secretion complex, the protein mediates the invasion of different cell types, in particular the nonphagocytic cells. Second, the SipB virulence factor induces apoptosis, which may be a prerequisite for the escape from bactericidal mechanisms of the host cells, especially the APC, and the spread from the Peyer’s patches to the primary lymph nodes. DCs would be likely candidates to carry the bacteria from the initial site of infection to the sites where specific immunity is induced. Therefore, the interaction of S. typhimurium with the DCs at this stage of infection is expected to be of critical importance for the in vivo progression or elimination of the pathogen.

Our findings in the human DCs suggest a central function of the sipB gene product not only in the induction of apoptosis but also in the stimulation of IL-18 secretion in the DC. It is likely that other bacterial virulence factors that control infection and/or intracellular survival influence the effects that are mediated by the SipB protein. Among the attenuated vaccine strains PhoPc, PhoP-, and AroA, the PhoPc mutant most efficiently induced apoptosis and IL-18 secretion. Although the expression of the SipB protein would not be expected to be increased in the PhoPc mutant [44 ], this strain is characterized by a particularly high infectious efficacy in human DCs [20 ], which might have influenced its impact as compared with the other attenuated strains. It is likely that the combined effect of the Salmonella virulence factor SipB on apoptosis and IL-18 secretion might also be observed for SipB protein homologues in other intracellular pathogens. Shigella produces the IpaB virulence factor that induces apoptosis in the host cell [45 ]. It has been shown recently that this effect is a result of activation of caspase-1 [46 ]. However, the effects of IpaB or other bacterial virulence factors on the release and/or the activation IL-18 have not yet been investigated. The induction of apoptosis by M. tuberculosis was also found to be mediated by caspase-1 [47 ], but a mycobacterial factor that activates caspase-1 has not been identified to date.

The unique ability of DCs to induce and sustain primary immune responses makes them ideal targets for vaccine vectors. The role of IL-18 in the induction of innate and acquired immunity has been demonstrated previously [3 4 5 6 7 8 ]. In the in vitro study presented here, we have found that the bacterial vector S. typhimurium, which might provide the basis for a human live vaccine [48 ], efficiently triggers the secretion of active IL-18 in the DCs. It appears plausible that in vivo, DCs secreting IL-18 have more impact on the immune response than other cell types, because of their privileged contact with the effector cells. Although live PhoPc induce apoptosis in a substantial part of the DCs [20 ], our results reveal that they stimulate DCs much more efficiently to induce IFN-{gamma} than heat-killed bacteria do. The more virulent S. typhimurium strains kill and reinfect DCs and T cells very efficiently, which prevents antigen presentation in these cells. Therefore, the virulent strains cannot be used in the AMLR to assess their effect on the induction of IFN-{gamma} in T cells. However, when we used PhoPc, we were able to see that up to one-third of the IFN-{gamma} secretion induced by this strain was dependent on IL-18, as shown by antibodies against the IL-18-receptor. The remaining production was probably related to the IL-12 and/or other stimulatory cytokines that were released by the infected DCs.

It has been suggested that bacteria that provoke cell death through the activation of caspase-1 induce a particular form of apoptosis, which involves the release of proinflammatory cytokines [49 ], and that the induction of such "inflammatory apoptosis" may increase the efficacy of vaccine vectors [50 ]. Here, we demonstrate that the activation and the release of IL-18 in human DCs are triggered by the proapoptotic virulence factor SipB. It is likely that more immunostimulatory cytokines, for example other IL-1 family proteins that are also processed by caspase-1, are activated and released along with IL-18 when the apoptotic cascade is triggered by the bacteria. This mechanism may constitute an important element of the innate-immune defense against pathogens that induce cell death, as it would allow the infected cells to signal the danger before being neutralized. Future investigation may be aimed at finding ways to dissociate the IL-18-activating action of live vaccine vectors from their proapoptotic effects.


   ACKNOWLEDGEMENTS
 
This work was supported by the Swiss National Foundation Grants #3100-047073.96/1, #32-52638.97, #4 037 055 164, and #4 037 061 164. D. D. received support from the Lancardis Foundation. C. O. was supported by a fellowship grant from the European Respiratory Society. We thank Elisabeth Bühlmann for excellent assistance with cell culture, Ursula Gerber for technical assistance with TEM, and Dominique Wohlwend for his advice with flow cytometry. We thank Drs. J. J. Mekalanos, C. Lee, M. Y. Popoff, J-M. Clement, and S. Falkow for kindly supplying bacterial strains or plasmids.

Received February 22, 2002; revised May 31, 2002; accepted June 17, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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