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(Journal of Leukocyte Biology. 2001;69:583-589.)
© 2001 by Society for Leukocyte Biology

Genetic background of attenuated Salmonella typhimurium has profound influence on infection and cytokine patterns in human dendritic cells

Donatus Dreher*, Menno Kok{dagger}, Laurence Cochand*, Stephen Gitahi Kiama{ddagger}, Peter Gehr{ddagger}, Jean-Claude Pechère{dagger} and Laurent Pierre Nicod*

* Division of Pneumology, University Hospital of Geneva, Switzerland;
{dagger} Department of Genetics and Microbiology, University of Geneva, Geneva, Switzerland; and
{ddagger} Institute of Anatomy, University of Berne, Berne, Switzerland

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


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ABSTRACT
 
Salmonella typhimurium (ST) can cause infection in man, and attenuated strains are under consideration as live vaccine vectors. However, little is known about the interaction of ST with human dendritic cells (DC). Here, we compared the consequences of exposure of human, monocyte-derived DC with different attenuated strains of ST. Infection was observed with all four strains tested (wild type, PhoP-, PhoPc, and AroA), but the PhoPc strain was by far the most efficient. Intracellular persistence of wild type and PhoP- was longer than that of PhoPc and AroA, both of which were largely eliminated within 24 h. Most DC survived infection by the attenuated strains, although apoptosis was observed in a fraction of the exposed cells. All strains induced DC maturation, independent from the extent of infection. Although all strains stimulated secretion of TNF-{alpha} and IL-12 strongly, PhoPc induced significantly less IL-10 than the other three strains and as much as 10 times less IL-10 than heat-killed PhoPc, suggesting that this mutant suppressed the secretion of IL-10 by the DC. These data indicate that infectivity, bacterial elimination, and cytokine secretion in human DC are controlled by the genetic background of ST.

Key Words: host defense • vaccine vectors • antigen-presenting cells • apoptosis • interleukin-10 • interleukin-12


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INTRODUCTION
 
Dendritic cells (DC) are the most potent antigen-presenting cells (APC) of the human body. They capture particulate or soluble antigens (Ag) at the site of pathogen entry, and after activation by microbial signals, migrate to the local lymphatic organs where they present the processed Ag in the context of lymphocyte co-stimulatory molecules to naive and primed T cells [1 , 2 ]. The development and proliferation of the T cells depend on the cytokine microenvironment created by the activated DC. It was demonstrated recently that DC control the differential activation of T cells toward a T helper cell type (Th)1- or Th2-directed immune response [3 ]. Th1 lymphocytes stimulate cell-mediated immunity predominantly, whereas Th2 cells stimulate humoral immune responses primarily. Hence, the potential to control Th1- versus Th2-immune stimulation is critical to the success of protective immunization [4 ].

Attenuated strains of the facultative, intracellular bacterium Salmonella typhimurium (ST) have been tested extensively as vaccine vectors in animal models [5 6 7 8 9 10 ], where they can induce T cell- and B cell-mediated immune responses [11 ]. Salmonella infect murine DC efficiently [12 ] and are capable of gene transfer into mammalian APC [6 , 7 ]. In addition, infection with Salmonella can induce maturation and cytokine production in murine DC [12 ], programming the cells for Ag presentation and stimulation of T cells. However, experimental data on the interaction between human DC and these microbial vaccine vectors are scarce. Very recently, the consequences of infection of human DC by Listeria monocytogenes have been shown [13 ]. The interaction of human DC with ST, however, has not been investigated yet.

When confronted with an intracellular pathogen, such as Salmonella, DC have to assume a dual role: 1) They become themselves a target of infection by the pathogen, and 2) they must initiate and regulate the immune defence against this pathogen [2 , 14 ]. As a first step toward the elucidation of these two roles, which are relevant to vaccine research and our understanding of Salmonella-related disease, we have studied the interaction between human, monocyte-derived DC and live ST in vitro. We demonstrate that ST can infect human DC with relatively small impact on viability, induce maturation, and stimulate the secretion of inflammatory cytokines. It is interesting that the different attenuating mutations of the ST vaccine strains did not only influence the infectious properties of the bacteria profoundly but also the cytokine-response pattern of the DC.


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MATERIALS AND METHODS
 
Culture of DC from peripheral blood monocytes
Human DC were cultured from peripheral blood mononuclear cells (PBMC) as described by Sallusto and Lanzavecchia [15 ]. PBMC were obtained from healthy donors by FICOLL-Hypaque (AP Biotech, Uppsala, Sweden) density-gradient centrifugation of buffy coats, as described previously [16 ]. After adherence for 1 h, culture dishes were rinsed with Hanks’ balanced salt solution (HBSS), and adherent cells were incubated overnight in RPMI 1640 (Life Technologies, Inc., Rockville, MD) supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin (complete culture medium, CCM). Loosely adherent monocytes were recovered with three rinses of HBSS. DC were obtained from the monocytes by culture during 7 days in CCM with granulocyte-macrophage colony-stimulating factor (GM-CSF; 10 ng/ml; Immugenex Corp., Los Angeles, CA) and interleukin (IL)-4 (10 ng/ml; R&D Systems, Minneapolis, MN) [15 ]. We used immature DC at day 7 post-isolation for infection with ST, when their ability to phagocytize bacteria is at a maximum [17 ].

Bacteria and growth conditions
We studied the the ST wild-type strain ATCC14028, which is highly virulent in mice (LD50<5 colony-forming units by intraperitoneal injection; unpublished results), and three attenuated strains of ST that are under consideration as vaccine vectors [5 6 7 8 9 10 ]: 1 and 2) two mutants in the phoPQ virulence regulatory system that are derived from this wild type: one (PhoP-) with a deletion in the major virulence regulator gene phoP [18 ] and another (PhoPc) with a mutation in the phoQ gene (phoQ24) that leads to constitutive activation of phoP and deregulation of virulence genes [19 ]; and 3) a ST strain with a mutation in the aroA gene (AroA) that is avirulent as a result of its incapability to synthesize aromatic amino acids and para-aminobenzoic acid, which cannot be acquired from mammalian sources [20 ]. Prior to the infection of human DC, 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 a measurement of 450 nm optical density. When the optical density reached values between 3 and 5 x 108 cfu/ml, the cultures were diluted in pre-warmed RPMI to obtain a suspension of 5.0 x 107 cfu/ml.

Infection of DC with Salmonella, rates of infection, and intracellular survival
For infection with ST at day 7, DC were washed, resuspended in CCM without antibiotics, and seeded in wells (1 ml containing 2x105 cells/well). DC were infected by the addition of 100 µl of the bacterial suspensions to obtain a multiplicity of infection (M.O.I.) of 25 bacteria/cell. Infection was allowed to proceed for 30 min, after which extracellular bacteria were killed by the addition of 60 µg/ml gentamicin (Sigma Chemical Co., St. Louis, MO).

To evaluate the number of infected cells, the DC were recovered in trypsin-ethylenediaminetetraacetate (EDTA) 3 or 24 h after infection, and the cell suspension was diluted in RPMI medium before plating on LB-agar. One colony-forming unit was considered to correspond to one infected cell. To estimate the total number of intracellular bacteria, the initial cell suspension in RPMI was lysed with 0.05% Triton X-100 and diluted in 10 mM MgCl2 before plating.

Transmission electron microscopy (TEM) of DC exposed to Salmonella
To process the infected DC for TEM, cells were washed in HBSS and resuspended in phosphate-buffered (pH 6.8) glutaraldehyde (2.5%) solution. The glutaraldehyde-fixed cells were centrifuged, 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 (70%, 80%, 96%, 100%, and 100%) 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 200-mesh 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.

Analysis of apoptotic versus necrotic DC by flow cytometry
Apoptotic cells were labeled with annexin V, which detects the translocation of phosphatidylserine to the outer layer of the cell membrane in the early stages of apoptosis [21 ]. Cells were washed and exposed during 10 min at room temperature to phycoerythrin (PE)-labeled annexin V in annexin-binding buffer (10 µg/ml; PharMingen, San Diego, CA), followed by exposure to propidium iodide (PI; 1 µg/ml; PharMingen) immediately before analysis, which penetrates and stains necrotic cells. Two-color flow cytometry of PI versus PE was performed on a FACScan (Becton Dickinson, Mountain View, CA).

Analysis of maturation marker CD86 and phagocytosis of dextran by flow cytometry analysis
Maturation of DC was determined 48 h after infection with ST by analysis of the expression of the surface marker CD86 (B7.2) and the down-regulation of phagocytic activity of inert particles (dextran). Cell staining was performed with the CD86 MAB (IT2.2) and control isotype monoclonal antibodies (mAbs), followed by fluorescein isothiocyanate (FITC)-conjugated, goat anti-mouse Ab. The samples were analyzed on a FACS (Coulter, EPICS XL-MCL, Beckman Coulter, Fullerton, CA). Dead cells were gated out on the basis of their light-scattering properties. The results are shown as mean fluorescence intensity (MFI). No MFI changes were observed with the isotype-matched control antibody for CD86.

Uptake of FITC-dextran was performed as described previously [16 ]. Briefly, FITC-dextran (Molecular Probes, Eugene, OR) was added to the cell suspension at the final concentration of 1 mg/ml. The cells were incubated for 1 h at 4°C (nonspecific binding) or at 37°C, washed with cold CCM containing 0.01% NaN3, and analyzed on the FACS.

Secretion of cytokines by DC after infection with Salmonella
Cytokine secretion by DC was measured in the culturesupernatant 24 h after ST infection with enzyme-linked immunosorbent assay (ELISA) kits using mAb against tumor necrosis factor {alpha} (TNF-{alpha}; Biosource, Camarillo, CA), IL-10 (Biosource), and the active form of IL-12 [p70] (R&D Systems), respectively.

Statistical analysis
Results are shown as means ± SE of independent experiments performed with cells from separate donors. Using the nonparametric Friedman and Kruskal-Wallis tests for paired and nonpaired data, respectively, multiple comparisons were carried out when the overall testing for differences among groups was significant. Tests were done with the StatsDirect software, Version 1.5 (www.statsdirect.com).


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RESULTS
 
Infection of DC by Salmonella
The gram-negative bacteria ST can infect, survive, and replicate within many cell types in vitro, including murine DC [12 ] and human macrophages [22 ]. To determine how potential vaccine strains of ST would interact with DC of human origin, we exposed DC derived from peripheral blood monocytes to the ST wild-type strain and to the attenuated strains PhoP-, PhoPc, and AroA. The proportion of infected cells was evaluated after 3 h, as described in Materials and Methods. Infection of human DC was observed with all strains (Fig. 1A ). However, infection rates were significantly higher for PhoPc (54±10%) than for the wild type (15±4%, p<0.001), PhoP- (9±2%, p<0.001), or AroA (4±2%, p<0.001).



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  		Figure 1. The attenuated S. typhimurium strain PhoPc infects human dendritic cells most efficiently. After 24 h, PhoPc and AroA are killed largely by the DC. Means of six independent experiments ± SE are shown. (A) Efficiency of infection was estimated from the percentage of cells that contained live bacteria 3 h after exposure to the different strains (25 bacteria/cell). (B) Intracellular survival of the different strains was evaluated by the bacterial growth index, defined as the number of cells still containing live bacteria at 24 h after exposure, divided by the number of cells containing live bacteria at 3 h. *, P < 0.05 as compared with PhoPc.

Intracellular survival of Salmonella
An ideal vaccine strain should be highly infectious for APC but also well-susceptible to antibacterial defense mechanisms of the host cell to avoid uncontrolled multiplication of the pathogen. To evaluate the survival of the different ST strains in human DC, we followed bacterial growth after infection. The growth index was defined as the number of DC containing live bacteria at 24 h divided by the number of DC 3 h after infection. As shown in Figure 1B , the PhoPc and AroA strains were eliminated efficiently by the DC in vitro (bacterial growth index, 0.25±0.06 and 0.32±0.07, respectively), whereas most of the wild type and the PhoP- bacteria survived within the cells (0.56±0.25 and 1.08±0.15, respectively). The differences in intracellular growth of PhoPc or AroA compared with wild type or PhoP- were all statistically significant (p<0.05). The fact that host-cell death did not increase significantly between 3 and 24 h of infection (see below, Fig. 3 ) suggested that the bacterial growth index was low for PhoPc and AroA because of the bactericidal activity of the DC rather than because of lysis of the host cells. For all strains, except the wild type, bacterial survival decreased after more than 24 h of infection (unpublished results). Our microbiological methods were accurate to follow intracellular survival of the attenuated strains up to 24 h after infection, because at that time point, necrosis and lysis of DC induced by these strains appeared to be negligible, and no excess necrotic cell death was observed compared with the controls (see Fig. 4 ). However, we could not use these methods to follow the intracellular behavior of the wild-type strain over 48 h, because this strain caused progressive cell lysis during prolonged infection (see below), with some bacteria escaping the effect of the extracellular antibiotic and reinfecting adjacent DC. This particular behavior explains the relatively low intracellular growth index of the wild type (Fig. 1B) and the observation that it killed more cells at 48 h than it had infected initially (see below).



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  		Figure 3. Viability of DC does not decrease significantly between 3 and 24 h after exposure to S. typhimurium. The number of viable cells was determined by the exclusion of TB. Means of four independent experiments ± SE are shown. {circ}, P < 0.05 as compared with medium; *, P < 0.05 as compared with PhoPc.



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  		Figure 4. Apoptosis or necrosis is induced in only a minor fraction of the DC at 24 h after exposure to S. typhimurium. Two-color flow cytometry was performed using PE-annexin V and PI. Apoptosis = cells positive for PE-annexin V but negative for PI; necrosis = all PI-positive cells. Means of three independent experiments ± SE are shown. {circ}, P < 0.05 as compared with medium; *, P < 0.05 as compared with PhoPc.

TEM of DC after infection with Salmonella
To analyze the nature of DC infection by ST morphologically, infected cells were examined by TEM, which confirmed that the attenuated strains and the wild-type strain of ST were phagocytized by the DC. Figure 2 shows the intracellular localization of the bacteria, 3 h after infection by the attenuated strain PhoPc. For all ST strains, the number of cell profiles with intracellular bacteria and the total number of bacteria profiles inside 100 randomly chosen cell profiles were counted at 3 h. Thus, in a characteristic series, the wild-type strain was found in 28% of all profiles randomly analyzed, whereas 12%, 45%, and 19% of all cell profiles contained bacteria after infection with the strains PhoP-, PhoPc, and AroA, respectively. The number of bacterial profiles divided by the number of cell profiles amounted to 0.43, 0.19, 1.0, and 0.24, for the wild type, PhoP-, PhoPc, and AroA, respectively. The results from this analysis confirm the intracellular localization of the ST and corroborate the high infection rates of PhoPc that were detected with the microbiological methods (see above). In contrast to these, TEM did not distinguish between live and dead bacteria.



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  		Figure 2. Intracellular localization of S. typhimurium strain PhoPc in DC by TEM 3 h after exposure to the bacteria. Several bacteria are seen enclosed in separate phagosomes (arrows; N=cell nucleus; original scale bar=2 µm).

Viability of DC after infection
To determine to which degree infection with ST induces cell death in human DC, we assessed viability by microscopy and flow cytometry with a dye-exclusion method and by detecting apoptosis with flow cytometry. Figure 3 shows viability of DC at 3 h and at 24 h after infection that was determined microscopically by the exclusion of Trypan blue (TB). At 3 h after infection, the viability of DC was significantly higher for AroA than for all the other three strains (p<0.01). At 24 h, excess cell death compared with the control ranged from 8% (AroA) to 28% (PhoP-), and a significant difference persisted between AroA and PhoPc (p<0.05). For none of the tested ST strains, a significant difference between DC viability at 3 h versus 24 h after infection was found, indicating that no substantial killing of DC had occurred beyond the first 3 h of infection during this time period. However, at 48 h after infection, the impact on viability became more important, in particular in DC exposed to the wild type, where 58% (range, 56–59%) of all cells stained TB-positive at 48 h. In DC infected with the attenuated strains, cell death at 48 h was 45% for PhoP-, 35% for PhoPc, and 18% for AroA.

Apoptosis versus necrosis in DC
Salmonella has been shown to induce apoptosis in infected macrophages [23 ]. To test whether ST would also induce apoptosis in human DC, we detected apoptosis with flow cytometry by binding annexin V to the cell membrane [21 ]. In the same experiment, necrosis was assessed by PI staining. Figure 4 shows the percentages of apoptotic (annexin V-positive but PI-negative) and necrotic (PI-positive) cells at 24 h after infection with the different ST strains. The proportion of apoptotic cells was increased significantly for all strains, ranging from 8.1% (AroA) to 21.3% (PhoPc) compared with 5.2% in the control and 3.1% for heat-killed PhoPc. The fact that DC exposed to PhoPc showed a significantly higher percentage of apoptotic cells than those exposed to PhoP- or AroA (p<0.01) might be explained by the high infection rates of PhoPc compared with PhoP- or AroA (see Fig. 1A ). It is interesting that the overall increase in cell death (apoptosis or necrosis) induced by the strain PhoPc appeared to be about five times smaller than the proportion of cells that was infected. As compared with the assessment of DC viability by TB, flow cytometry using annexin-V had the advantage of detecting early apoptosis that would not be seen by the TB method. Conversely, flow cytometry using PI would not recognize necrotic cells that have already lost their typical DC morphology and would be gated out by the FACS, and these cells would be seen by the TB method. Yet the results of both methods show consistently that ST had a significant impact on DC viability, which was limited to the minority of cells at 24 h after infection and appeared to be more important for wild type, PhoP-, and PhoPc than for AroA.

Maturation of DC after infection
Monocyte-derived DC present two distinct stages of maturation. Immature DC obtained after 7 days of culture in the presence of GM-CSF and IL-4 are very effective in capturing and processing antigens. Further maturation of these cells in the presence of stimuli such as lipopolysaccharide (LPS) or TNF-{alpha} reduces their capacity to capture the antigens, whereas the cells increase their immunostimulatory capacity [24 ]. To follow DC maturation after infection by ST, we measured the expression of CD86 (B7.2) on the cell surface and the ability of the DC to phagocytize the inert particle dextran [16 ]. We chose CD86 as a marker of maturation because of its significance as a co-stimulatory molecule, which improves the T cell stimulatory capacity of DC, and because of our observation that its expression was less experiment-dependent than that of CD83. The effect of ST on maturation was assessed 48 h after infection for all the attenuated strains but not for the wild type, which killed the majority of the cells at this time point (see above). In Figure 5 , the results for CD86 expression and phagocytosis of dextran are expressed as changes relative to the control, i.e., the cells that were not exposed to LPS or bacteria and thus remained in their immature state. CD86 was up-regulated by a factor 2.3 ± 0.3, 4.7 ± 1.1, and 4.9 ± 2.1 after infection with the strains PhoP-, PhoPc, and AroA, respectively (Fig. 5A) , whereas dextran phagocytosis was down-regulated by a factor 7.9 ± 0.9, 7.1 ± 0.4, and 10.5 ± 1.2, respectively (Fig. 5B) . Thus, all ST strains stimulated DC maturation markedly. In addition, heat-killed bacteria (PhoPc) and LPS induced maturation similarly. Statistical comparison showed no difference between the induction of CD86 by heat-killed bacteria, LPS, or the different live strains. These findings suggest that LPS released by the bacteria might have been responsible for most of the observed maturation effects.



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  		Figure 5. Maturation of DC is induced effectively at 48 h after exposure to S. typhimurium. MFI was determined by flow cytometry. Means of three to five independent experiments ± SE are shown. (A) Expression of cell-surface marker CD86 (=co-stimulatory molecule B7.2). (B) Phagocytosis of inert particles (FITC-labeled dextran). Overall testing for differences between groups is not significant.

Cytokine secretion by DC in response to infection
The amplitude and the nature of T cell stimulation are influenced strongly by the cytokine microenvironment in which antigen presentation takes place. Salmonella dublin was demonstrated to induce production of IL-1, IL-6, IL-10, IL-12 [p70], and TNF-{alpha} in murine DC [12 ]. In this study, we show the secretion of TNF-{alpha}, IL-12 [p70], and IL-10 by human DC after infection with ST. These cytokines play crucial roles in the induction of the inflammatory, humoral, and cellular immune responses, respectively [25 26 27 ]. Our results showed that TNF-{alpha} was induced strongly by all live ST strains (Fig. 6A ). LPS and heat-killed bacteria (PhoPc) also induced TNF-{alpha} secretion but at significantly lower levels than all the live strains (p<0.01). Induction of TNF-{alpha} by the AroA strain was significantly higher than for all the other strains (p<0.01), and the wild type and PhoP- induced more TNF-{alpha} than PhoPc (p<0.05 and p< 0.01, respectively). All live bacteria stimulated IL-12 secretion, whereas LPS or heat-killed bacteria did not induce significant amounts of this cytokine (Fig. 6B) . The wild type and the PhoP- strain induced significantly more IL-12 than PhoPc or AroA (p<0.05). IL-10 secretion was stimulated to different degrees by all live bacteria, LPS, and heat-killed PhoPc (Fig. 6C) . The wild-type and the attenuated strains PhoP- and AroA induced significantly more IL-10 than PhoPc (P<0.01). It is interesting that induction of IL-10 by heat-killed PhoPc was significantly higher than for live PhoPc (p<0.001, ratio 12.5±4.3), suggesting that the PhoPc mutant might repress IL-10 secretion actively in the DC.



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  		Figure 6. All S. typhimurium strains stimulate the secretion of TNF-{alpha} and IL-12 in DC during the 24 h following exposure to the bacteria. As compared with the other strains or with heat-killed bacteria (PhoPc), the secretion of IL-10 is repressed with PhoPc. Means of four independent experiments ± SE are shown. (A) TNF-{alpha}. (B) Interleukin-12 [p70]. (C) Interleukin-10. {circ}, P < 0.05 as compared with medium; *, P < 0.05 as compared with PhoPc.


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DISCUSSION
 
Given the key role of DC in the control of acquired immunity [1 ], there is considerable interest in targeting these cells with vaccination vectors. Among the bacterial vectors, the facultative, intracellular pathogen ST is one of the most promising [28 ], because ST is capable of introducing recombinant proteins or DNA coding for antigens into mammalian APC [9 ] and induces T cell and B cell responses [11 ] against heterologous antigens. However, the effects of infection by Salmonella in human DC have not been investigated previously. Here, we have exposed human DC to the wild type and to three attenuated, potential vaccine strains and a wild-type strain of ST to assess the influence of the genetic background of these bacteria on infection and intracellular survival, as well as viability, maturation, and cytokine production of the DC.

Our results show that the wild type and the three attenuated ST strains infect human DC efficiently in vitro. Concerning the wild type, our finding was not unexpected, because ST is a frequent cause of food-borne infections in humans, making DC natural targets in the infection as well as early players in acquired immunity against Salmonella [17 ], although we were surprised by the striking differences with respect to infection and intracellular survival among the attenuated strains. Comparing the three attenuated strains PhoPc, PhoP-, and AroA with respect to infection and persistence in the DC, we observed three distinct patterns. With the PhoPc mutant, high infection was obtained, but the bacteria were killed efficiently by the host cell within the first 24 h of infection. In contrast, the PhoP- mutant did not enter the DC as readily, but the intracellular bacteria persisted to a greater extent. Similar to PhoP-, AroA did not enter the DC readily, but similar to PhoPc, it was killed effectively by the host cell within 24 h.

We cannot explain fully the observation that the attenuated strain PhoPc infected human DC much more efficiently than any other of the ST strains. The deletion in phoP (phenotype PhoP-) and the mutation phoQ24 (phenotype PhoPc) affect the two components phoP and phoQ, which co-regulate bacterial invasion genes and an unknown number of virulence genes [29 ]. It is interesting that ST invasion genes localized in Salmonella pathogeneity Island I (SpI-1), required for entry into nonprofessional phagocytes, are repressed in the PhoPc strain [19 ], whereas in the PhoP- strain, the expression of these genes is stimulated [18 ]. We hypothesize that early events in the infection of DC may be favored by the PhoPc phenotype, such as initial cell adhesion or survival and/or replication in the phagosome. These events are undetectable in the gentamicin protection assay that we used in our studies to determine invasion efficiency. However, the TEM images of infected DC did not indicate enhanced bacterial multiplication in the early stages of infection by PhoPc, because, typically, we observed a single occupancy of the phagosomes by these bacteria (see Fig. 2 ). Therefore, the more likely explanation for the enhanced-infection efficacy of PhoPc would be an early interaction with the host that would improve phagocytosis or prevent early destruction of ST.

Intracellular survival of ST after infection of the human DC varied strongly. The strains PhoPc and AroA were eliminated efficiently by the DC during the first day of infection. This finding does not imply necessarily that they would be safe for human vaccine applications, because other cell types, such as macrophages, might serve as a reservoir for the replication of ST. Our results showed that the three attenuated ST strains had only a limited impact on cell viability or apoptosis in the human DC. After one day of infection, i.e., at a time point where intracellular PhoPc and AroA were already eliminated largely by the DC, only a minor fraction of the cells had become apoptotic or necrotic. Unexposed controls or cells exposed to heat-killed bacteria also showed some apoptosis and necrosis. The wild-type strain did not kill many DC within the first 24 h of infection but, in contrast with the attenuated strains, eliminated a large proportion of cells during the subsequent 24–48-h period. Our results in human DC contrast with studies on murine macrophages, where apoptosis of 80% was found as soon as 2 h after infection with ST [23 ].

Maturation of DC includes a coordinate series of changes that are necessary to trigger migration to the lymphatic organs and to induce the full, T cell-stimulatory capacity of the APC [1 , 24 , 30 ]. One of the main advantages of using bacterial vectors for the transfer of proteins or genes coding for antigens into the APC is that bacteria are potent inducers of DC maturation [31 ]. We observed that all live ST strains, as well as heat-killed PhoPc or highly purified LPS, induced maturation of DC, as evidenced by the strong up-regulation of the co-stimulatory molecule CD86 (B7.2) and down-regulation of the phagocytic capacity for inert particles [24 ]. Our results suggest that contact with LPS alone, in purified form or released from the bacterial surface, was likely sufficient to induce maturation, confirming the important role of LPS in DC maturation by Gram-negative bacteria [24 , 32 ].

In addition to direct, cell-to-cell contacts, T cell stimulation is regulated by cytokines secreted by the DC [3 ]. In the mouse model, IL-12 has been found to be a key cytokine for Th1-type immune responses [26 ]. Conversely, IL-10 appears to be an important Th2-type cytokine that up-regulates humoral [27 ] and down-regulates cell-mediated, immune responses [33 34 ]. In the mouse model, IL-10 secretion has been correlated negatively with resistance to ST infection [35 ]. Here, we demonstrate in vitro infection of human DC with ST-stimulated secretion of TNF-{alpha}, of the IL-12 active form, and of IL-10. The induction of IL-12 in murine DC by intracellular pathogens has been demonstrated previously [36 ]. However, we found important differences in the balance between IL-12 and IL-10 secretion after infection with the different ST strains. In particular, DC infected with the PhoPc strain produced only minor amounts of IL-10, and the three other ST strains as well as the heat-killed PhoPc induced the secretion of IL-10 strongly. The mechanism of the apparent suppression of IL-10 production by PhoPc remains to be shown. Differential repression of LPS-induced IL-10 secretion through the activation of protein kinase C has been demonstrated previously in human macrophages [37 ] and might be involved here as well.

In conclusion, we have shown that wild-type and attenuated ST infect immature, human, monocyte-derived DC efficiently and that infection with the attenuated strains had little consequences for DC viability up to a time point where most PhoPc and AroA mutants were killed inside the cells. Moreover, all ST strains induced the maturation of the DC effectively. These results appear to confirm the hypothesis that DC infected by bacteria may avoid apoptosis and undergo maturation instead to stimulate the specific immune defense against that pathogen [2 , 31 ]. Moreover, in our experiments, Salmonella stimulated the secretion of IL-12 and IL-10, which have been associated in vivo with the stimulation of cellular and humoral immune responses, respectively [26 , 27 ]. The relative levels of IL-10 and IL-12 induction were dramatically different and suggested that the PhoPc strain might be a more effective inducer of cell-mediated immunity in vivo than the other strains. The induction by PhoPc of a cytokine pattern that might be expected to favor type-1 T cell stimulation is in line with the in vivo finding that invasiveness, but not intracellular survival, of ST was required for Th1 dominance in the immune response [8 ]. The responses of DC to attenuated ST strains demonstrated here using primary human cells in vitro may help us to understand how these mutants might induce distinct immunological responses in vivo and could lead the way to bacterial vaccine vectors capable of stimulating specifically cellular or humoral immune defenses.


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ACKNOWLEDGEMENTS
 
This work was supported by the Swiss National Research Foundation, grants #32-40844.94, #3100-047073.96/1, #32-52638.97, and #4 037 055 164/1. D. D. was supported by the Lancardis Foundation. We thank Elisabeth Bühlmann and Ursula Gerber for excellent technical assistance in cell culture and transmission electron microscopy, respectively, and Dominique Wohlwendt for expert assistance in flow cytometry. We thank John J. Mekalanos and Jean-Marie Clement for kindly supplying bacterial strains.

Received July 27, 2000; revised November 17, 2000; accepted November 22, 2000.


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