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Originally published online as doi:10.1189/jlb.1204721 on August 4, 2005 Originally published online as doi:10.1189/jlb.1204721 on July 20, 2005

Published online before print July 20, 2005
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(Journal of Leukocyte Biology. 2005;78:909-920.)
© 2005 by Society for Leukocyte Biology

Activation, cytokine production, and intracellular survival of bacteria in Salmonella-infected human monocyte-derived macrophages and dendritic cells

Taija E. Pietilä*,1, Ville Veckman*, Päivi Kyllönen{dagger}, Kaarina Lähteenmäki{dagger}, Timo K. Korhonen{dagger} and Ilkka Julkunen*

* Department of Viral Diseases and Immunology, National Public Health Institute, Helsinki, Finland; and
{dagger} General Microbiology, Faculty of Biosciences, University of Helsinki, Finland

1Correspondence: Department of Microbiology, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland. E-mail: taija.pietila{at}ktl.fi


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Salmonella enterica serovar typhimurium (S. typhimurium) is an intracellular pathogen causing localized gastroenteritis in humans. Macrophages (M{phi}s) and dendritic cells (DCs) play an important role in innate immunity against Salmonella. In this report, we have compared the consequences of infection of human M{phi}s and DCs with wild-type S. typhimurium and an isogenic PgtE-defective strain. PgtE is an outer membrane protein hypothesized to have a role in intracellular survival of Salmonella. We observed that DCs undergo full maturation in response to Salmonella infection, as indicated by up-regulation of cell-surface marker proteins CD80, CD83, CD86, and human leukocyte antigen class II. CC chemokine ligand 5 (CCL5), CXC chemokine ligand 10, tumor necrosis factor {alpha}, interleukin (IL)-12, and IL-18 gene expression and protein production were readily induced by Salmonella-infected M{phi}s and DCs. CCL20 was preferentially produced by M{phi}s, whereas DCs secreted higher levels of CCL19 as compared with M{phi}s. DCs and M{phi}s infected with S. typhimurium also produced high levels of interferon-{gamma} (IFN-{gamma}). Cytokine neutralization and stimulation experiments suggest that the production was partly regulated by Salmonella-induced type I IFNs, IL-12, and IL-18. DC cytokine production induced by Salmonella was much higher as compared with the responses induced by Salmonella lipopolysaccharide or flagellin. M{phi}s and DCs were capable of internalizing and harboring Salmonella for several days. S. enterica PgtE provided no survival advantage for the bacteria in human M{phi}s or DCs. Our results demonstrate that although M{phi}s and DCs share similar functions, they may have different roles during Salmonella infection as a result of differential production of certain chemokines and cytokines.

Key Words: innate immunity • IFN-{gamma} • PgtE


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Salmonella enterica serovar typhimurium (S. typhimurium) is a facultative, intracellular pathogen, which is a common cause of localized gastroenteritis in humans [1 ]. S. typhimurium infection in genetically susceptible mice results in a severe, systemic infection resembling that of human typhoid fever [1 ]. Invasive Salmonella exploits mainly M cells located in Peyer’s patches to traverse the epithelial cell barrier, but also enterocytes have been implicated in this process [2 ]. Recent data also suggest a potential role of dendritic cells (DCs) in mediating bacterial transport across the epithelium [3 ]. Underneath the epithelial cell layer, bacteria can interact with the cells of the innate immune system such as macrophages (M{phi}s) and DCs, which are on alert for invading pathogens [2 , 4 ]. The intracellular life of Salmonella in M{phi}s has been studied in detail, and several virulence factors needed for its intracellular survival have been identified [1 ].

PgtE of S. enterica is an outer membrane protein homologous to Yersinia pestis Pla, which is an important factor in bacterial migration during plague infection [5 , 6 ]. PgtE is an aspartic protease, which in vitro, activates human plasma plasminogen [5 ], inactivates the antiprotease {alpha}2-antiplasmin [7 ], and cleaves antimicrobial peptides [8 ], but the role of these activities has not yet been established in salmonellosis. Up-regulation of pgtE inside murine M{phi}-like cells [9 ] and via activation of the PhoP/Q regulatory system, which responds to intracellular conditions [8 , 10 ], has led to the hypothesis that PgtE may be functional in Salmonella inside phagocytic cells or after bacterial release from dying M{phi}s. Supporting this, S. typhimurium expresses functional PgtE after growth inside murine M{phi}s [7 ].

M{phi}s and DCs are important cell types regulating host innate and adaptive immune responses. When these cells come in contact with microbes, they are activated, and they secrete various cytokines [11 , 12 ]. Both cell types also function as antigen-presenting cells (APC), thus providing a link between the innate and adaptive immunity. DCs have a high capacity to present captured antigens and activate naïve T cells [12 ]. In peripheral tissues, immature DCs capture foreign antigens such as bacteria, after which DCs undergo a coordinated maturation process. DC maturation is characterized by enhanced expression of cell-surface costimulatory and human leukocyte antigen (HLA) molecules, down-regulation of antigen capture capacity, and enhanced cytokine and chemokine production [12 ].

Cytokines play an important role in regulating host immune responses in Salmonella infection [13 ]. Particularly, tumor necrosis factor {alpha} (TNF-{alpha}) and cytokines driving T helper cell type 1 (Th1) immunity, such as interleukin (IL)-12, IL-18, and interferon- {gamma} (IFN-{gamma}), are important in the early stages of murine salmonellosis [13 14 15 ]. In monocytes and M{phi}s, Salmonella infection induces the production of a wide array of cytokines, and bacterial surface constituents such as lipopolysaccharide (LPS), flagella, and certain outer membrane proteins have been identified as components responsible for the induction [13 , 16 ]. Murine DCs can be infected with Salmonella, leading to DC maturation, enhanced cytokine production, and T cell proliferation [17 ]. Experimental data on the interaction of human DCs with Salmonella are still limited. It has been reported that stimulation of human DCs with S. typhimurium leads to the production of TNF-{alpha}, IL-12 p70, and IL-10 [18 ]. In addition, IL-18 secretion by human DCs is triggered by Salmonella virulence factor SipB [19 ]. At present, no information is available on Salmonella-induced chemokine gene expression in human DCs.

In the present study, we have analyzed the interaction of S. typhimurium with human monocyte-derived M{phi}s and DCs. We show that Salmonella induces DC maturation and that human M{phi}s and DCs have a somewhat differential cytokine/chemokine production pattern in Salmonella infection. Furthermore, Salmonella survives equally well in both phagocytic cell types, and outer membrane protein PgtE had no significant effect on the intracellular survival of bacteria. Our findings also show that Salmonella-infected M{phi}s and DCs produce high levels of IFN-{gamma}, which is likely to contribute to the activation of host immune responses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacteria and culture conditions
The strains of bacteria used in this study were the virulent wild-type strain of S. typhimurium ATCC 14028 and an isogenic mutant strain 14028-1, which lacks the pgtE gene as a result of in-frame deletion [20 ]. Bacteria were cultivated overnight in Luria broth at 37°C with shaking. This culture was diluted 1:100 in Luria broth and grown for 2.5 h, as above, to obtain logarithmic growth-phase bacteria. The bacteria were collected by centrifugation and washed with phosphate-buffered saline (PBS). The multiplicity of infection (MOI) was adjusted by reading the culture density at the optical density of 600 nm. The MOI was confirmed by plating serial dilutions onto Luria plates.

M{phi} and DC cultures
Monocytes were purified from freshly collected, leukocyte-rich buffy coats obtained from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki) as described previously [21 ]. In brief, human peripheral blood mononuclear cells were isolated by a density gradient centrifugation over a Ficoll-Paque gradient (Amersham Biotech, Uppsala, Sweden) followed by subsequent purification of monocytes in Percoll gradient (Amersham Biotech) centrifugation. Monocytes were collected from the top layer of the gradient, and remaining T and B cells were depleted using anti-CD3 and anti-CD19 magnetic beads (Dynal, Oslo, Norway). Purified monocytes were allowed to adhere onto six-well plates (2.5x106 cells/well; Falcon, Becton Dickinson, Franklin Lakes, NJ) for 1 h at 37°C in RPMI-1640 serum-free medium (SFM) supplemented with 0.6 µg/ml penicillin, 60 µg/ml streptomycin, 2 mM L-glutamine, and 20 mM HEPES. For bacterial survival assays, 24-well plates were used (1.25x106 cells/well). Plastic-adhered monocytes were washed with PBS. For M{phi} differentiation, the cells were grown in M{phi} SFM (Life Technologies, Grand Island, NY) supplemented with 10 ng/ml granulocyte M{phi}-colony stimulating factor (GM-CSF; Leucomax, Schering-Plough, Innishannon, Ireland), 0.6 µg/ml penicillin, and 60 µg/ml streptomycin. The media was replaced every 2 days, and after 6–7 days of differentiation, more than 90% of the cultured cells were mature M{phi}s, as determined by their typical morphology and cell-surface CD14 expression (data not shown). To differentiate monocytes into immature DCs, the cells were grown in RPMI-1640 medium with supplements as above, 10% fetal calf serum (FCS; Integro BV, Dieren, The Netherlands), 10 ng/ml recombinant human GM-CSF, and 20 ng/ml recombinant human IL-4 (R&D Systems, Abingdon, UK). Fresh media were added every 2 days, and the cells were used in experiments at 6–7 days after cultivation. Cultured cells were CD1a+, CD14–, CD80+, CD83–, and CD86+, and they showed a typical DC morphology (data not shown).

Cytokines, antibodies, and pathogen-associated molecular patterns (PAMPs)
Human IFN-{alpha} and IFN-{gamma} were obtained from the Finnish Red Cross Blood Transfusion Service and used at 100 IU/ml. Recombinant human TNF-{alpha} and IL-12 were purchased from R&D Systems and were used at 5 ng/ml and 10 ng/ml, respectively. Recombinant human IL-18 was from Biosite (Täby, Sweden) and used at 30 ng/ml. Neutralizing antibodies against human IFN-{alpha}/ß and anti-IL-18 antibodies have been described previously [22 , 23 ]. Anti-IL-12 antibody was from R&D Systems. In cytokine neutralization assays, antibodies were added 1 and 10 h after addition of the bacteria.

S. typhimurium smooth LPS preparation was from Sigma Chemical Co. (St. Louis, MO). Purified FliC, flagellin protein of S. typhimurium, was purchased from Alexis Biochemicals (Lausen, Switzerland).

Stimulation experiments
To minimize interindividual variation, all experiments were performed with cells obtained from three to four blood donors. Stimulation experiments were conducted in RPMI-1640 medium (supplements as above) with 10% FCS. In Northern blot analysis, cells were stimulated with live bacteria at a 5:1 bacteria:host cell ratio. After stimulation, cells and cell culture supernatants from different donors were collected and pooled or kept separate when indicated. Cells were used for the isolation of total cellular RNA or for flow cytometric analysis (fluorescein-activated cell sorter). Supernatants were stored at –20°C and used for cytokine determinations.

RNA isolation and analysis
For isolation of total cellular RNA, stimulated cells were collected, washed once with cold PBS, and lysed in guanidium isothiocyanate [24 ], followed by centrifugation through a CsCl cushion [25 ]. RNA was quantitated photometrically, and samples containing equal amounts of RNA (10 µg) were size-fractionated on 1% formaldehyde-agarose gels and transferred onto Hybond-N membranes (Amersham Biotech). To control equal RNA loading, ethidium bromide staining was used. The following cDNA probes for hybridizations were used: human CC chemokine ligand 5 (CCL5), kindly provided by Dr. Alberto Mantovani (Istituto di Ricerche Farmacologiche, Milan, Italy), CXC chemokine ligand 10 (CXCL10) [26 ], CCL19, and CCL20, provided by Dr. Zlotnik [27 ], human TNF-{alpha} (American Type Culture Collection, Manassas, VA), IFN-ß [28 ], IL-12 p35 and IL-12 p40 [23 , 29 ], IL-18 [30 ], and IFN-{gamma} [31 ]. The probes were labeled with [{alpha}-32P]deoxy-adenosine 5'-triphosphate (3000 Ci/mmol, Amersham Biotech) using a random-primed DNA labeling kit. Hybridizations were performed in Ultrahyb buffer (Ambion, Austin, TX) at 42°C. The membranes were washed three times with 1x saline sodium citrate/0.01% sodium dodecyl sulfate at 42°C for 30 min and once at 65°C for 30 min. The membranes were exposed to Kodak X-Omat AR films (Eastman Kodak, Rochester, NY) at –70°C with intensifying screens.

Cytokine-specific enzyme-linked immmunosorbent assays (ELISAs)
Cytokine and chemokine levels from cell culture supernatants were analyzed by a sandwich ELISA method. TNF-{alpha}, CCL5/regulated on activation, normal T expressed and secreted, and CXCL10/IFN-inducible protein 10 levels were determined with antibody pairs and standards obtained from BD PharMingen (San Diego, CA). IL-12 p70 and IFN-{gamma} levels were determined with the Elipair kit (Biosite) and CCL19/M{phi}-inflammatory protein (MIP)-3ß and CCL20/MIP-3{alpha}, with a Duoset kit (R&D Systems). IL-18 ELISA was purchased from Biosite (Medical and Biological Laboratories, Nagoya, Japan).

Detection of cell-surface markers by flow cytometry
DCs were stimulated with live bacteria for 24 h as described above. The cells were collected and washed once with cold PBS and fixed with 3% paraformaldehyde for 15 min. After fixation, the cells were washed twice with PBS and suspended in PBS + 2% FCS. The expression of cell-surface marker proteins was analyzed with FACScan flow cytometer by using Cell Quest software (Becton Dickinson). Fluorescein isothiocyanate-conjugated monoclonal antibodies (mAb) against CD80, CD83, CD86, HLA class II, and isotype-matched control antibodies were purchased from Caltag Laboratories (Burlingame, CA). Paraformaldehyde-fixed DCs were stained with appropriate dilutions of mAb for 30 min at +4°C. The cells were washed twice with PBS + 2% FCS, suspended in the same solution, and analyzed.

Intracellular bacterial survival assay
Before infection, human monocyte-derived M{phi}s and DCs were cultured for 2 h in complete RPMI medium (10% FCS) without antibiotics. For pretreatment of cells, 100 IU/ml IFN-{gamma} and/or 5 ng/ml TNF-{alpha} were added to the cells 16 h before the infection. The cells were infected with nonopsonized, logarithmic growth-phase S. typhimurium at a MOI of ~1. The cells were incubated with bacteria for 1 h followed by substitution of the medium with RPMI containing 100 µg/ml gentamicin to kill extracellular bacteria. After another 1 h of incubation at 37°C, the medium was replaced with RPMI medium containing 20 µg/ml gentamicin. The cells were maintained in this medium throughout the assay. The efficiency of bacterial entry into host cells was determined at 2 h after infection. Cells were washed with PBS, and bacteria were harvested by adding 0.5 ml 0.2% Triton-X-100 in distilled water to each well in 24-well plates. The lysates were pipeted vigorously, after which serial tenfold dilutions were performed in PBS. Aliquots were plated onto Luria agar to assess bacterial colony-forming units (CFU).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of bacterial dose on DC maturation
DCs undergo a maturation process in response to bacterial stimulation. This is characterized by up-regulation of cell-surface marker proteins and production of cytokines [12 ]. To determine the optimal bacterial dose for stimulation experiments, we infected DCs with different amounts of wild-type S. typhimurium 14028 for 24 h and analyzed the expression of CD80, CD83, CD86, and HLA class II by flow cytometry. The cells from different blood donors were analyzed separately. Stimulation of cells with live bacteria induced the expression of all cell-surface molecules studied (Fig. 1A ). Maximal up-regulation of HLA class II was observed at a bacteria:DC ratio of 3:1 and at ratio 10:1 for CD80, CD83, and CD86. Even the lowest dose, 0.3 bacteria/cell, was sufficient to induce up-regulation of the marker proteins (Fig. 1A) . Stimulation of DCs with the PgtE-defective Salmonella 14028-1 maturated DCs equally well as the wild-type (data not shown). A clear, bacterial, dose-dependent increase in CD86 expression was seen, whereas for the other marker proteins, already, the lower bacterial dose of MOI 1 caused a submaximal induction (Fig. 1B) . A Salmonella:DC ratio of 5:1 was selected for further experiments.



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  		Figure 1. Effect of Salmonella dose on DC maturation. Human monocyte-derived DCs were stimulated with indicated bacteria:DC ratios for 24 h. Cells obtained from different blood donors were analyzed separately for cell-surface expression of CD80, CD83, CD86, and HLA class II. (A) Flow cytometric analysis of one representative experiment performed with the cells of one donor is shown. Dotted lines indicate respective isotype controls. (B) The expression of cell-surface marker proteins in Salmonella-infected DCs. MOI values 1 and 10 are shown. Results are mean fluorescent intensities (MFIs) from one representative experiment (out of two) performed with cells from four different donors. The bars and error bars indicate the means ± SD. The differences between groups were assessed with the Student’s t-test (paired, two-tailed), and P values are shown in the figure.

 
Salmonella-induced cytokine mRNA expression in M{phi} and DC
M{phi}s are considered to be important in Salmonella infection, as they are known to produce cytokines that induce antimicrobial activity against intracellular pathogens such as Salmonella [13 , 16 ]. However, Salmonella-induced cytokine production in human M{phi}s and DCs has been poorly studied. To compare cytokine gene expression profiles in M{phi}s and DCs, the cells were infected with Salmonella 14028 (Fig. 2 ) and 14028-1 (data not shown). To obtain comparable data, Northern blot hybridizations and autoradiograms were performed at the same time for M{phi} and DC membranes. Stimulation of M{phi}s with S. typhimurium resulted in enhanced mRNA expression of CCL5, CCL20, CXCL10, TNF-{alpha}, IFN-ß, and IL-18 genes, already at 3 h postinfection (Fig. 2A) . IL-12 p35 and p40 mRNA expression was enhanced at 9 h after infection. It is interesting that enhanced IFN-{gamma} and CCL19 mRNA expression was seen in M{phi}s at 24 h after infection.



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  		Figure 2. Salmonella-induced mRNA expression of chemokine and cytokine genes. M{phi}s (A) or DCs (B) were stimulated with wild-type Salmonella 14028 strain at a MOI value of 5 for indicated times. mRNA expression of CCL5, CCL19, CCL20, CXCL10, TNF-{alpha}, IFN-ß, IL-12 p35, IL-12 p40, IL-18, and IFN-{gamma} was analyzed by Northern blotting. Northern blot analyses for M{phi} and DC mRNAs were carried out at the same time to enable direct comparison of signal intensities. Ethidium bromide staining was used to control equal sample loading. Results from one representative experiment out of two are shown.

 
As compared with M{phi}s, cytokine mRNA expression patterns were found to be somewhat different in DCs (Fig. 2B) . Similar to M{phi}s, stimulation of DCs with Salmonella resulted in a rapid up-regulation of CCL5, CCL20, CXCL10, TNF-{alpha}, and IL-18 mRNA expression, starting at 3 h after infection. However, in contrast to M{phi}s, IL-12 p40 was detectable already at 3 h postinfection, and only weak IFN-ß mRNA expression was seen in DCs at 3 h postinfection. IL-12 p35 mRNA in DCs was detectable at 9 h post-infection, unlike in M{phi}s, where the induction kinetics was faster. In DCs, enhanced expression of CCL19 mRNA was seen at 9 h postinfection, whereas IFN-{gamma} mRNA induction was seen at later time-points of 24 and 48 h. The ability of 14028-1 {Delta}pgtE Salmonella to induce cytokine and chemokine gene expression did not differ from that of the wild-type S. typhimurium (results not shown).

Salmonella-induced chemokine and cytokine production in M{phi} and DC
To further characterize the kinetics and quantity of Salmonella-induced chemokine and cytokine production, M{phi}s and DCs were stimulated with wild-type or {Delta}pgtE mutant strain Salmonella at a 5:1 bacteria:host cell ratio. Cell culture supernatants were collected at different time-points, and chemokine and cytokine levels were determined by ELISA. Both cell types produced large amounts of CCL5 and CXCL10 in response to Salmonella infection (Fig. 3A ). CCL19 was more prominently produced by DCs, whereas CCL20 production was higher in M{phi}s. Uninfected cells produced low levels of chemokines.



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  		Figure 3. Salmonella-induced chemokine (A) and cytokine (B) production. M{phi}s or DCs were stimulated with live wild-type (solid bars) and {Delta}pgtE (shaded bars) Salmonella at a MOI of 5. Cell culture supernatants were collected at indicated times, and cytokine and chemokine levels were measured by ELISA. Results are the means of two independent experiments performed with cells from four different donors. Error bars represent SDs of the means.

 
Next, we analyzed the production of TNF-{alpha}, IL-12, IL-18, and IFN-{gamma} in Salmonella-infected M{phi}s and DCs. M{phi}s were found to produce TNF-{alpha} and low levels of IL-18 early, at 3 h after infection, whereas IL-12 and IFN-{gamma} production was clearly detectable at later times of infection (Fig. 3B) . The kinetics of cytokine production in Salmonella-infected DCs was delayed compared with M{phi}s, and TNF-{alpha} and IL-18 secreted by DCs remained at lower levels as compared with M{phi}s (Fig. 3B) .

Regulation of IFN-{gamma} production in M{phi} and DC
We observed that Salmonella infection in human monocyte-derived M{phi}s and DCs leads to significant production of IFN-{gamma}. As IFN-{gamma} mRNA expression and protein production were detectable only at late times of infection, we hypothesized that the production is regulated by other cytokines. Natural killer (NK) and T cells have long been considered to be the major cell types producing IFN-{gamma}, and high cytokine-induced IFN-{gamma} production from these cells is a result of synergistic stimulation by IL-18 with IL-12 or type I IFNs [32 33 34 35 ]. Therefore, to determine the role of type I IFNs, IL-12, and IL-18 on IFN-{gamma} gene expression in M{phi}s and DCs, neutralizing anti-IFN-{alpha}/ß, anti-IL-12, and anti-IL-18 antibodies were used. As shown in Figure 4A , Salmonella-induced IFN-{gamma} production in M{phi}s (range 320–1900 pg/ml) was reduced to 13% (P<0.05) with the addition of anti-IFN-{alpha}/ß together with anti-IL-12 and anti-IL-18 antibodies (range 15–310 pg/ml). In DCs, Salmonella-induced IFN-{gamma} production (range 280–11,500 pg/ml) was reduced to 54% (P<0.05) with treatment of anti-IL-18 antibodies alone (range 100–8900 pg/ml). Combination of anti-IL-12 and anti-IL-18 antibodies was more efficient and reduced IFN-{gamma} production to 31% (range 220–6000 pg/ml) of that of untreated cells. Neutralizing anti-IFN-{alpha}/ß antibodies did not further reduce Salmonella-induced IFN-{gamma} production in DCs.



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  		Figure 4. Regulation of Salmonella-induced IFN-{gamma} production in M{phi}s and DCs. (A) Cells were infected with live wild-type Salmonella at a MOI of 5, and neutralizing antibodies against IFN-{alpha}/ß, IL-12, and IL-18 were added at 1 and 10 h postinfection. Cell culture supernatants were collected at 24 h postinfection, and IFN-{gamma} levels were determined by ELISA. Bar graph shows the percentual reduction of IFN-{gamma} production when cells infected with Salmonella in the absence of antibodies are marked as 100%. Data are from two independent experiments performed in triplicates. The differences between groups were assessed with the Student’s t-test (paired, two-tailed), *, P < 0.05. (B) Cells were stimulated with purified natural human IFN-{alpha} (100 IU/ml), recombinant human IL-12 (10 ng/ml), and IL-18 (30 ng/ml) for 24 or 48 h, after which cell culture supernatants were collected and analyzed for IFN-{gamma} production by ELISA.

 
Next, we studied whether recombinant cytokines alone could also induce IFN-{gamma} production in M{phi}s and DCs. Cells were stimulated with IFN-{alpha}, IL-12, or IL-18 or their combinations, and IFN-{gamma} production was determined by ELISA. As shown in Figure 4B , stimulation of M{phi}s with IFN-{alpha}, IL-12, and IL-18 led to a significant production of IFN-{gamma}. In DCs, addition of IL-12 and IL-18 was sufficient for maximal IFN-{gamma} production. However, cytokine-induced IFN-{gamma} production was tenfold lower as compared with that induced by Salmonella.

Host cell responses to Salmonella PAMPs versus whole bacteria
APC, including DCs, express receptors that can recognize molecular structures of the microbes PAMPs. The recognizing receptors are collectively known as pattern recognition receptors (PRRs), which include the family of Toll-like receptors (TLRs). Salmonellae possess a range of protein and nonprotein structures that function as PAMPs. LPS is the main component of the outer membrane of the cell wall of salmonellae, and the conserved PRR involved in LPS signaling is TLR4 [36 ]. Flagellin, the structural constituent of motility apparatus of Gram-negative bacteria, signals through TLR5 [37 ]. As TLR signaling leads to the activation of innate immune responses in host cells, we compared the ability of TLR ligands LPS and flagellin to induce maturation and cytokine production in human DCs versus whole live bacteria. Human monocyte-derived DCs were stimulated with wild-type Salmonella 14028 strain, purified LPS, or flagellin (both derived from S. typhimurium). After 24 h, we observed an efficient maturation of treated DCs, as demonstrated by an increase in the surface expression of CD86 (Fig. 5A ). However, stimulation with whole bacteria led to higher expression of CD86 as compared with the induction caused by TLR ligands. As shown in Figure 5B , the ability of TLR ligands to stimulate the expression of proinflammatory TNF-{alpha} and CXCL10 was clearly lower as compared with whole bacteria. Furthermore, LPS and flagellin stimulation produced only negligible amounts of IL-12 p70 or IFN-{gamma}. These results suggest that whole bacteria are more effective at inducing DC maturation and cytokine production as compared with TLR ligands.



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  		Figure 5. Activation of DCs in response to whole bacteria versus TLR ligands LPS and flagellin. Human DCs were stimulated with live wild-type Salmonella at indicated MOIs or with different concentrations of purified LPS or flagellin for 24 h. (A) Flow cytometric analysis of CD86 expression on DCs. Unstimulated control cells (ctrl) are marked as 1, and CD86 expression of stimulated cells is shown relative to control cells. (B) Cytokine and chemokine levels from cell culture supernatants were measured by ELISA. The results are the means (±SD) of stimulated DCs obtained from three individual blood donors. One representative experiment out of two is shown.

 
Survival of wild-type and PgtE-defective Salmonella in M{phi}s and DCs
Murine and human DCs have been shown to harbor S. typhimurium [17 , 18 ], and intracellular survival in phagocytic cells represents an important pathogenetic mechanism for Salmonella. We were thus interested to compare the ability of S. typhimurium to survive in human M{phi}s and DCs. In addition, we studied the effect of PgtE on bacterial survival, as recent evidence suggests that it contributes to bacterial survival in murine M{phi}s [7 ]. Human M{phi}s and DCs were infected with Salmonella 14028 and 14028-1 at a MOI of 1, and intracellular bacterial counts were determined at various times postinfection by a viable count assay. Approximately 2% of the inoculated wild-type Salmonella 14028 was internalized by M{phi}s, whereas the pgtE-negative 14028-1 was internalized at a higher efficiency of 10% (Fig. 6A ). In M{phi}s, 14028 and 14028-1 Salmonella showed an increased number of viable bacteria during the first 24 h of infection (Fig. 6A) . Thereafter, viable bacterial counts declined 1.5–2 logs/day, being ~102 CFU per 5 x 105 M{phi}s at 72 h postinfection. Bacterial uptake into DCs occurred at a similar range as in M{phi}s, and 3.1% of the wild-type and 7.2% of the pgtE-negative bacteria were internalized. As in M{phi}s, DCs were found to take up Salmonella relatively efficiently and harbor them for a long period of time (Fig. 6B) . In contrast to M{phi}s, viable counts started to decrease already during the first 24 h, but yet, 101–102 CFU per 5 x 105 cells were found 3 days after the infection. M{phi}s and DCs initially phagocytosed the pgtE-defective Salmonella strain more efficiently than the wild-type strain. However, the lack of PgtE did not seem to decrease survival of the bacteria in M{phi}s or DCs.



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  		Figure 6. Survival of S. typhimurium in human M{phi}s (A) and DCs (B). Cells were infected with live Salmonella at a MOI value of 1. Infection was allowed to last for 1 h, after which noninternalized bacteria were killed by gentamicin treatment (100 µg/ml) for 1 h. The cells were then maintained in media containing a lower concentration of gentamicin (20 µg/ml) throughout the experiment. Top panels: Bacterial uptake into the cells is shown as the percentage of inoculated bacteria surviving gentamicin treatment. Solid bars refer to wild-type and open bars, to {Delta}pgtE Salmonella. All bottom panels: Bacterial CFU per 5 x 105 cells (y-axis) at different times after infection (x-axis) were determined. Where indicated, the cells were pretreated with IFN-{gamma} (100 IU/ml) and/or TNF-{alpha} (5 ng/ml) for 16 h prior to infection with wild-type ({diamondsuit}) or pgtE mutant ({square}) S. typhimurium. Results are the means ± SD of one representative experiment performed with cells of three different donors. Two independent experiments were performed with similar results.

 
IFN-{gamma} and TNF-{alpha} are known to activate M{phi}s and inhibit the growth of intracellular bacteria [38 ]. It has also been reported that antimicrobial effects are activated in murine DCs by IFN-{gamma} [39 ]. Thus, we carried out IFN-{gamma} and/or TNF-{alpha} pretreatment experiments. In M{phi}s, cytokine pretreatment decreased the number of internalized bacteria, especially in the cells pretreated with IFN-{gamma} and TNF-{alpha} (Fig. 6A , top panel). In our model cell system, cytokine pretreatment did not significantly enhance bacterial killing. Comparable numbers of viable bacteria were found in cytokine-pretreated and untreated M{phi}s at 72 h after infection (Fig. 6A) . In DCs, cytokine pretreatment had a more pronounced effect on the internalization of bacteria. Only 0.04% of inoculated wild-type bacteria was phagocytosed when cells had been pretreated with IFN-{gamma} and TNF-{alpha} (Fig. 6B , top panel). This may result from cytokine-stimulated maturation of DCs, leading to a dramatic decrease in their endocytic capacity. As in M{phi}s, cytokine pretreatment of DCs did not further enhance bacterial killing, taking into account the number of bacteria that was initially internalized (Fig. 6B) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have investigated host cell responses to pathogenic S. typhimurium in human M{phi}s and DCs. Besides the commonly studied virulent 14028 strain, we also used the 14028-1 isogenic mutant that lacks the pgtE surface protease gene. Stimulation of human DCs with live Salmonella led to maturation of the cells, as characterized by enhanced expression of HLA class II and cell-surface costimulatory molecules CD80, CD83, and CD86. Stimulation of DCs with PgtE-defective bacteria induced their maturation in a similar manner (data not shown).

M{phi}s and DCs are able to produce various cytokines and chemokines when they come in contact with pathogenic bacteria. We observed a somewhat differential chemokine and cytokine expression pattern in human M{phi}s and DCs. Wild-type and PgtE-defective S. typhimurium induced the production of inflammatory chemokines CCL5 and CXCL10 in M{phi}s and DCs. However, M{phi}s appeared to be more efficient producers of these chemokines than DCs. CCL5 recruits monocytes and effector T cells to the site of inflammation, whereas the CXCL10 receptor, CXCR3, is found in NK and Th1 cells [40 ]. CCL19 and CCL20 are important chemokines regulating DC migration. CCL20 attracts immature DCs to the inflammatory site [41 ]. In contrast, mature DCs produce CCL19, which binds to CC chemokine receptor 7 found on mature DCs and naïve T cells [41 , 42 ]. It has been reported that mature murine DCs harboring Salmonella are chemoattracted toward CCL19 and CCL21, secreted in secondary lymphoid organs [43 ]. Thus, it is likely that CCL19 production by human DCs also has a role in the dissemination of Salmonella. Our findings indicate that human M{phi}s and DCs are able to produce CCL19 and CCL20 in Salmonella infection. Based on Northern blot and ELISA data, CCL20 was preferentially produced by M{phi}s and CCL19, by DCs.

Cytokines are essential in the host innate immune response against Salmonella. Particularly, the production of proinflammatory cytokines is important. Here, we demonstrate that in vitro infection of human M{phi}s and DCs with Salmonella leads to the production of TNF-{alpha}, IL-12, IL-18, and IFN-{gamma}. Our data are consistent with the data of Dreher and co-workers [18 , 19 ], who reported the production of TNF-{alpha}, IL-12, and IL-18 in Salmonella-infected human monocyte-derived DCs. Unlike the above authors [18 ], we did not observe any IL-10 production in our DC model system. IL-12 is a key Th1 cytokine in the mouse model, although its production in Salmonella-infected bone marrow-derived or splenic DCs is limited [44 ]. In our experimental system, we detected IL-12 mRNA expression and secretion of biologically active IL-12 p70 (Figs. 2 and 3) . IL-18 also plays an important role in the innate and acquired immunity against intracellular pathogens. Biologically active IL-18 is produced post-translationally by caspase-1-mediated cleavage. Consistent with previous studies [19 , 45 ], we observed Salmonella-induced IL-18 mRNA and protein production in M{phi}s and DCs (Fig. 3) .

Recent studies about innate immune recognition have focused on the interaction of host cell PRRs with bacterial PAMPs. As DCs express a wide repertoire of TLRs, we studied whether LPS and flagellin, TLR4 and TLR5 ligands, respectively, can activate innate immune responses in our DC model system. We found out that although LPS and flagellin readily induced DC maturation, the cytokine production was limited. Flagellin-stimulated DCs failed to produce detectable levels of TNF-{alpha}, CXCL10, IL-12, and IFN-{gamma}. LPS induced low levels of IL-12 and IFN-{gamma}, and TNF-{alpha} and CXCL10 levels were also somewhat lower than those induced by live Salmonella. The results are consistent with a recent report by Means and co-workers [46 ], who observed maturation but the lack of IL-12 and CXCL10 production in flagellin-stimulated, human monocyte-derived DCs. Our findings suggest that the biological consequences in DCs encountering live bacteria versus bacteria-derived components such as LPS or flagellin clearly differ from each other.

IFN-{gamma} plays a central role in host defense against Salmonella. Perhaps the most important function of IFN-{gamma} in Salmonella infection is its ability to activate M{phi}s to kill intracellular bacteria [13 ]. IFN-{gamma} expression was long considered to be restricted to T and NK cells. However, recently, it has become evident that IFN-{gamma} production can also occur in other cell types, including murine and human monocytes/M{phi}s [47 ] and murine DCs [48 ]. Here, we show that human M{phi}s and DCs are able to produce IFN-{gamma} in response to S. typhimurium infection.

Neutralizing anti-IFN-{alpha}/ß, anti-IL-12, and anti-IL-18 antibodies were used to investigate the role of secreted cytokines in Salmonella-induced IFN-{gamma} production. It was of interest that although type I IFNs and IL-18 appeared to be preferentially involved in IFN-{gamma} production in M{phi}s, IL-12 together with IL-18 seemed to be more important in DCs. Cytokine neutralization experiments correlated well with observed cytokine mRNA expression patterns and protein production in M{phi}s and DCs (Figs. 2 and 3) . It is likely that other cytokine-independent Salmonella-induced mechanisms are also involved in IFN-{gamma} production by M{phi}s and DCs, as Salmonella-induced IFN-{gamma} production was at least tenfold higher as compared with that obtained by different cytokine combinations. It is also possible that other Th1 cytokines (such as IL-23 and IL-27), apart from those we have analyzed, are involved in the regulation of IFN-{gamma} production in human M{phi}s and DCs.

The important role of M{phi}s in Salmonella infection is well established, whereas DCs as targets of Salmonella have been recognized only recently [3 , 18 ]. Here, we show that S. typhimurium survives equally well in M{phi}s and DCs. At early times of infection (at 24 h), DCs seemed to be more efficient than M{phi}s at eliminating bacteria. However, after 72 h, equal amounts of bacteria were harvested from M{phi}s and DCs. In our experimental model, pretreatment of cells with TNF-{alpha}, IFN-{gamma}, or their combination did not further enhance the intracellular killing of Salmonella. In DCs, however, cytokine pretreatment resulted in a dramatic decrease in bacterial internalization, which is related to cytokine-induced maturation of DCs.

Many studies report that internalized Salmonella survive inside DCs and M{phi}s [4 , 17 ], but Salmonella has also been reported to rapidly kill the host cells [19 , 49 , 50 ]. To minimize bacterial cytotoxicity, we used low bacterial doses (MOI 1), which are significantly smaller than the amounts causing severe host cell cytotoxicity [19 , 49 ]. The reason for the higher uptake of the pgtE-negative S. typhimurium 14028-1 into DCs and M{phi}s is probably not directly a result of PgtE-mediated functions, as PgtE is poorly expressed in bacteria grown in rich culture medium but is induced during residence of Salmonella inside murine M{phi}s [7 , 9 ]. In Escherichia coli, the expression of certain outer membrane proteins and type 1 fimbriae is conversely regulated [51 , 52 ]. We observed that the pgtE-negative S. typhimurium 14028-1 expressed more type 1 fimbriae compared with the wild-type 14028 (data not shown), which may contribute to the increased uptake of PgtE-defective Salmonella, as type 1 fimbriae are known to promote contact between bacteria and phagocytic cells [53 ].

In summary, in the present work, we have compared M{phi} and DC cytokine responses during S. typhimurium infection. Both cell types responded to Salmonella infection by activating the expression of multiple cytokine and chemokine genes. M{phi}s appeared to produce higher levels of proinflammatory chemokines CCL5, CCL20, and CXCL10 and cytokines TNF-{alpha} and IL-18 as compared with DCs. Somewhat higher production levels of the major Th1 cytokine IL-12 were seen in DCs. CCL19, an important chemoattractant of mature DCs and naïve T cells, was also produced in significant amounts by DCs. Although no drastic differences in the ability of M{phi}s and DCs to internalize and kill Salmonella were found, it is likely that M{phi}s mediate proinflammatory responses during the infection, whereas DCs contribute more to stimulating Th1 adaptive immune responses to Salmonella.


   ACKNOWLEDGEMENTS
 
This work was supported by the Medical Research Council and Bioscience and Environmental Research Council of the Academy of Finland (Project Numbers 80666, 201967, 202505, and 205644 and the Microbes and Man Program), the University of Helsinki, and the Sigrid Juselius Foundation. We thank Mari Aaltonen, Hanna Valtonen, and Teija Westerlund for expert technical assistance.

Received December 7, 2004; revised May 4, 2005; accepted June 16, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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