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* Departments of Microbiology, Immunology and Pathology and
Department of Clinical Sciences, Colorado State University, Fort Collins, CO 80523
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and IL-6, from DC. However, F. tularensis infection did elicit production of the potent immunosuppressive cytokine, TGF-
. Furthermore, F. tularensis actively suppressed the ability of DC to secrete cytokines in response to specific TLR agonists. Finally, we also found that infection of DC and macrophages in the lungs appears to actually increase the severity of pulmonary infection with F. tularensis. For example, depletion of airway DC and alveolar macrophages before infection resulted in significantly prolonged survival times. Together, these data suggest F. tularensis is able to selectively uncouple Ag-presenting functions from proinflammatory cytokine secretion by critical APCs in the lungs, which may serve to create a relatively immunosuppressive environment favorable to replication and dissemination of the organism. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Previous work has demonstrated that F. tularensis readily infects macrophages both in vivo and in vitro and that these cells appear to be a major site of replication for the bacterium in the host (1, 2). Macrophages are also able to aid in the control of F. tularensis infections by several mechanisms, including production of reactive nitrogen and oxygen species, such as NO and superoxide, after exposure to at least two proinflammatory cytokines, IFN-
and TNF- (2, 3, 4, 5). In turn, F. tularensis has developed several mechanisms by which to escape early protective responses in the host. For example, F. tularensis avoids degradation by macrophages via escape of the phagolysosome within the first few hours of infection (6). Furthermore, it has recently been shown that F. tularensis produces at least one protein that inhibits macrophage responsiveness to a TLR agonist for production of proinflammatory cytokines. The 23-kDa F. tularensis inhibits production of TNF- after exposure of infected cells with LPS (7).
Although macrophages clearly play an important role in the course of infection with F. tularensis, the interaction of F. tularensis with dendritic cells (DC)3 has not been described previously. DC act as sentinels for the immune system (8). For example, DC are often the first cells to encounter and engulf pathogens. This, in turn, triggers a cascade of events designed to limit early pathogen replication and in some cases to initiate the process of Ag presentation for development of T cell-mediated immunity to the pathogen (8). These initial events are marked by rapid secretion of proinflammatory cytokines, followed shortly by increased expression of cell surface receptors associated with Ag presentation and DC migration. Thus, DC serve as an important constituent of and link between innate and adaptive immune responses.
Many pathogens successfully infect and replicate within DC; recently, it has been suggested that several pathogens target DC early in infection as a means of disabling and evading host immune responses (9, 10, 11). Given the intracellular nature of F. tularensis and its immunomodulatory activity in macrophages, we sought first to determine whether F. tularensis could infect DC. In addition, we determined what functional consequences infection might have on DC and how these responses might differ compared with those of macrophages. In this report we demonstrate productive infection of DC in vivo and in vitro by F. tularensis. Interestingly, we also find that airway DC are the primary cell infected in the lungs immediately after pulmonary challenge.
However, despite the fact that F. tularensis infected both DC and macrophages in the lungs, we did not observe complete activation of the cells, nor did we observe a lack of response to infection. Rather, we observed a novel partial activation of these two cell types to infection with F. tularensis, both in vivo and in vitro. This state of partial or aberrant activation was characterized by up-regulation of MHC class II (MHC II) and CD86 expression, without concomitant secretion of proinflammatory cytokines. The lack of cytokine release was associated with an active suppression of cytokine production, as indicated by the inability of F. tularensis-infected DC to respond to two potent TLR agonists, LPS and zymosan. Furthermore, F. tularensis infection elicited TGF- secretion by infected DC and macrophages after either in vivo or in vitro infection. Finally, depletion of airway DC and alveolar macrophages before infection significantly increased survival times in mice given a lethal pulmonary infection with F. tularensis. Together, these data demonstrate a dynamic interaction between DC and F. tularensis in which the bacterium manipulates the host immune response by simultaneously suppressing cytokine production while still allowing up-regulation of molecules associated with Ag presentation. These two processes are believed to play important roles in the virulence of F. tularensis and the ability of the organism to establish lethal pulmonary infections.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Specific pathogen-free, 6- to 8-wk-old female C57BL/6 mice were purchased from The Jackson Laboratory. All mice were housed in sterile microisolater cages in the laboratory animal resources facility at Colorado State University and were provided sterile water and food ad libitum. All research involving animals was conducted in accordance with animal care and use guidelines, and animal protocols were approved by the animal care and use committee at Colorado State University.
Bacteria
F. tularensis live vaccine strain (LVS) and Yersinia pestis strain A1122 were provided by Drs. M. Chu and Jeannine Peterson (Centers for Disease Control, Fort Collins, CO). Y. pestis A1122 is a Pgm– that has also been cured of the low calcium response plasmid by selection at 37°C on magnesium oxalate agar (12) A challenge dose of 5 x 104 LVS organisms delivered either intratracheally (i.t.) or intranasally (i.n.) was found to induce 100% mortality in BALB/c, C57BL/6, 129, and ICR mice, with a mean time to death in all strains of 5 days (data not shown). LVS was cultured in modified Mueller-Hinton broth at 37°C with constant shaking overnight, aliquoted into 1-ml samples, frozen at –80°C, and thawed just before use as previously described (13). Frozen stocks were titrated by enumerating viable bacteria from serial dilutions plated on modified Mueller-Hinton agar as previously described (13). Y. pestis A1122 was cultured in brain-heart infusion broth at 37°C with constant shaking overnight, aliquoted into 1-ml samples, frozen at –80°C, and thawed just before use. Frozen stocks were titrated by enumerating viable bacteria from serial dilutions plated on blood agar plates. For both Y. pestis and F. tularensis, the number of viable bacteria in frozen stock vials varied <5% over a 10-mo period.
Preparation of labeled bacteria
F. tularensis was labeled with CFSE (Molecular Probes), a vital dye that irreversibly stains intracellular proteins of living cells, immediately before use. Freshly thawed bacteria were washed twice and resuspended in sterile PBS to remove brain-heart infusion medium. CFSE was reconstituted in DMSO as a 10-mM stock, then added to the bacteria at a final concentration of 10 µM. Bacteria were incubated for 30 min at 37°C in 5% CO2, centrifuged once, and resuspended in fresh, sterile PBS. Bacteria were incubated for an additional 30 min, washed three times to remove excess CFSE, and resuspended to a final concentration of 1 x 108 or 4 x 107 CFU/ml in sterile PBS. We found that this technique did not result in appreciable CFSE labeling of nonviable bacteria (data not shown).
Generation of bone marrow-derived macrophages (BMM
) and DC
BMM and bone marrow-derived DC (BMDC) were generated as previously described (13, 14). Briefly, bone marrow was flushed from femurs of healthy C57BL/6 mice with HBSS/2% FBS. Cells were washed, and RBC were lysed with buffered NH4Cl solution. Cells were washed again, and viable cells were enumerated by trypan blue exclusion. For generation of BMM, a single-cell suspension was prepared by gentle pipetting, and cells were plated at 2 x 106/ml in 24-well plates (Costar) in DMEM (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich), 10% L-929 conditioned medium, 0.2 mM L-glutamine, 1 mM HEPES buffer, and 0.1 mM nonessential amino acids (all from Invitrogen Life Technologies; cDMEM), supplemented with 50 IU/ml penicillin and 50 µg/ml streptomycin (Invitrogen Life Technologies), and incubated at 37°C in 5% CO2. After 1 day of incubation, the medium was replaced with antibiotic-free cDMEM, and the cells were incubated for an additional 7 days at 37°C in 5% CO2. The medium was replaced with fresh, gentamicin-free cDMEM every 2 days during the 8-day incubation period. For BMDC, freshly isolated bone marrow cells were resuspended at 1 x 106/ml in MEM (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS (Sigma-Aldrich), 2 mmol/l L-glutamine, 1% nonessential amino acids, 50 IU/ml penicillin, 50 µg/ml streptomycin (all from Invitrogen Life Technologies; cMEM), and 3% B78-Hi (a gift from P. Marrack, National Jewish Medical Research Center, Denver, CO) supernatant (GM-CSF). After medium was removed, every 2 days of incubation, fresh cMEM/GM-CSF without antibiotics was added throughout the 8-day culture period.
Collection of airway cells
Airway cells were obtained by bronchoalveolar lavage (BAL) as previously described (15). Briefly, mice were killed by cervical dislocation, and an 18-gauge catheter was immediately inserted into the trachea. Approximately 1.5 ml of ice-cold PBS was injected and then aspirated from the lungs. This was repeated three times. BAL cells from each mouse were pooled and then centrifuged at 1200 rpm for 5 min at 4°C. BAL cells were then resuspended in either cMEM or FACS buffer (PBS with 2% FBS and 0.05% sodium azide) before analysis.
Enrichment of airway DC
Airway DC were enriched by negative and positive selections first using biotinylated rat anti-mouse-CD11b (eBioscience), followed by biotinylated rat anti-mouse DEC-205 (Cedarlane Laboratories) and streptavidin-coated microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. Briefly, BAL fluid was harvested and pooled from five mice. Cells were washed twice in ice-cold PBS/2 mM EDTA/0.5% BSA (enrichment buffer) and resuspended with biotinylated anti-mouse CD11b Ab. Cells were incubated for 20 min at 4°C and then washed once. Cells were resuspended, streptavidin microbeads were added, and the bead/cell mixture was incubated for 15 min at 4°C. The cell/bead mixture was washed once, followed by resuspension in enrichment buffer, then added to a mass spectrometry separation column (Miltenyi Biotec); the column was placed in the magnetic field, and unbound cells were collected in the flow-through. DEC-205-positive cells were enriched from the unbound cells using biotinylated rat anti-mouse DEC-205 Ab following the technique as described above with the following adjustments. After addition of the bead/cell mixture to the column, placement of the column in the magnetic field, and collection of the effluent, the column was removed from the magnet, and the bound cells were allowed to detach from the column. The resulting cells were routinely >95% CD11c+/DEC-205+/CD11b–/GR-1– as assessed by flow cytometry using this technique.
Intratracheal infections
Before i.t. infections, mice were anesthetized with 200 µl of a 2.5% solution of Avertin (Sigma-Aldrich) administered i.p. Unlabeled or CFSE-labeled bacteria were diluted in PBS and stored on ice immediately before use. For i.t. infection, 50 µl of a 1 x 106 CFU/ml stock of F. tularensis LVS (5 x 104 total CFU/mouse) or 50 µl of a 4 x 107 CFU/ml stock of Y. pestis A1122 (5 x 104 total CFU/mouse) was administered, using a steel 22-gauge gavage needle (Fine Science Tools) passed blindly into the trachea. The dose of viable LVS or A1122 inoculated was confirmed by plating serial dilutions of the inoculum on modified Mueller-Hinton or blood agar, respectively. Statistical significance in survival experiments was determined by Kaplan-Meier test.
Culture of airway DC
One hour after i.t. infection, airway DC were enriched from the BAL fluid as described above. Airway DC were adjusted to 105 cells/ml in MEM supplemented with 10% heat-inactivated FBS, 2 mmol/l L-glutamine, 1% nonessential amino acids, 50 IU/ml penicillin, and 50 µg/ml streptomycin (all from Invitrogen Life Technologies; cMEM) and plated at 104 cells/well in 96-well tissue culture plates. At designated time points, cells (three wells per time point) were pelleted, and the supernatant was removed and stored at –80°C. Cells were washed three times and then lysed with sterile distilled water for 3 min to release intracellular bacteria. Cell lysates were serially diluted and plated onto modified Mueller-Hinton agar. Plates were incubated for 48–72 h at 37°C, and bacterial colonies were enumerated.
Infection and stimulation of BMM, BMDC, and BAL cells in vitro
BMM and BMDC were generated as described above. BAL cells were harvested as described above. BMM and BMDC were infected at a multiplicity of infection (MOI) of 50 with F. tularensis. Approximately 2 x 105 BMM were present in each well of a 24-well plate after 8 days of culture; thus, before infection, BMDCs were removed from their original cultures and pelleted, and their numbers were adjusted to 2 x 106/ml in reserved BMDC medium. BAL cells were adjusted to 107 cells/ml and cMEM. BMDC were harvested from their original cultures, pelleted, and adjusted to 2 x 107/ml in reserved BMDC medium. F. tularensis was added at an MOI of 50 to BMM, BMDC, or BAL cells. Cells treated with medium alone served as negative controls. Cells were incubated at 37°C in 5% CO2 for 1 h, washed once, then incubated with 50 µg/ml gentamicin (Invitrogen Life Technologies) to kill extracellular bacteria. Then cells were washed extensively and counted. BAL cells were adjusted to 105 cells/ml in cMEM and then plated at 104 cells/well in 96-well tissue culture plates. BMDC were adjusted to 2 x 105 cells/ml and then plated at 1 ml/well in 24-well tissue culture plates. As indicated, ultrapure LPS (5 µg/ml) or zymosan (5 µg/ml; both from InvivoGen) was added immediately after plating. Cultures were incubated for 24–72 h at 37°C in 5% CO2. Supernatants were collected at each time point and stored at –80°C. Infection and intracellular replication of F. tularensis were monitored as described above. Experimental and control groups consisted of two wells per time point or treatment each. The SEM and statistical significance between treatment groups were determined by ANOVA, followed by Tukey-Kramer’s comparison of means.
Flow cytometry and analysis of BMM, BMDC, and BAL cells
BMM, BMDC, and macrophage, monocyte, granulocyte, and DC populations in BAL fluid were assessed by immunostaining and flow cytometry. Directly conjugated Abs for these analyses were purchased from Cedarlane Laboratories, Serotec, BD Pharmingen, or eBiosciences. The following Abs in various combinations were used for flow cytometric analysis: anti-CD11c (allophycocyanin or PE; clone N418), anti-CD11b (PE-Cy5 or allophycocyanin-Cy7; clone M1/70), anti-GR-1 (Pey7; clone RB6-8C5), anti-B220 (allophycocyanin-Cy7; clone RA3-6B2), anti-CD8 (FITC or PE; clone 53-6.7), anti-CD4 (allophycocyanin; clone RM4-5), anti-CD3 (allophycocyanin-Cy7; clone 145-2C11), anti-I-A/I-E (MHC II, biotin or PE; clone M5/114.15.2), anti-CD86 (PE; clone GL1), anti-DEC-205 (biotin; clone NLDC-145), and anti-CD40 (Pey5; clone 1C-10). Typical flow cytometric assessment of BMM
, BMDC, or airway cells and were designed to include all viable cell populations. Of note, the side and forward scatter settings were reduced from those typically used for analysis of lymphocytes to include macrophages and DC, which have high forward and side scatter properties. Approximately 25,000 (BAL samples) and 100,000 (BMM and BMDC) events were analyzed for each sample.
DC were defined as CD11c+/DEC-205+ cells that expressed variable levels of other surface determinants, including CD11b, Gr-1, B220, MHC II, CD86, and F4/80, as well as by high side and forward scatter properties, as previously reported (16, 17). Alveolar macrophages were identified as CD11b+/F480+/CD11c–/DEC-205– cells with high forward and side scatter properties. BMM were defined as CD11b+/CD11c+/F4/80+/DEC-205–/MHC II+/–. BMDC were defined as CD11c+/DEC-205+/MHC II+. Monocytes were identified as CD11b+/GR-1+/–/MHC II+/– cells that had forward and side scatter properties more characteristic of lymphocytes. Granulocytes were identified as GR-1+/MHC II–/CD11b+/– cells that displayed an elongated side scatter profile typical of granulocytic cells in blood. Isotype control Abs were included when analyses and panels were first being performed to assure specificity of staining, but were not routinely included with each experiment, with the exception of intracellular cytokine staining (see below). Data were analyzed using Summit software (DakoCytomation). The percentage of each cell population was determined, then total cell numbers for each population were calculated from the total number of viable cells collected. Experimental and control groups consisted of three to five animals each. The SEM and statistical significance between treatment groups were determined by ANOVA, followed by Tukey-Kramer’s comparison of means.
Depletion of airway DC and alveolar macrophages with liposomal clodronate and enumeration of bacteria after infection
Mannosylated liposomal dichloromethylene bisphosphonate (clodronate; Sigma-Aldrich) was prepared as previously described (18) Briefly, lipid films comprised of phosphotidylcholine and cholesterol (Avanti Polar Lipids) and p-aminophenyl--D-mannopyranoside (Sigma-Aldrich; molar ratio, 7:2:1) were resuspended in either 10 ml of LPS-free PBS (Invitrogen Life Technologies; control liposomes) or 10 ml of PBS containing 1.89 g of clodronate (liposomal clodronate), washed extensively to remove unincorporated clodronate, then stored under argon at 4°C before use. Before i.t. administration of liposomes mice were anesthetized with 200 µl of a 2.5% solution of avertin i.p. Liposomes were resuspended by shaking immediately before use, and 50 µl of either liposome solution was injected i.t. 18 h before bacterial infection. At the designated time points after infection, BAL fluid was aseptically collected, and the lung, mediastinal lymph node, and spleen were aseptically removed. Organs were homogenized in sterile PBS using a Seward Stomacher 80 Biomaster tissue homogenizer (Brinkmann Instruments). Then homogenates and BAL fluid were serially diluted, plated onto modified Mueller-Hinton agar plates, and incubated at 37°C for 48–72 h for enumeration of colonies.
Intracellular cytokine staining
Intracellular cytokines were detected as previously described (19). Briefly, at the indicated time points, airway DC were resuspended in cMEM to which 5 µg/ml brefeldin A (Sigma-Aldrich) was added. Cells were then incubated at 37°C in 5% CO2 for 4–6 h. After incubation, cells were washed once and resuspended in FACS buffer. Cells were first surface stained with anti-CD11c (allophycocyanin) and anti-CD11b (Pe/Cy5) Abs. After washing, cells were fixed in 2% paraformaldehyde in HBSS for 10 min at 37°C and washed twice more in Perm buffer (FACS buffer supplemented with 0.25% saponin (Sigma-Aldrich)). Next, cells were incubated at room temperature with anti-TNF- (PE; clone MP6-XT22) or rat IgG1 (PE) as an isotype control (both from eBiosciences). Cells were incubated with cytokine or isotype control Abs for 20 min at room temperature. Cells were washed twice in perm buffer, fixed in 1% paraformaldehyde for 30 min, then resuspended in FACS buffer at 4°C until analysis. Cells were analyzed using a Cyan flow cytometer as described above. Each experimental or control group consisted of three to five animals. The SD and significance between cytokine production in groups of mice were determined by Student’s paired t test.
Determination of secreted cytokines
Culture supernatants were assayed for the presence of TNF-, IL-6, IL-10, MCP-1, IL-12p70, IFN-, TGF-, and IL-12p40 using a cytometric bead array (CBA Inflammation kit; BD Pharmingen) according to the manufacturer’s instructions using a Coulter EPIC XL flow cytometer or by ELISA using commercially available kits according to the manufacturer’s instructions (R&D Systems). For cytometric bead arrays, sample analysis was accomplished using BD Cytometric Array software (BD Pharmingen). For ELISAs, sample analysis was accomplished using a Multiskan Ascent ELISA plate reader and Ascent software (ThermoLabsystems). Each experimental or control group consisted of three to five animals. The SD and significance between cytokine production in mice were determined by ANOVA, followed by Tukey-Kramer’s comparison of means.
Determination of apoptosis
BMDC were infected with F. tularensis as described above. Twenty-four hours after infection, cells were washed and stained with biotin-annexin V, followed by streptavidin-Alexa 488 (Molecular Probes) according to the manufacturer’s instructions (R&D Systems). Cells were analyzed by flow cytometry for bound annexin V staining using a Cyan MLE and Summit software as described above (DakoCytomation). Significance between uninfected and infected cells was determined by Student’s paired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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F. tularensis has been shown previously to infect and replicate in macrophages, both in vitro and in vivo (1, 2). However, the interaction of F. tularensis with DC has not been previously described. DC are often the major phagocytic cells present at sites of infection and may serve as targets for F. tularensis infection during the first few hours after inoculation. Thus, interactions of F. tularensis with DC could be an integral event in the pathogenesis of tularemia. To determine whether DC are susceptible to F. tularensis infection, BMDC were inoculated with F. tularensis in vitro. BMDC were readily infected with F. tularensis and supported logarithmic growth of the bacterium over the course of 3 days of infection (Fig. 1A). Ninety-six hours after infection, significant cell death was noted, and the numbers of bacteria retrieved from BMDC cultures began to decline (data not shown). Therefore, like macrophages, BMDC were able to support the growth of F. tularensis.
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Despite replicating intracellularly, F. tularensis does not induce a strong inflammatory response in macrophages after infection (7, 13). Nevertheless, proinflammatory cytokines, especially TNF-, are critical for controlling F. tularensis infections (4). Therefore, the inability of host cells to produce proinflammatory cytokines in response to F. tularensis infection is an important evasion mechanism used by the bacterium. Given the ability of F. tularensis to infect and replicate in BMDC (Fig. 1A), we next determined whether F. tularensis could elicit secretion of several important proinflammatory cytokines. Equal numbers of BMM and BMDC were infected with F. tularensis, and 48 h later culture supernatants were tested for TNF-, IL-6, and IL-10 concentrations. Neither BMDC nor BMM secreted TNF-, IL-6, or IL-10 in response to F. tularensis infection (Fig. 1B and data not shown). Furthermore, these cytokines were not detected throughout the 72-h culture period (our unpublished observations). The absence of these proinflammatory cytokines was not due to an inability of the BMDC and BMM cultures to produce cytokines, because LPS elicited large amounts of TNF- production (Fig. 1B). This result suggested, therefore, that either BMDC and BMM simply do not detect F. tularensis infection or that the bacterium may actively suppress the production and secretion of proinflammatory cytokines in APCs.
F. tularensis induces phenotypic maturation of BMDC
In addition to secretion of proinflammatory cytokines, DC prime the immune response by increasing the expression of MHC and costimulatory molecules. These receptors are integral for Ag presentation to T cells in the local lymph nodes. Increased expression of these receptors is often linked to the presence of cytokines such as TNF-. Some pathogens are known to interfere with the activation of DC by blocking both cytokine release and phenotypic maturation (9, 10, 11, 20). Because F. tularensis did not elicit the production of proinflammatory cytokines often associated with maturation of DC (Fig. 1B), we next examined the effect F. tularensis on phenotypic maturation of BMDC. BMDC and BMM were infected with F. tularensis, and changes in MHC II and CD86 expression were followed over time. Surprisingly, BMDC increased the expression of MHC II within the first 24 h after infection in response to F. tularensis infection despite the lack of proinflammatory cytokines to drive the maturation (Fig. 2B). The first change in phenotypic maturation of BMDC in response to F. tularensis infection was increased overall expression (increased mean fluorescence intensity) of MHC II on BMDC. This was followed next by an increase in the number of BMDC positive for expression of MHC II (Fig. 2, A and B). The number of BMDC expressing CD86 did not significantly increase until 72 h after infection (Fig. 2C). In contrast, BMM did not increase the expression of MHC II until 48 h after infection and did not significantly increase CD86 throughout the course of infection (Fig. 2, A, C, and D). Therefore, despite the absence of proinflammatory cytokines, BMDC underwent rapid phenotypic maturation. This result indicates that BMDC are not completely unresponsive to F. tularensis infection. Rather, phenotypic maturation and release of proinflammatory cytokines appear to be uncoupled in DC in response to F. tularensis infection.
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Airway DC have been recently recognized to play a key cell in eliciting inflammation and development of adaptive immunity in the lung (16, 17). Although F. tularensis is infectious by multiple routes, the most lethal manifestation of F. tularensis infection is primary pneumonic tularemia. Therefore, airway DC may serve as an important primary target cell after introduction of F. tularensis into the lung. Previous reports have shown that F. tularensis infects alveolar macrophages in vitro (3). However, the interaction of this bacterium with airway DC has not been previously described. To determine whether F. tularensis targeted airway DC in vivo, we infected mice i.t. with CFSE-labeled F. tularensis. CFSE did not appreciably label nonviable bacteria and has been used by our laboratory to label and track other bacteria, such as Y. pestis in vivo (our unpublished observations). Airway DC were characterized as CD11c+/DEC-205+/GR-1–/CD11b–/MHC II+/–, whereas alveolar macrophages were characterized as CD11b+/DEC-205–/GR-1–/CD11c–/MHC II–. Additional studies performed in our laboratory have also shown that these airway DC have functional characteristics associated with classic DC. These characteristics include support of allogeneic MLRs and macropinocytosis of labeled dextran (our unpublished observations). Furthermore, macropinocytosis by airway DC decreased upon maturation of these cells with LPS (our unpublished observations).
One hour after pulmonary infection, labeled F. tularensis was found primarily associated with airway DC (Fig. 3A). Approximately 18% of all airway cells were found to be airway DC that were associated with labeled F. tularensis (Fig. 3A). The airway DC that contained labeled F. tularensis represented 
50% of all airway DC and contained 95% of all labeled F. tularensis organisms. In contrast, <1% of all gated cells were alveolar macrophages associated with labeled F. tularensis (Fig. 3A). The alveolar macrophages that contained labeled F. tularensis represented 60% of all alveolar macrophages, but these cells contained only 5% of all labeled bacteria. Thus, airway DC were the primary target cell for uptake of F. tularensis after pulmonary infection by i.t. inoculation.
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The airways are a dynamic environment that possess a variety of molecules that may interfere with uptake of particles, activation of local cells, and, potentially, replication of pathogens introduced by inhalation (21, 22). Although, F. tularensis replicates in the airways and lungs after infection, it is not known whether airway DC are among the cells in which the bacterium can replicate. To determine whether airway DC merely bound, but did not internalize, F. tularensis; became infected with F. tularensis, but did not permit replication of the bacterium; or became infected and supported growth of the bacterium, we examined replication of F. tularensis in airway DC after in vivo infection. Airway DC infected with F. tularensis by i.t. inoculation were isolated and cultured as described in Materials and Methods. Consistent with observations in BMDC (Fig. 1), airway DC supported logarithmic growth of F. tularensis after in vivo infection (Fig. 3B). Therefore, airway DC serve as a primary target cell and site of replication by F. tularensis within the first few hours after pulmonary infection.
F. tularensis does not induce production of proinflammatory cytokines in airway DC
An unusual hallmark of clinical pulmonary F. tularensis infection is the absence of marked pathology in the lung (23). A major regulator of inflammation in the lungs is liberation of proinflammatory cytokines after infection (24, 25). Although F. tularensis did not induce production of proinflammatory cytokines in BMDC, it was important to confirm this result in airway DC. Therefore, the effect of F. tularensis infection on production of proinflammatory cytokines in airway DC was assessed after in vivo infection. Y. pestis, another Gram-negative bacterium that causes lethal pulmonary disease, was used as a positive control. One hour after in vivo infection, airway DC were isolated, cultured, and examined for production of intracellular cytokines as described in Materials and Methods. In striking contrast to Y. pestis, F. tularensis induced barely measurable production of TNF- and immeasurable concentrations of IL-6 and IL-10 from airway DC (Fig. 3C and data not shown). Alveolar macrophages infected with F. tularensis also failed to produce intracellular TNF- (Fig. 3C). Furthermore, intracellular TNF- in DC and macrophages was not detected for the first 72 h after in vivo infection and was only observed at the end stages of disease (data not shown). Thus, consistent with in vitro infection results, F. tularensis did not induce production of proinflammatory cytokines by infected APCs after in vivo infection.
F. tularensis induces early phenotypic maturation of airway DC, but not alveolar macrophages, after in vivo infection
Although F. tularensis induced phenotypic maturation of BMDC in vitro, infection failed to elicit production of proinflammatory cytokines. Additionally, BMDC responded faster to F. tularensis in terms of increased expression of MHC II and CD86 compared with BMM. Therefore, we hypothesized that a similar scenario may occur in the airways of mice given a pulmonary challenge of F. tularensis. DC and macrophages were therefore harvested from the airways and lungs at various time points after i.t. F. tularensis infection as described in Materials and Methods and assessed for phenotypic changes by flow cytometry. As observed in BMDC, airway DC increased the expression of both MHC II and CD86 within 24 h of infection (Fig. 4). The number of lung DC expressing MHC II also increased at this time point (Fig. 5). Airway and lung DC maintained elevated expression of MHC II and CD86 over the first 72 h of infection (data not shown). However, the increase in MHC class II and CD86 expression did not appear to be due to an influx of new DC, because the numbers of both airway DC and lung DC were significantly lower in infected animals than in control animals 48 and 72 h after infection (Fig. 6). In contrast, alveolar and lung macrophages did not increase the expression of either MHC II or CD86 within 24 h after infection (Figs. 4 and 5). Changes in MHC II and CD86 expression by alveolar macrophages also were not observed until 48 and 72 h after infection (data not shown). However, the increases in MHC II and CD86 expression by alveolar macrophages by 72 h after infection were most likely due to the influx of new cells into the airways. This conclusion is based on the fact that there were significantly more alveolar macrophages present in the airways of infected mice than control mice 72 h after infection (Fig. 6). Together, these data suggest that although DC and macrophages are not stimulated by F. tularensis to produce proinflammatory cytokines in vivo, they are capable of increasing the expression of at least two receptors critical for Ag presentation, MHC II and CD86. Furthermore, DC respond more rapidly than macrophages to F. tularensis infection.
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by DC
The lack of production of TNF- and other proinflammatory cytokines after F. tularensis infection could result from the inability of DC and macrophages to detect the bacterium. However, the fact that infection induced phenotypic activation of DC and macrophages suggests that this interpretation is incorrect. Alternatively, it may be that F. tularensis infection actively suppresses cytokine production by DC. For example, a recent report shows that F. tularensis actively suppresses the ability of macrophages to respond to LPS, a TLR4 agonist (7). To determine whether active suppression of cytokine production also occurred in F. tularensis-infected DC, BMDC and freshly isolated airway DC were infected with F. tularensis in vitro, followed by exposure to different TLR agonists, LPS (TLR4) and zymosan (TLR2). Both LPS and zymosan induced the production of TNF- from control DC, and F. tularensis infection alone did not elicit TNF- production by either population of DC (Fig. 7). However, in marked contrast to DC treated with TLR agonist alone, DC infected with F. tularensis failed to secrete TNF- in response to either LPS or zymosan (Fig. 7). This suggested that F. tularensis actively suppresses the production of TNF- and that the mechanism responsible for suppression of cytokine production involved least one pathway used by multiple TLR agonists.
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production by DC and macrophages
In addition to active suppression of host cell responsiveness, pathogens have developed mechanisms to exploit the endogenous immunosuppressive mechanisms used by the host to control immune responses. TGF- and IL-10 are important immunosuppressive cytokines that play key roles in maintaining homeostasis and regulating immune responses in the host (26). Recently, it has been shown that T. gondii promotes TGF- responses as one mechanism of evading the host immune response (27, 28). Given the relative lack of inflammatory responses during the first few days of experimental pulmonary F. tularensis infection, we hypothesized that the bacterium may use a similar strategy to evade immune defenses. Using either in vitro or in vivo infection models, we did not detect an increase in IL-10 production from F. tularensis-infected DC or macrophages (data not shown). These supernatants were also analyzed for TNF-, and as previously observed, none was found. However, the situation with F. tularensis and production of TGF- was quite different. In this study we observed that in vivo infection with F. tularensis induced the secretion of significant quantities of TGF- from DC and macrophages (Fig. 8A). To assess the relative responsiveness of DC and macrophages to F. tularensis-induced production of TGF-, BMM and BMDC were infected in vitro with F. tularensis. We found that both BMM and BMDC produced TGF- in response to F. tularensis infection, but that only BMM secreted significantly more TGF- in response to infection than control cultures. Therefore, induction of TGF- may be an additional immunosuppressive mechanism used by F. tularensis to evade host immune response.
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As another possible means of inducing immune suppression, we assessed the ability of F. tularensis to induce DC apoptosis. BMDC were infected with F. tularensis, and induction of apoptosis was assessed by flow cytometric analysis of annexin V binding. Within 24 h of infection, we observed a significant increase in apoptosis in BMDC infected with F. tularenesis compared with uninfected controls DC (p = 0.0188; Fig. 9). Thus, it is also possible that in part the lack of production of proinflammatory cytokines after infection with F. tularensis may result from rapid induction of apoptosis in infected DC.
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The preceding results suggested that both DC and macrophages in the lungs play an important role in early F. tularensis infection. Furthermore, F. tularensis infection of both DC and macrophages blocked the secretion of proinflammatory and instead promoted the secretion of immunosuppressive TGF-. Thus, both cell types are likely to play critical roles in F. tularensis infection. To directly assess the roles of these cells in pulmonary infection, depletion experiments were conducted. DC and macrophages were depleted from the airways and lungs by i.t. instillation of mannosylated liposomal clodronate (clodronate). This approach has been used previously for in vivo depletion of macrophages and DC (29, 30). Intratracheal administration of clodronate resulted in the depletion of >90% of all APCs in the airways and lungs 18 h after instillation, and depletion persisted for up to 5 days in uninfected mice (data not shown). Eighteen hours after treatment with clodronate, mice were infected i.t. with F. tularensis. Remarkably, mice depleted of APCs in the airways and lungs before infection survived significantly longer (p = 0.0446) than untreated control animals (Fig. 10). Furthermore, clodronate-treated mice had significantly fewer bacteria in the airways, lung, lymph node, and spleen for the first 12 h after infection and significantly fewer bacteria in the lungs and airways 48 and 72 h after infection compared with untreated controls (p < 0.01; Fig. 10B). There were no significant differences in the magnitude or type of inflammatory infiltrate in the lungs and airways of clodronate-treated mice compared with untreated mice after infection (data not shown). These results therefore suggest that DC and macrophages play an important role in the establishment, dissemination, and ultimately ability of the host to control pulmonary infection with F. tularensis.
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Immune evasion via manipulation of DC function has been described for other pathogens. For example, both ebola and Marburg viruses replicate in DC without inducing any phenotypic maturation (11, 31). Furthermore, both these viruses also actively suppress type I IFN responses in DC, thereby effectively removing a critical first line of defense in the infected host (11, 32). Some mycobacteria suppress DC function by manipulation of signaling via a variety of TLRs and at least one C-type lectin, DC-SIGN (33, 34). However, we are unaware of previous reports demonstrating the exact pattern of aberrant activation of DC that we describe in this paper.
In addition to direct modulation of the proinflammatory response in DC and macrophages, we demonstrate for the first time that F. tularensis induces the production of a key immunosuppressive cytokine, TGF-. TGF- is known to play an important role in immune homeostasis (35). However, other recent reports have highlighted the role this cytokine plays in exacerbating several different infectious diseases. For example, the direct inhibitory effect of TGF- on T cell function is associated with the inability of mice to clear Leishmania infections (36). In another example, overexpression of TGF- in the lungs increased susceptibility to pulmonary Cryptococcus neoformans infections (37).
F. tularensis is an intracellular pathogen capable of causing disease after infection by multiple different routes. However, inhalation of the bacterium with resulting pneumonic tularemia is considered the most deadly manifestation of the disease. Despite the high mortality rate, the acute nature of pneumonic tularemia, and the potential for F. tularensis to be used as an effective aerosol bioweapon, very little is known about the innate immune response after infection, especially in the lung.
DC are recognized as key sentinel cells for the immune system. These cells are necessary for both initiation of the innate immune response as well as bridging the transition to adaptive immunity. Recently, the critical role of DC in the lungs and airways has been recognized (16, 17, 38). In the lungs, DC are organized in a structural arrangement in terms of DC subset distribution within the airways and lung parenchyma. Within the normal mouse lung parenchyma, there are several different CD11c+ DC populations that express CD11b, CD8, or B220 (39). In contrast, airway DC represent a more homogeneous population, which may reflect their relative immaturity, compared with DC in the lung parenchyma (16, 40, 41). Airway DC are ideally suited for sampling inhaled microorganisms and noninfectious particulate matter and are functional and able to present soluble Ags after i.n. or i.t. delivery (16, 42).
Interactions between pathogens and airway and lung DC have only been studied very recently. For example, i.n. administration of M. bovis was recently shown to trigger recruitment of DC into the lung, and these newly recruited DC promoted the development of a Th1 immune response (15, 43). In another report, transport of Aspergillum hyphae to draining lymph nodes, and thus dissemination of the fungus, was also shown to be dependent on pulmonary DC (44). However, despite these recent observations, little is understood about exactly how pathogens interact with DC in the lungs and what role these DC play in the control or exacerbation of infectious diseases of the lungs, including pneumonic F. tularensis.
Therefore, we investigated the roles of lung DC and macrophages in the pathogenesis of pulmonary F. tularensis infections. The ability of F. tularensis to suppress macrophage function has been reported previously (7). Similarly, we have provided data that F. tularensis suppresses the responsiveness of DC to TLR agonists. There are several explanations for this suppression. The observed lack of cytokine production in F. tularensis-infected DC may be a result of the DC undergoing maturation, rendering them refractory to additional stimuli. However, we did not observe phenotypic maturation of DC in the first 4 h of infection, and in the studies we report in this paper, the TLR agonists were added immediately after infection. Therefore, it seems unlikely that the remarkable suppression of TLR responsiveness by F. tularensis-infected cells, presented in Fig. 7, could be attributed to refractory cells. Another possible explanation for the lack of cytokine production after F. tularensis infection is that the bacterium is inducing apoptosis in infected cells. For example, we observed significant increases in F. tularensis-mediated apoptosis in infected DC, although this occurred 24 h after infection (Fig. 9). Thus, we do not believe that this is the sole mechanism for the lack of cytokine production. Although we cannot exclude that a combination of DC maturation and apoptosis of infected cells is responsible for attenuated responsiveness to TLR agonists, our new data suggest that F. tularensis also actively suppressed DC function similar to that previously observed in macrophages. This leads us, therefore, to hypothesize that F. tularensis may actually prefer to use airway DC and alveolar macrophages as a portal of entry in the host. Suppression of proinflammatory cytokine release together with stimulation of TGF- production may allow the bacterium to undergo several initial rounds of replication before eliciting immunological recognition. Furthermore, the induction of early phenotypic activation of DC may facilitate dissemination of the bacterium to the draining lymph node and systemic spread before a T cell response can be mounted. In this scenario, after infection by inhalation, DC and macrophages may actually contribute to the progression of tularemia, instead of helping to resolve the infection. Our results are consistent with this theory. In support of this idea, we observed that depletion of DC and macrophages before infection actually decreased survival times significantly (see Fig. 10). In contrast, activation of DC and macrophages in the lungs before infection may help control pulmonary F. tularensis infection. For example, our preliminary data suggest that activation of airway DC and macrophages with TLR agonists before infection can increase survival after pulmonary challenge with F. tularensis (our unpublished observations).
Together, the data presented in this study demonstrate a dynamic interaction between F. tularensis and pulmonary APCs, especially airway DC. F. tularensis appears to have developed an array of mechanisms to subvert the protective functions of lung DC. These suppressive mechanisms include interference with TLR signaling and induction of TGF-, a potent immunosuppressive cytokine. Additional studies of the early interactions between pathogens and lung DC are likely to lead to the development of new strategies to treat and/or prevent infections with F. tularensis and other important pulmonary pathogens.
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1 This work was supported by a grant from the Colorado State University College Research Council and National Institutes of Health Grant U01AI056487-01. 
2 Address correspondence and reprint requests to Dr. Steven W. Dow, 1619 Campus Delivery, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523. E-mail address: sdow{at}colostate.edu
3 Abbreviations used in this paper: DC, dendritic cell; BAL, bronchoalveolar lavage; BMDC, bone marrow-derived DC; BMM, bone marrow-derived macrophage; i.n., intranasally; i.t., intratracheally; LVS, live vaccine strain; MHC II, MHC class II; MOI, multiplicity of infection.
Received for publication June 3, 2005. Accepted for publication August 30, 2005.
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-stimulated murine alveolar macrophages. J. Immunol. 153:1238.-1245. [Abstract]
and IL-1 from murine macrophages. Cell. Microbiol. 5:41.-51. [Medline]
expression in human retinal pigment epithelial cells is enhanced by Toxoplasma gondii: a possible role in the immunopathogenesis of retinochoroiditis. Clin. Exp. Immunol. 128:372.-378. [Medline]
. Annu. Rev. Immunol. 16:137.-161. [Medline]
-induced inhibition of T helper type 1 differentiation. J. Exp. Med. 195:14991505.
prevents eosinophilic lung disease but impairs pathogen clearance. Microb. Infect. 7:365.-374. [Medline]
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) increase the expression of MHC II at later time points (48 and 72 h) after infection. No changes in CD86 expression were noted in F. tularensis-infected BMM
