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The Journal of Immunology, 2007, 178: 4445-4454.
Copyright © 2007 by The American Association of Immunologists, Inc.

Francisella tularensis Induces IL-23 Production in Human Monocytes1

Jonathan P. Butchar*, Murugesan V. S. Rajaram*, Latha P. Ganesan*, Kishore V. L. Parsa{dagger}, Corey D. Clay{ddagger}, Larry S. Schlesinger{ddagger} and Susheela Tridandapani2,*

* Department of Internal Medicine, {dagger} Biochemistry Program, and {ddagger} Center for Microbial Interface Biology, Division of Infectious Diseases, Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, OH 43210


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Francisella tularensis, the causative agent of tularemia, is phagocytosed by immune cells such as monocytes and macrophages. Instead of being destroyed in the phagolysosome, the bacterium escapes the phagosome and replicates within the host cytosol. Recent studies indicate that phagosomal escape may have a major impact on the nature of the inflammatory cytokine response to infection. To better understand the host cell response to Francisella infection, we exposed human peripheral blood monocytes to Francisella novicida and analyzed transcriptional changes using high-density oligonucleotide microarrays. Results showed a nearly 300-fold up-regulation of transcripts for the p19 subunit of IL-23, and a nearly 18-fold up-regulation for the p40 subunit of IL-12. IL-23 is formed by the heterodimerization of p19 and p40, and is an important cytokine of the innate immune response. Up-regulation of p19 and p40 was confirmed at the protein level by Western blotting and ELISA analyses, and was found to be largely dependent on PI3K and NF-{kappa}B activity. Studies using medium from infected monocytes with or without a p19 blocking Ab showed that the secreted IL-23 induced IFN-{gamma} production from NK cells, suggesting a potential biologically important role for IL-23 in host defense. Finally, infection of human monocytes by the highly virulent Francisella SCHU S4 strain likewise led to IL-23 production, suggesting that the IL-23 response may be relevant during tularemia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Gram-negative Francisella tularensis is a facultative bacterium that is responsible for causing the zoonotic disease tularemia (1). The two primary human pathogens are F. tularensis subspecies tularensis (type A strain) and F. tularensis subspecies holarctica (type B strain). F. novicida, a close relative of type A strain F. tularensis, is a much less frequent cause of infection in humans than either type A or type B (2). Nevertheless, F. novicida has been used as a model bacterium because it causes a lethal systemic infection in mice when inoculated by most routes (3) and has been reported to have a similar intracellular lifestyle to the more virulent human strains (4).

Many routes of infection exist for Francisella, with the most dangerous being inhalation. Following infection of the host, one of the key targets of Francisella is the monocyte/macrophage. These cells readily phagocytose the bacteria via receptor-mediated entry pathways (5), encapsulating them in the phagosome. However, Francisella subsequently disrupts the normal process of phagosome-lysosome fusion that leads to pathogen destruction and escapes into the host cell cytosol where bacterial replication occurs. Following cell lysis, a cycle of naive phagocyte infection ensues (1).

Francisella-infected monocytes send signals (cytokines/chemokines) to neighboring cells during the innate immune phase of the infection thereby influencing the nature of the inflammatory response. Hence, an understanding of how Francisella affects monocyte cytokine response is critical.

Recent studies have shown that monocytes/macrophages release both proinflammatory and anti-inflammatory cytokines in response to Francisella infection (6, 7, 8, 9, 10, 11, 12). Attachment of Francisella appears to be sufficient to initiate signaling mechanisms leading to the biosynthesis and release of most inflammatory cytokines (10, 11). However, there is emerging evidence that internalization of the bacilli, phagosomal escape, and recognition by intracellular host cell sensors are required for the posttranslational processing and release of certain proinflammatory cytokines such as IL-1beta (6, 10). In contrast, the release of other proinflammatory cytokines such as TNF-{alpha} is dampened after phagosomal escape (8, 12). Interestingly, the anti-inflammatory cytokine IL-10 is up-regulated after phagosomal escape, and this response has been linked to a decrease in Akt activation (12). From these and other studies, it is clear that much remains to be learned about the nature of the monocyte inflammatory response to Francisella.

In this study, we used microarray analysis to help identify novel cytokine/chemokine genes that respond to Francisella infection of human peripheral blood monocytes (PBM).3 Analysis of results showed that the p19 subunit of IL-23 was up-regulated almost 300-fold following infection by F. novicida. Western blot and ELISA analyses verified this up-regulation at the protein level, and assays using conditioned medium from infected monocytes showed IFN-{gamma}-inducing activity when applied to NK cells. Production of IL-23 depended on NF-{kappa}B and PI3K activity, and to a lesser extent on Erk activation. To extend these findings, we infected human monocytes with the virulent SCHU S4 strain of Francisella. Results showed that SCHU S4 also elicited an IL-23 response, suggesting that IL-23 production is a general response to Francisella infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells, Abs, and reagents

THP-1 human monocytic cells were obtained from American Type Culture Collection and maintained in RPMI 1640 with 5% heat-inactivated FBS. Human IL-2-dependent NK-92 cells were a gift from Dr. H. Klingemann (Rush Cancer Center, Chicago, IL) and were maintained in RPMI 1640 supplemented with 20% heat-inactivated FBS and 150 IU/ml recombinant human IL-2 (Hoffmann-LaRoche). Abs specific for phospho-Erk, phospho-p38, and phospho-Ser Akt were purchased from Cell Signaling Technology. Actin, IL-23p19, and Akt Abs were from Santa Cruz Biotechnology. PI3K and Erk inhibitors were purchased from Calbiochem. The NF-{kappa}B inhibitor SN50 was from Biomol. F. novicida U112 (JSG1819), and an mglA mutant of F. novicida U112 (JSG2250), were used in all experiments. The SCHU S4 strain of Francisella, a Centers for Disease Control and Prevention isolate, was provided by Dr. R. Lyons (University of New Mexico, Albuquerque, NM). Bacteria were grown overnight on Chocolate II agar plates (BD Biosciences) at 37°C.

Isolation of PBM

CD14-positive PBM were isolated as previously described (13). Briefly, PBMCs were first isolated by density gradient centrifugation over Histopaque (Sigma-Aldrich). Monocytes were then purified from the PBMCs by negative selection using the MACS Monocyte Isolation kit (Miltenyi Biotec). For purification, PBMCs were first treated with Fc receptor blocking reagent (hIgG), followed by a Hapten-Antibody Cocktail (mixture of monoclonal hapten-conjugated CD3, CD7, CD19, CD45RA, CD56 and anti-IgE Abs). The labeled cells were further treated with MACS anti-hapten magnetic microbeads that were conjugated to a monoclonal anti-hapten Ab. The cells were than passed over a MACS column, and the effluent was collected as the negative fraction representing enriched monocytes. The purity of the monocytes obtained by this method is >97% as determined by flow cytometry using CD14 Abs.

Cell stimulation, lysis, and Western blotting

PBM/THP-1 cells were infected with F. novicida or F. novicida mutant mglA that were scraped from plates, resuspended and diluted in RPMI 1640 (10). Uninfected and infected cells were lysed in TN1 buffer (50 mM Tris (pH 8.0), 10 mM EDTA, 10 mM Na4P2O7, 10 mM NaF, 1% Triton X-100, 125 mM NaCl, 10 mM Na3VO4, 10 µg/ml each aprotinin and leupeptin). Postnuclear lysates were boiled in Laemmli sample buffer and were separated by SDS-PAGE, transferred to nitrocellulose filters, probed with the Ab of interest, and developed by ECL (Amersham Biosciences).

Cells infected with SCHU S4 strain of Francisella were pelleted and then boiled for 10 min in 500 µl of SDS sample buffer containing 1.5% 2-ME. After boiling, 100 µl from each was plated to verify that no viable Francisella remained.

Western blot data quantitation

The ECL signal was quantitated using a scanner and a densitometry program (Scion Image), as previously described (14). To quantitate the p19-specific signal in the infected samples, we first subtracted background, normalized the signal to the amount of actin in the lysate, and plotted the values. A paired Student’s t test was used for each statistical comparison and a value for p ≤ 0.05 was considered significant.

ELISA measurement of cytokine production

PBM/THP-1 cells were infected with F. novicida or F. novicida mutant mglA for varying time points. Cell supernatants were harvested, centrifuged to remove dead cells, and analyzed by ELISA using cytokine-specific kits from R&D Systems and eBioscience.

Supernatants from cells infected by SCHU S4 strain of Francisella were centrifuged for 15 min at 4000 x g in a Spin-X 0.22-µm filter tubes (Corning) to remove any remaining bacilli. Samples from the filtered supernatants were plated to verify that no viable Francisella remained before the ELISA analysis was performed. A paired Student’s t test was used for each statistical comparison and a value of p ≤ 0.05 was considered significant.

Microscopy assays of Francisella association with and phagocytosis by host cells

THP-1 cells were infected with F. novicida or the SCHU S4 strain (multiplicity of infection (MOI) of 100). At 2 h postinfection, cells were washed twice in warm RPMI 1640, and then fixed using 2% paraformaldehyde while in suspension. Following fixation, cells were washed three times in PBS. Cells were diluted by a factor of 10, and spun onto coated Cytospin slides (Thermo Fisher Scientific) in a Shandon Cytospin 4 (Thermo Fisher Scientific) at 800 rpm for 5 min at low acceleration. Half of the slides were permeabilized with 100% methanol. Immunostaining was performed using a monoclonal mouse anti-F. tularensis LPS Ab (diluted 1/1000; Abcam) or monoclonal mouse anti-F. novicida LPS Ab (diluted 1/100; Immuno-Precise Antibodies) (5). Alexa Fluor 488-conjugated goat anti-mouse IgG was used as secondary Ab (Molecular Probes). Bacteria attached to the surface of cells were counted on non-methanol-permeabilized cells, and bacteria associated (both phagocytosed and attached bacteria) with cells were counted on methanol-permeabilized cells using a x100 oil immersion objective with a wide bandwidth 570-nm dichroic mirror on a BX51 Olympus fluorescence microscope. Three separate sets of infections were analyzed. A total of 100 host cells were analyzed per sample. A paired Student’s t test was used for each statistical comparison and a value of p ≤ 0.05 was considered significant.

Intracellular survival assay

THP-1 cells were infected with F. novicida or SCHU S4 strain (100 MOI). At 1.5, 11.5, or 23.5 h postinfection, cells were incubated with 50 µg/ml gentamicin for 30 min at 37°C and 5% CO2. After washing cells at 2, 12, and 24 h, 0.1% deoxycholate was added to resuspended cells for 5' at 37°C to lyse macrophages. Immediately, 10-fold serial dilutions were made and appropriate dilutions were plated on Chocolate II agar plates to determine CFU. Three separate infections were performed for each test group.

Bio-Plex measurement of cytokine production

A Bio-Plex Human x-Plex Assay kit (Bio-Rad) was used to measure the following cytokines/chemokines following human monocyte infection with F. novicida: IL-6, IL-8, IL-10, GM-CSF, IFN-{gamma}, TNF-{alpha}, IL-12p70, MIP-1beta, G-CSF, MCP-1 (CCL3), MIP-1{alpha}, and RANTES. IL-8, IL-6, and TNF-{alpha} were also tested with ELISA (R&D Systems). The Bio-Plex assay was done following the manufacturer’s instructions.

Microarray analysis

PBM were isolated from four donors and aliquoted into polypropylene tubes at 5 million cells in 1 ml of RPMI 1640 containing 5% heat-inactivated FBS. F. novicida bacteria were added at an MOI of 100 to the treatment tubes. The tubes were then gently shaken to mix the monocytes and bacteria, and incubated at 37°C for 24 h. RNA was extracted using TRIzol Reagent (Invitrogen Life Technologies), column-purified using RNeasy columns (Qiagen), and then hybridized to Human Genome U133 Plus 2.0 Array chips (Affymetrix). Uninfected and infected PBM from each donor were hybridized for a total of 8 chips and compared. Expression values were calculated using the gcrma package in the BioConductor project (www.bioconductor.org) and the resulting data were analyzed for differential expression using the limma package (15). Genes with values adjusted for p ≤ 0.05 were counted as significantly different. These were ranked by fold change.

Serum IL-23 measurement

Age-matched FVBN mice were i.p. injected with F. novicida (~200 CFU) or with PBS and returned to their cages, as previously described (12). The mice were sacrificed 24 or 48 h postinfection and blood was collected from the posterior vena cava (16). To obtain serum, blood was centrifuged at 13,000 rpm for 10 min at 4°C, and analyzed by ELISA for IL-23. All mouse experiments were performed with Institutional Animal Care and Use Committee approved protocols. A paired Student’s t test was used and a value of p ≤ 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
F. novicida induces production of cytokines/chemokines

Monocytes are key components of the host innate immune system, and a critical function of these cells is the secretion of inflammatory mediators when encountering a pathogen. We performed a microarray screen in an attempt to identify newly described cytokines/chemokines in response to F. novicida infection. Analysis of the array data from uninfected and infected PBM isolated from four donors showed that many secreted mediators were up-regulated after infection (Fig. 1A). We verified several of these at the protein level using ELISAs and BioPlex assays (Fig. 1, B and C). The magnitude of induction of the cytokine/chemokine protein did not always correspond to the mRNA data obtained from the arrays, but much of this discrepancy could be due to additional regulation at the protein level. Genes that were shown to not be up-regulated by the array data were also tested. Of note, IL-12p70 showed no increase, which is in agreement with the lack of IL-12p35 up-regulation in the array data.


Figure 1
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  		FIGURE 1. Cytokine/chemokine production in human PBM uninfected (UT) and infected with F. novicida. A, Cytokine/chemokine gene response to F. novicida (FN) in human PBM. B and C, ELISA and Bio-Plex assays confirmed the up-regulation of these genes, as well as the lack of increase in IL-12p35 (via p70 assay) and MIP-1{alpha}.

 
F. novicida induces IL-23 production in human monocytes

Although there was no increase in IL-12p35, the p19 subunit of IL-23 was up-regulated ~300-fold (Fig. 1A). The p19 subunit of IL-23 is reported to combine with the p40 subunit of IL-12 to form functional IL-23 (17). To verify whether IL-23 was indeed being produced, Western blots against p19 and ELISA analysis for IL-23 were performed. For these analyses, PBM were infected with F. novicida for 24 h. Protein-matched cell lysates were analyzed by Western blotting with IL-23p19 Ab (Fig. 2A, top), followed by a reprobe of the same membrane with actin Ab to ensure equal loading of protein in the lanes (Fig. 2A, bottom). The results demonstrate a clear induction of p19 in infected monocytes. In parallel, cell supernatants were analyzed by ELISA for IL-12/23 p40 and IL-23. The latter ELISA uses a capture Ab specific for the p19 subunit of IL-23 and a detection Ab specific for the p40 subunit of IL-12/23. The IL-23 response shown in Fig. 2C virtually mirrors the increase in IL-12/23 p40 (Fig. 2B). This response, in conjunction with the lack of IL-12 p35 response seen in the array data (Fig. 1A) and the lack of IL-12p70 seen with the Bio-Plex assay (Fig. 1C), suggests that much of the IL-12p40 heterodimerizes with p19 to form IL-23. A similar induction of IL-23 was seen in the human monocytic cell line THP-1 infected with F. novicida (Fig. 2, D–F), indicating that THP-1 cells are a good cell line model to study the human monocyte response to infection.


Figure 2
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  		FIGURE 2. IL-23 is induced in human monocytes uninfected (UT) and infected with F. novicida (FN). PBM (A–C) and THP-1 cells (D–F) were uninfected or infected for 24 h with 100 MOI F. novicida. Protein-matched lysates were analyzed by Western blotting with IL-23p19 Ab (A and D, top). The same membranes were reprobed with actin Ab to ensure equal loading of protein (A and D, bottom). Cell supernatants were analyzed by ELISA for the p40 subunit of IL-12/23 (B and E) and IL-23 (C and F). Data from six independent experiments were analyzed by Student’s t test. Error bars indicate SD. *, p ≤ 0.05.

 
Dose response and kinetics of IL-23 induction

We next examined the influence of F. novicida dosage on IL-23 induction. As shown in Fig. 3, there was no detectable induction of IL-23 at an MOI of 1. However, an MOI of 10 elicited a robust IL-23 response. The response at 10 MOI was consistently greater than the response in cells infected at an MOI of 100. Although these differences did not reach statistical significance in the case of the individual subunits p19 and p40 (Fig. 3, A and B), IL-23 production was significantly different (Fig. 3C).


Figure 3
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  		FIGURE 3. IL-23 response to F. novicida is dose-dependent. THP-1 cells were infected for 24 h with F. novicida (FN, 1, 10, or 100 MOI). A, Protein-matched cell lysates were analyzed by Western blotting with IL-23p19 Ab (top blot), followed by a reprobe with actin Ab (bottom blot). Subunit p19 band intensities (from three independent experiments) were normalized to actin in the corresponding lanes and expressed as fold increase over untreated (lower graph). B and C, Cell supernatants were assayed by ELISA for IL-12/23p40 and IL-23. Data from three independent experiments were analyzed by Student’s t test. The error bars indicate SD. *, p ≤ 0.05, indicates significant difference between the response at 10 and 100 MOI.

 
To examine the time required for IL-23 secretion, THP-1 cells were infected for 2, 4, 8, and 24 h. Cell lysates and supernatants from infected and uninfected cells were analyzed for the induction of the IL-23 p19 subunit, IL-12/23 p40 subunit and IL-23. The results shown in Fig. 4 demonstrate that IL-23 secretion begins at 4 h. However, we did not detect any IL-12/23 p40 at the 4-h time point. We speculate that this result may simply be due to differences in the sensitivity of our detection methods for IL-12/23 p40 and IL-23.


Figure 4
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  		FIGURE 4. Kinetics of IL-23 production in response to F. novicida infection. THP-1 cells were infected for the indicated time points with F. novicida (FN, MOI of 100). A, Protein-matched cell lysates were analyzed by Western blotting with IL-23p19 Ab (top), followed by a reprobe with actin Ab (bottom). B and C, Cell supernatants were assayed by ELISA for IL-12/23p40 and IL-23. The graphs indicate data from three independent experiments. The error bars indicate SD.

 
IL-23 production does not require phagosomal escape

The kinetics of IL-23 secretion correspond roughly to the time required for Francisella to escape from the phagosome and begin replicating in the cytosol (18). This role opened the possibility that phagosomal escape may be required for IL-23 induction. It has been previously reported that certain host cell responses such as the processing of IL-1beta require phagosomal escape of F. novicida and potential interaction of the bacteria or bacterial proteins with host cell machinery (6, 10). To test whether phagosomal escape is required for IL-23 production, we infected THP-1 cells with the mglA mutant of Francisella. MglA is a critical transcription factor regulating the IglC operon of Francisella, and IglC is important for phagosomal escape (19, 20). As shown in Fig. 5, both p19 (Fig. 5A) and IL-23 (Fig. 5C) are produced following infection with this mutant strain. In fact, the IL-23 response was significantly greater in cells infected with F. novicida mutant mglA than in cells infected with F. novicida. These results indicate that phagosomal escape is not required for IL-23 induction.


Figure 5
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  		FIGURE 5. Phagosomal escape is not required for IL-23 production. THP-1 cells were infected for 24 h with F. novicida (FN) or F. novicida mutant mglA (MOI of 100). A, Protein-matched cell lysates were analyzed by Western blotting with IL-23p19 Ab (top blot), followed by a reprobe with actin Ab (bottom blot). Subunit p19 band intensities (from three independent experiments) were normalized to actin in the corresponding lanes and expressed as fold increase over untreated (lower graph). B and C, Cell supernatants were assayed by ELISA for IL-12/23p40 and IL-23. Data from three independent experiments were analyzed by Student’s t test. Error bars indicate SD. *, p ≤ 0.05.

 
IL-23 production depends on the activation of PI3K and NF-{kappa}B

We next examined the molecular mechanism of IL-23 production. Previous studies from our group and others have demonstrated that signaling molecules such as Erk MAPK and PI3K are activated in response to Francisella infection (8, 11, 12). To determine which signaling pathways were required for IL-23 production, we pretreated THP-1 cells with inhibitors of PI3K (LY294002) or MAPK kinase (U0126) followed by infection with F. novicida. Cell lysates and supernatants were harvested 24 h postinfection and analyzed by Western blotting for p19 and by ELISA for IL-12/23 p40 and IL-23 production. The results shown in Fig. 6A indicate that inhibition of PI3K or Erk did not completely abolish, but significantly dampened the production of the p19 subunit of IL-23. To ensure that the inhibitors used were indeed effective parallel Western blots of protein-matched lysates were performed to examine the phosphorylation status of Akt, as an indicator PI3K activity (Fig. 6B, upper) and the phosphorylation status of Erk (Fig. 6B, middle). Likewise, inhibition of PI3K or Erk resulted in a down-regulation of IL-12/23 p40 and IL-23 production (Fig. 6, C and D).


Figure 6
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  		FIGURE 6. Production of IL-23 in F. novicida-infected cells is PI3K-dependent. THP-1 cells were pretreated for 1 h with either DMSO or with inhibitors of the PI3K (FN-LY) or the Erk (FN-U0) pathways, and subsequently infected for 24 h with F. novicida (FN, MOI of 100). A, Protein-matched cell lysates were analyzed by Western blotting with IL-23p19 Ab (top blot), followed by a reprobe with actin Ab (bottom blot). p19 band intensities (from three independent experiments) were normalized to actin in the corresponding lanes and expressed as fold increase over untreated (lower graph). B, Protein-matched lysates were analyzed by Western blotting with Abs specific for phosphorylated Akt (upper), phosphorylated Erk (middle), and reprobed with actin Ab (lower). C and D, Cell supernatants were assayed by ELISA for IL-12/23 p40 and IL-23. Data from three independent experiments were analyzed by Student’s t test. The error bars indicate SD. *, p ≤ 0.05.

 
We extended these studies to ask whether NF-{kappa}B activation was required for IL-23 production. For this experiment, THP-1 cells were pretreated with SN50, an inhibitor of NF-{kappa}B (Fig. 7). Although this inhibition did not block the production of p19 (Fig. 7A), both IL-12/23 p40 and IL-23 were significantly lower in cells treated with the NF-{kappa}B inhibitor (Fig. 7, B and C).


Figure 7
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  		FIGURE 7. Production of IL-23 in F. novicida-infected cells is NF-{kappa}B-dependent. THP-1 cells were pretreated for 1 h with either dH2O or an inhibitor of NF-{kappa}B (SN50), and subsequently infected for 24 h with F. novicida (FN, MOI of 100). A, Protein-matched cell lysates were analyzed by Western blotting with IL-23p19 Ab (top), followed by a reprobe with actin Ab (bottom). Subunit p19 band intensities (from three independent experiments) were normalized to Actin in the corresponding lanes and expressed as fold increase over untreated (lower graph). B and C, Cell supernatants were assayed by ELISA for IL-12/23 p40 and IL-23. Data from three independent experiments were analyzed by Student’s t test. The error bars indicate SD. *, p ≤ 0.05.

 
F. novicida-induced IL-23 is functional

IL-23 is reported to induce NK cells to produce IFN-{gamma} (21). Therefore, to examine whether the IL-23 produced was biologically active, we tested the ability of F. novicida-induced IL-23 to elicit NK cell IFN-{gamma} production. For this test, conditioned medium from uninfected and F. novicida-infected PBM (Fig. 8A) or THP-1 cells (Fig. 8B) were applied to NK-92 cells with or without a p19-blocking Ab. NK cell supernatants were harvested 14 h later and tested for IFN-{gamma} production by ELISA. As shown in Fig. 8, treatment with medium from infected cells prompted an IFN-{gamma} response in the NK cells, which was largely blocked by the anti-p19 Ab (Fig. 8B). This result provides evidence that human monocytes produce functional IL-23 following infection with F. novicida.


Figure 8
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  		FIGURE 8. F. novicida-induced IL-23 from human monocytes is bioactive. PBM and THP-1 cells were infected with F. novicida for 24 h. Cell supernatants were harvested and designated as conditioned medium. A, NK-92 cells were incubated for 14 h in medium alone or in conditioned medium from uninfected and F. novicida-infected PBM (UT PBM sup. and FN PBM sup., respectively). NK-92 supernatants were subsequently analyzed by ELISA for IFN-{gamma} production. B, NK-92 cells were incubated for 14 h in medium alone, conditioned medium from uninfected THP-1 cells, or infected THP-1 cells in the presence of an isotype control or a p19 blocking Ab. NK92 supernatants were analyzed for IFN-{gamma} production. Data from three independent experiments were analyzed by Student’s t test. The error bars indicate SD. *, p ≤ 0.05.

 
The type A virulent Francisella strain SCHU S4 induces IL-23 production from human monocytes

Although F. novicida is highly virulent in mice and serves as a good model of Francisella infection, the question remained as to whether human monocytes would show the same IL-23 response when challenged with a virulent strain of Francisella. To answer this, we infected both PBM and THP-1 cells with the SCHU S4, a type A strain of F. tularensis for 24 h (MOI of 10 and 100). Cell lysates were analyzed by Western blotting for the p19 subunit of IL-23 and supernatants were analyzed by ELISA for IL-12/23 p40 and IL-23 production. As shown in Fig. 9, SCHU S4 also elicits an IL-23 response. These results suggest that IL-23 production by monocytes may be relevant to Francisella infection in humans.


Figure 9
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  		FIGURE 9. IL-23 is induced in human monocytes infected with F. tularensis SCHU S4. PBM (A–C) and THP-1 cells (D–F) were uninfected (UT) or infected for 24 h with F. tularensis SCHU S4 (MOI of 10 or 100). Protein-matched lysates were analyzed by Western blotting with IL-23p19 Ab (A and D, top). The same membranes were reprobed with actin Ab to ensure equal loading of protein (bottom). Cell supernatants were analyzed by ELISA for the p40 subunit of IL-12/23 (B and E) and IL-23 (C and G). Data from three independent infections were analyzed by Student’s t test. The error bars indicate SD. *, p ≤ 0.05.

 
We extended these findings to compare the attachment, phagocytosis and replication rates of F. novicida and SCHU S4 in THP-1 cells. First, attachment of bacteria to nonpermeabilized host cells was measured by fluorescent Ab labeling of F. novicida or SCHU S4 strain at 2 h postinfection followed by microscopy. Results indicated that both strains bound THP-1 cells comparably (0.16 and 0.12 bacteria/cell for F. novicida and SCHU S4 respectively; p = 0.07). Second, phagocytosis was measured by fluorescent Ab labeling of permeabilized THP-1 cells 2 h postinfection, and subtracting the number of organisms counted in the nonpermeabilized samples from the number of organisms counted in the permeabilized samples. Interestingly, these analyses revealed a significantly higher level of phagocytosis of F. novicida compared with SCHU S4 (0.25 and 0.026 bacteria/cell respectively; p = 0.04). Finally, intracellular replication rates were measured in THP-1 cells infected with 100 MOI F. novicida or SCHU S4. In this study, CFU assays were performed at 2, 12, and 24 h postinfection. These data from three independent infections indicated that the average replication rates between F. novicida and SCHU S4 are comparable (13.4- vs 10.6-fold increase between 2 and 12 h; 2.38- vs 2.78-fold increase between 12 and 24 h, respectively).

Minimal IL-23 production in F. novicida-infected mice

A recent study by Elkins et al. (22) showed that neutralization of p40, but not IL-12 p70, adversely affected host resistance to Francisella in a murine model of infection. It was proposed that this effect may be due either to the loss of IL-23 or some other independent function of IL-12 p40. Thus to test whether F. novicida infection would induce IL-23 production in vivo, mice were injected i.p. with PBS alone or with 200 CFU F. novicida. Serum samples, harvested 24 and 48 h postinfection, were analyzed by ELISA for IL-23. Only 4 of the 14 infected mice had detectable IL-23 in the serum (ranging from 80 to 155 pg/ml). One of seven mice injected with PBS showed detectable IL-23 (93 pg/ml).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study we have shown that human PBM, as well as the monocytic THP-1 cell line, secrete IL-23 in response to both F. novicida and the virulent SCHU S4 strain. This induction is largely dependent on PI3K and NF-{kappa}B activation. Furthermore, IL-23 production is not a consequence of phagosomal escape as has been shown for IL-1beta processing and release (6, 10) because the F. novicida mglA mutant strain also led to IL-23 production and release. In fact, IL-23 production in cells infected with F. novicida mutant mglA was significantly higher than in cells infected with F. novicida. This finding suggests that phagosomal escape, the ensuing bacterial replication, or some other MglA-dependent bacterial event may in fact down-regulate IL-23 production. This latter observation is consistent with the studies of Telepnev et al. (8) that demonstrated an enhanced host cell TNF-{alpha} response in cells infected with an IglC mutant of F. tularensis LVS compared with cells infected with wild-type bacteria. Also consistent with the latter suggestion, we have recently reported that murine macrophages infected with F. novicida mglA display enhanced NF-{kappa}B activation and enhanced proinflammatory cytokine production compared with cells infected with F. novicida (12).

Our array data and protein assays showed that IL-23 and not IL-12p70 was induced in human monocytes following F. novicida infection. IL-23 is an important component of the immune system, and works both hand in hand with IL-12p70 and on its own (17). Specifically, it can enhance the IFN-{gamma} response in NK cells in similar manner to IL-12p70 (21). IFN-{gamma}, in turn, has been shown to help prevent Francisella from escaping from the phagosome (4, 23, 24). Our data clearly demonstrate that the IL-23 produced by F. novicida-infected human monocytic cells is functional and can induce IFN-{gamma} production by NK cells (Fig. 8).

IL-23 has been shown to protect against pathogens by additional mechanisms as well. For example, IL-23 also independently promotes an IL-17 response in memory T cells (25), which serves to enhance the immune response by promoting production of proinflammatory cytokines and synergizing with other active proinflammatory activities. IL-23 enhances resistance against pathogens such as Cryptococcus neoformans (26), Klebsiella pneumoniae (27), and Mycobacterium tuberculosis (28), yet has also been implicated in autoimmune diseases (reviewed in Refs. 29 , 30). IL-23 was found to be preferentially increased in a rat model of autoimmunity (31), and has been linked to diseases such as brain inflammation (32), encephalomyelitis (33), colitis (34), multiple sclerosis (35), and joint inflammation (36), and possibly sepsis (37).

Within the context of infection with Francisella, human monocytes appear to secrete IL-23, but not IL-12. This response is also seen with the highly virulent SCHU S4 strain of F. tularensis (Fig. 9). Interestingly, human monocytic cell phagocytosis of F. novicida was significantly higher than that of SCHU S4, although intracellular replication rates were similar between the two strains. The difference in phagocytosis may be related to genetic differences between the two strains of Francisella and may account for the lower level of IL-23 production in SCHU S4-infected monocytic cells. Because our experiments were performed in RPMI 1640 containing heat-inactivated FBS, the difference in phagocytosis may also reflect a higher dependence on serum opsonins such as complement for SCHU S4 phagocytosis by these cells.

In contrast to the human host cell, murine bone marrow macrophages infected with an MOI of 100 F. novicida did not produce any detectable IL-23 (data not shown). Likewise, infection of mice with F. novicida also did not lead to detectable increases in serum IL-23 levels. We speculate that the inability of the murine host to produce sufficient amounts of IL-23 may account at least in part for the virulence of F. novicida in mice. We are currently investigating this notion.

To our knowledge, this report is the first in which IL-23 is produced by human monocytes in response to Francisella infection. This IL-23 is biologically active and induces the production of IFN-{gamma} from NK cells and may thus be important in host defense against human infection with F. tularensis. Our data further demonstrate that IL-23 is induced in a manner that is dependent on the activation of PI3K and NF-{kappa}B, and to a lesser extent on Erk. This study enhances our understanding of host response following Francisella infection and identifies a new target for further study.


   Acknowledgments
 
We thank Dr. John Gunn for the availability of Francisella isolates and Dr. Melissa Hunter for assistance with Bio-Plex assays.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by Grants U54-AI-057153, R01 AI059406, and P01 CA095426 from the National Institutes of Health. J.P.B. is supported by Institutional Training Grant T32CA090223 from the National Institutes of Health. Back

2 Address correspondence and reprint requests Dr. Susheela Tridandapani, Room 405B Davis Heart and Lung Research Institute, Department of Internal Medicine, The Ohio State University, 473 West 12th Avenue, Columbus, OH 43210. E-mail address: tridandapani.2{at}osu.edu Back

3 Abbreviations used in this paper: PBM, peripheral blood monocyte; MOI, multiplicity of infection. Back

Received for publication September 20, 2006. Accepted for publication January 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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