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The Journal of Immunology, 2008, 181, 5560 -5567
Copyright © 2008 by The American Association of Immunologists, Inc.
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Yersinia pestis Evades TLR4-dependent Induction of IL-12(p40)2 by Dendritic Cells and Subsequent Cell Migration1

Richard T. Robinson*, Shabaana A. Khader2,*, Richard M. Locksley{dagger}, Egil Lien{ddagger}, Stephen T. Smiley* and Andrea M. Cooper3,*

* Trudeau Institute, Saranac Lake, NY 12983; {dagger} Howard Hughes Medical Institute, Department of Medicine and Microbiology and Immunology, University of California, San Francisco, CA 94143; and {ddagger} Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
At the temperature of its flea vector (~20–30°C), the causative agent of plague, Yersinia pestis, expresses a profile of genes distinct from those expressed in a mammalian host (37°C). When dendritic cells (DC) are exposed to Y. pestis grown at 26°C (Y. pestis-26°), they secrete copious amounts of IL-12p40 homodimer (IL-12(p40)2). In contrast, when DCs are exposed to Y. pestis grown at 37°C (Y. pestis-37°), they transcribe very little IL-12p40, which is secreted as IL-12p40 monomer (IL-12p40). Y. pestis-26° also induces migration of DCs to the homeostatic chemokine CCL19, whereas Y. pestis-37° does not; migratory DCs are positive for IL-12p40 transcription and secrete mostly IL-12(p40)2; DCs lacking IL-12p40 do not migrate. Expression of acyltransferase LpxL from Escherichia coli in Y. pestis-37° results in the production of a hexa-acylated lipid A, also seen in Y. pestis-26°, rather than tetra-acylated lipid A normally seen in Y. pestis-37°. The LpxL-expressing Y. pestis-37° promotes DC IL-12(p40)2 production and induction of DC migration. In addition, absence of TLR4 ablates production of IL-12(p40)2 in DC exposed to Y. pestis-26°. The data demonstrate the molecular pathway by which Y. pestis evades induction of early DC activation as measured by migration and IL-12(p40)2 production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Yersinia pestis, a Gram-negative facultative intracellular bacterium, is the causative agent of plague (1). Although it is primarily a rodent pathogen, Y. pestis can be transmitted intradermally to humans through the bite of an infected flea (2). It is believed that through this mode of transmission and also aerosol transmission, Y. pestis was responsible for the Black Death in the Middle Ages (3, 4). Plague is a global disease and poses an increased threat to world health due to the rise of both drug resistance and the ability to aerosolize this pathogen and create a biological weapon (5, 6, 7, 8). Understanding the biology of Y. pestis is therefore important, and recent work has resulted in the sequencing of a number of virulent Y. pestis strains (9) and definition of the complete Y. pestis virulence proteome (10). Currently, there is no safe and efficacious vaccine against plague (11, 12), and rational design of such a vaccine depends on knowledge of the factors that impact immunity to Y. pestis. In this paper, we investigate the interaction of Y. pestis with dendritic cells (DC),4 the cells that are pivotal in the initiation of immune responses.

Although induction of humoral immunity has been the focus of most plague vaccine research, both cellular and humoral immunity can mediate protection (12). A critical step in the generation of both cellular and humoral responses is the activation of DCs to a state that allows them to migrate to the lymphoid follicle, wherein they promote acquired immune responses (13). This activation is initiated as a result of the DCs being exposed to microbial components, and DC migration is mediated by the chemokines CCL19 and CCL21, acting on their receptor, CCR7 (14, 15, 16, 17). We recently reported that DCs deficient in IL-12p40 fail to migrate toward CCR7 ligands upon exposure to bacterial stimuli and that migration is rescued by the addition of IL-12p40 homodimer (IL-12(p40)2) but not IL-12p70 or denatured IL-12(p40)2 (18). Cells that migrate after s.c. vaccination also express IL-12p40 alone, suggesting that expression of IL-12(p40)2 by DCs is a key component of the initiation of immunity (19). Herein we describe a mechanism for induction of both IL-12(p40)2 production and chemokine responsiveness in response to Y. pestis.

Because Y. pestis modulates its gene expression in a temperature-dependent manner (20) and targets DC on infection (21, 22), we compared the ability of Y. pestis grown at 26°C (Y. pestis-26°) or 37°C (Y. pestis-37°) to induce IL-12(p40)2 production and chemokine responsiveness in DCs. Whereas Y. pestis-26° is a potent inducer of IL-12(p40)2, Y. pestis-37°-stimulated DCs secrete only low levels of IL-12p40. Further, whereas Y. pestis-26° induces responsiveness of DCs to chemokine in an IL-12p40-dependent manner, Y. pestis-37° does not. The ability of Y. pestis-37° to evade both IL-12(p40)2 induction and chemokine responsiveness is overcome when Y. pestis-37° is forced to express hexa-acylated rather than only tetra-acylated lipid A. Finally, the induction of IL-12(p40)2 secretion and chemokine responsiveness is dependent on TLR4. We have therefore demonstrated a molecular pathway by which Y. pestis evades DC activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

All mice were bred at the Trudeau Institute (Saranac, NY) and were treated according to National Institutes of Health (Bethesda, MD) and Trudeau Institute Animal Care and Use Committee guidelines. C57BL/6 (B6) and B6-il12b mice were originally purchased from The Jackson Laboratory. IL-12p40-IRES-YFP reporter mice (Yet40 mice) expressing a yellow-fluorescent protein upon IL-12p40 transcription were a gift from Dr. Richard M. Locksley and have been described (19). Mice deficient in the TLR4 gene (hereafter referred to as tlr4–/– mice; Ref. 23) were purchased from Oriental BioService.

Bacteria

Pigmentation-negative Y. pestis strains KIM5 (pCD1+, pMT+, pPCP+) and KIM6 (pCD1–, pMT+, pPCP+) were obtained from Robert R. Brubaker (Michigan State University, Ann Arbor, MI). The pigmentation-negative strain of Y. pestis CO92{Delta}pgm and pigmentation-positive Y. pestis strain KIM10+caf1– (pCD1–, pMT+, pPCP–, F1–) were kindly provided by Celine Pujol and James B. Bliska (State University of New York, Stony Brook, NY). Y. pestis strain KIM5-pLpxL was created by transforming KIM5 with an pLpxL-containing plasmid as previously described (24). All Y. pestis strains were grown overnight at 26°C in heart infusion broth (Difco Laboratories) supplemented with 2.5 mM CaCl2. Subsequently, they were diluted to OD620 0.1 and grown at either 26°C or 37°C for 3 h. Then, bacteria were quantified by measuring the OD at 620 nm (1 OD unit = 5.8 x 108 CFU), washed with PBS, inactivated by heating at 60°C for 1 h, and frozen. Heat inactivation was performed to eliminate the ability of Y. pestis grown at 26°C to alter its gene expression/surface phenotype after its addition to a 37°C culture.

Bone marrow-derived DC (BMDC) culture

DCs were generated from the bone marrow of C57BL/6, Yet40, and B6.tlr4–/– mice basically as described previously (25). Briefly, cultures were harvested at day 6 and either sorted by CD11c expression using magnetic beads (Miltenyi Biotec; all protein detection experiments) or used without sorting (chemotaxis assays or Yet40 YFP expression experiments). Generation of CD11c+ immature DC (i.e., low expression of cell surface markers, CD80, CD86, I-Ab, CD40) was similar in terms of numbers and frequency between wild-type and B6.tlr4–/–-derived populations (data not shown).

In vitro Y. pestis exposure

BMDCs were resuspended in cDMEM at a concentration of 2.5 x 105/ml, and 2 ml were pipeted into individual wells of a 24-well plate (i.e., 5 x 105 total cells). Aliquots of Y. pestis (stock, 1 x 109/ml) were thawed and serially diluted in cDMEM; 2 ml were added to 2 ml of BMDCs to generate 40 multiplicity of infection (MOI), 20 MOI, 10 MOI, and 5 MOI cultures. Medium alone (2 ml) was used for 0 MOI cultures. After the indicated incubation period at 37°C, the supernatants were collected and used for ELISA or Western blot analysis. Cells were centrifuged and washed twice with FACs buffer or chemotaxis buffer, depending on the subsequent aims of each individual experiment. Some cells received IL-12(p40)2 (R&D Systems) as previously described (18).

Flow cytometry

All Abs used for flow cytometric analysis were purchased from BD Pharmingen or eBiosciences. After exposure to varying MOIs of Y. pestis, BMDCs were washed with FACS buffer (2% FCS in PBS), Fc blocked, and stained with Abs that recognize CD11c (clone HL3) and I-Ab (clone AF6-120.1). YFP expression by Yet40 DCs was determined by gating CD11c+I-Ab+ cells; distinguishing YFP expression from autofluorescence was performed using nontransgenic control BMDCs. For all surface markers, positive staining was established using appropriate isotype controls. Data were acquired using a FACSCalibur (BD Biosciences) and analyzed with FlowJo software (Tree Star).

Chemotaxis measurement

BMDCs were activated with varying MOIs of Y. pestis as indicated, and their ability to respond to the chemokine CCL19 (25 ng/ml; R&D Systems) was determined using the previously described in vitro transwell chemotaxis assay (26).

Measurement of secreted IL-12p40

ELISA was used to measure the levels of IL-12p40, IL-23, and IL-12p70 present in the BMDC supernatants. Supernatants for Western analysis were collected and concentrated using ultrafiltration centrifugation in iCon concentrators (Pierce) with a molecular weight cutoff of 9K. Nondenaturing sample buffer (Invitrogen) was added to each concentrate and then loaded onto a 4–16% gradient Bis-Tris gel. Electrophoresis and transfer to a polyvinylidene difluoride membrane was performed with standard procedures. Blots were subsequently blocked (10% powdered milk in Tris-buffered saline) and probed first with anti-IL-12p40 (clone C15.6) followed with polyclonal HRP-conjugated anti-rat IgG. Cell lysate from splenocytes stimulated for 18 h with 5 µg/ml Con A (Sigma-Aldrich) was used to provide size standards of IL-12p40, IL-23, IL-12p70, and IL-12(p40)2.

Statistical analysis

The significance of any difference between the means of experimental groups was determined using Student’s t test. Means were considered different if p was ≤0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Y. pestis-26°, but not Y. pestis-37°, induces IL-12(p40)2 production

Because Y. pestis-37° expresses a different transcriptional pattern than Y. pestis-26°, we compared the ability of these two preparations to activate DCs. To do this, we exposed BMDC from Yet40 mice to both Y. pestis-37° and Y. pestis-26° and assessed transcription of IL-12p40. BMDCs exposed to varying MOI of KIM5 Y. pestis-26°-expressed YFP indicating a significant increase in IL-12p40-transcription among CD11c+I-Ab+ cells compared with unstimulated controls (Fig. 1A). KIM5 Y. pestis-37°did not induce IL-12p40 transcription above baseline (Fig. 1A). These results were dose dependent and observable across multiple experiments as shown by the fold change in frequency of YFP expressing cells (Fig. 1B). IL-12p40 is a subunit of the cytokines IL-12p70 and IL-23 as well as being secreted as a homodimer, IL-12(p40)2. To assess which IL-12p40-dependent cytokines were produced by Y. pestis-exposed DCs, we sorted C57BL/6 BMDC based on CD11c expression, stimulated them with Y. pestis-26°, and measured cytokine in the cell supernatant by ELISA. Whereas a copious amount of IL-12p40 was induced in a time-dependent manner by Y. pestis-26°, only a small amount of IL-23 and no IL-12p70 were induced (Fig. 1C). The IL-12p40 response was much stronger in the Y. pestis-26° than in the Y. pestis-37°-stimulated cells (Fig. 1D) and although both Y. pestis-26° and Y. pestis 37° failed to induce significant IL-12p70 (Fig. 1E) both induced a small amount of IL-23 (Fig. 1F).


Figure 1
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  		FIGURE 1. KIM5 Y. pestis-26°, but not KIM5 Y. pestis-37°, elicits DC IL-12(p40)2 production. A, Yet40 BMDCs stimulated with increasing MOI of KIM5 Y. pestis cultured at either 26°C or 37°C. Cells were gated based on size and expression of CD11c and I-Ab, and representative histograms show the level of IL-12p40 (YFP) expression in CD11c+I-Ab+ cells as detected by flow cytometry. B, The fold change in frequency of CD11c+I-Ab+ DCs transcribing IL-12p40 (YFP) in response to Y. pestis-26°C ({blacksquare}) or Y. pestis-37°C ({square}) relative to unstimulated DCs ({Delta}YFP = [YFP expressed in response to Y. pestis]/[YFP expressed in response to media alone]) was determined by flow cytometry for 3 experiments. Data points show the mean change in frequency ± SEM for all three experiments; for the difference between induction by Y. pestis-37°C and Y. pestis-26°C, *, p = 0.05, **, p = 0.005 by Student’s t test. C, B6 CD11c+ BMDCs were cultured with 5 MOI of Y. pestis-26°, and the level of IL-12p40 protein in the supernatant was determined by ELISA over time. Data points represent the mean ± SD of duplicate readings and show one experiment representative of three; for the difference between induction of cytokine relative to the first time point (0.5 h) by Y. pestis-26°C, *, p = 0.05, **, p = 0.005, ***, p = 0.0005 by Student’s t test. D–F, B6 CD11c+ BMDCs were cultured with increasing MOIs of Y. pestis-26° or Y. pestis-37° for 3 h, and the level of IL-12p40 (D), IL-12p70 (E) and IL23 (F) was determined by ELISA. Points represent the mean ± SD of duplicate readings and show one experiment representative of three (D–F) for the difference between induction by Y. pestis-37°C and Y. pestis-26°C, *, p = 0.05, by Student’s t test. G, Proteins in concentrated supernatants from B6 CD11c+ BMDCs stimulated with Y. pestis-26°C (i) or Y. pestis-26°C and Y. pestis-37°C (ii) for 3 h were separated by size on a nondenaturing gel and probed with Ab to IL-12p40; cell lysates from Con A-treated B6 and IL-12p40–/– splenocytes were run simultaneously as size indicators and as a positive and negative control, respectively.

 
Although the IL-12p40 response was much stronger following Y. pestis-26° stimulation, IL-12p40 could be induced by Y. pestis-37° (Fig. 1E). To determine the nature of the secreted IL-12p40 after Y. pestis stimulation, we concentrated the DC supernatants, separated the proteins by size on a nondenaturing gel, and probed the subsequent blot with an anti-IL-12p40 mAb. IL-12(p40)2 was the dominant IL-12p40-containing cytokine produced by DC exposed to KIM5 Y. pestis-26°; in contrast, DCs stimulated with KIM5 Y. pestis-37° secrete IL-12p40 in monomeric form (Fig. 1Gii). The IL-23 and IL-12p70 (measurable by ELISA) was below the level of detection by this method (Fig. 1Gi). Lysate from Con A-stimulated B6 splenocytes was used to provide size markers for the IL-12p40-containing proteins IL-12p40, IL-12(p40)2, IL-12p70, and IL-23 (Fig. 1G); these were all absent from lysate of identically stimulated IL-12p40–/– splenocytes (Fig. 1Gi).

Y. pestis-26°, but not Y. pestis-37°, elicits CCL19-dependent chemotaxis of IL-12(p40)2-producing DCs

In addition to IL-12p40 production, DCs respond to bacterial exposure by becoming responsive to homeostatic chemokines (18). We therefore tested whether DC chemokine responsiveness was differentially induced by exposure to Y. pestis-26° and Y. pestis-37°. Stimulation of the DCs with KIM5 Y. pestis-26° significantly increased their migration toward CCL19 in a MOI-dependent manner (Fig. 2A). In contrast, regardless of the MOI used, DCs stimulated with KIM5 Y. pestis-37° failed to migrate toward CCL19 (Fig. 2A). DCs that migrated across the transwell were confirmed by flow cytometry to be mostly CD11c+ (data not shown). Y. pestis-26° therefore is able to stimulate DC chemokine responsiveness whereas Y. pestis-37° cannot. To address whether the migration was dependent on IL-12(p40)2, we used DCs from mice lacking il12b and therefore unable to generate IL-12p40 (18). DCs lacking il12b were unable to migrate to CCL19 when stimulated with Y. pestis-26°, however, responsiveness could be restored by the addition of IL-12(p40)2 but not denatured IL 12(p40)2 (Fig. 2B).


Figure 2
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  		FIGURE 2. KIM5 Y. pestis-26°, but not KIM5 Y. pestis-37°, elicits CCL19-dependent chemotaxis of IL-12(p40)2-producing DCs. A, DCs were stimulated with either KIM5 Y. pestis-26° ({blacksquare}) or KIM Y. pestis-37° ({square}) at varying MOIs and the ability of the stimulated DCs to migrate toward a CCL19 gradient was determined by measuring the number of DCs that migrate to CCL19 relative to the number that migrate to media alone (chemotaxis index). Data points represent the mean of duplicate values and show one experiment representative of 3 total; for the difference between chemotaxis index induced by Y. pestis-37° relative to Y. pestis-26°, *, p = 0.05, **, p = 0.005, by Student’s t test. B, DCs from B6 and B6.il12b–/– mice were stimulated with either KIM5 Y. pestis-26° ({blacksquare}, B6; •, B6.il12b–/–), KIM Y. pestis-37° ({square}, B6), KIM5 Y. pestis-26° plus IL-12(p40)2 ({circ}, B6.il12b–/–) or KIM5 Y. pestis-26° plus denatured IL-12(p40)2 ({diamond}, B6.il12b–/–). The chemotaxis index was determined as for A. Data points represent the mean and SD of duplicate values and show one experiment representative of two; for the difference between chemotaxis index induced by Y. pestis-26° in all experimental groups relative to B6.il12b–/– DCs, *, p = 0.05, **, p = 0.005 by Student’s t test. C, Yet40 BMDCs were stimulated with KIM5 Y. pestis-26° and exposed to a CCL19 gradient. The frequency of CD11c+ cells that were transcribing IL-12p40 (YFP+) was determined by flow cytometry for the cells before they were exposed to CCL19 (Figure 2), for the cells remaining in the upper chamber (i.e., nonresponsive; {square}) and for cells that had migrated toward CCL19 (i.e., lower chamber; {blacksquare}). Bars represent the mean of duplicate values and show one experiment representative of two; for the difference in values relative to prechemotaxis values, *, p = 0.05, **, p = 0.005, ***, p = 0.0005 by Student’s t test. D, Yet40 BMDCs were either left unstimulated (0 MOI) or stimulated with KIM5 Y. pestis-26° (20 MOI) for 3 h and placed in a CCL19 gradient for 90 min. The nature of the IL-12p40-containing protein released into the supernatant during the 90-min incubation in the upper and lower chamber was determined as described for Fig. 1G; data are from one experiment representative of two.

 
To determine whether the Y. pestis-stimulated DCs that migrated to CCL19 also transcribed IL-12p40, we challenged Yet40 DCs with the different bacterial strains and measured YFP in CD11c+ cells that had migrated to the CCL19 in the lower chamber. The increased percentage of CD11c+ YFP+ cells in the lower chamber confirmed that chemotaxis toward CCL19 resulted in an enrichment of IL-12p40-transcribing cells (Fig. 2C).

To determine the dominant form of secreted IL-12p40 among migratory DCs, we performed nondenaturing Western blot analysis of the chemotaxis buffer that remained in the upper and lower chambers of the transwell following the collection of Yet40 cells for the experiment shown in Fig. 2C. In the unstimulated Yet40 DC cultures (0 MOI; Fig. 2D), IL-12p40 alone was observed in both upper and lower chambers. However, stimulation with 20 MOI KIM5 Y. pestis-26° resulted in the appearance of IL-12(p40)2 in the buffer (20 MOI; Fig. 2D). When CCL19 was present in the lower chamber, IL-12(p40)2 was preferentially observed in the lower chamber. We conclude from that the dominant form of secreted IL-12p40 associated with migratory DCs is IL-12(p40)2.

The increase in CD11c+YFP+ cells was not due to an ability of CCL19 to increase IL-12p40 transcription because coincubation of Yet40 DCs with CCL19 did not significantly enhance YFP expression (data not shown).

KIM5 Y. pestis-37° evasion of DC IL-12p40 production occurs with other strains of Y. pestis and is independent of pCD1 and caf1

To determine whether the ability to evade DC activation was a general feature of Y. pestis, we compared the ability of KIM5 to induce IL-12p40 transcription with that of strain CO92{Delta}pgm, which derives from a distinct Y. pestis biovar. We found that CO92{Delta}pgm Y. pestis-37° induced a similar low level of IL-12p40 transcription (YFP expression) in CD11c+I-Ab+ cells as the KIM5 Y. pestis-37° (Fig. 3) and that this was significantly less than that seen for KIM5 Y. pestis-26° and CO92{Delta}pgm Y. pestis-26° (Fig. 3).


Figure 3
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  		FIGURE 3. Suppression of DC IL-12p40 production by Y. pestis-37° is independent of pCD1 and caf1. Yet40 BMDCs were stimulated with pCD1+ caf1+ Y. pestis strains KIM5 Y. pestis-26°, KIM5 Y. pestis-37°, CO92{Delta}pgm Y. pestis-26°, and CO92{Delta}pgm Y. pestis-37° (top), or the pCD1–caf1+ strain KIM6 Y. pestis-26°C, KIM6 Y. pestis-37°C or the pCD1–caf1– strain KIM10 Y. pestis-26°C, KIM10 Y. pestis-37°C (bottom). Cells were gated based on size and expression of I-Ab, and representative histograms show the level of IL-12p40 (YFP) expression in CD11c+I-Ab+ cells as detected by flow cytometry. Dot plots show representative values for three independent experiments.

 
Several genes important to Y. pestis pathogenesis are known to be transcriptionally up-regulated upon transition from 26°C to 37°C. These include a type III secretion system and the F1 capsular protein. The type III secretion system is encoded by the pCD1 plasmid and injects host cells with effector proteins that block phagocytosis and down-regulate proinflammatory signals (1, 22, 27). The F1 protein, on the other hand, is encoded by the caf1 gene on pMT1 and forms a protective capsule around Y. pestis and suppresses phagocytosis (1, 28, 29, 30, 31). To test whether either pCD1 or caf1 is required for Y. pestis-37° evasion of DC p40 production, we exposed Yet40 BMDCs to Y. pestis strains lacking pCD1 (KIM6) or lacking both pCD1 and a functional caf1 gene (KIM10+caf1–) grown at either 26°C or 37°C. The percentage of CD11c+I-Ab+YFP+ cells induced upon exposure was equally low among all strains grown at 37°C (Fig. 3, bottom) relative to the same strains grown at 26°C. These data suggest that KIM5 Y. pestis-37° evasion of DC IL-12p40 production is independent of pCD1 and caf1 and that this activity can be generalized to several strains of Y. pestis.

Y. pestis-37° evasion of DC IL-12p40 induction is overcome by expression of hexa-acylated lipid A

It has been demonstrated that upon transition from 26°C to 37°C, Y. pestis modifies the structure of its cell surface lipid A (24, 32, 33). Lipid A is the immunostimulatory moiety of LPS, and at lower temperatures the acyl transferase LpxP enables the biosynthesis of hexa-acylated lipid A. Upon transition to 37°C, Y. pestis LpxP is presumably not produced and/or is inactivated and lipid A becomes mostly tetra-acylated (24, 32, 33). Recently, a strain of Y. pestis (Y. pestis KIM5-pLpxL) expressing Escherichia coli acyltransferase LpxL was generated. The enzyme activity of E. coli LpxL in KIM5 appears temperature insensitive; thus, the production of tetra-acylated lipid A of Y. pestis KIM5-pLpxL does not occur upon growth at 37°C (24). To test whether LpxL expression alters the ability of the Y. pestis KIM5 to evade DC activation, we grew KIM5-pLpxL at 26°C and 37°C and examined the ability of the bacteria to activate DCs. CD11c+I-Ab+ cells exhibited up-regulation of IL-12p40 transcription when exposed to KIM5-pLpxL grown at 26°C (KIM5pLpxL Y. pestis-26°); this was at levels similar to that elicited by KIM5 Y. pestis-26° (Fig. 4A). When exposed to KIM5-pLpxL grown at 37°C (KIM5pLpxL Y. pestis-37°), CD11c+I-Ab+ cells remained able to transcribe IL-12p40 (Fig. 4A). When BMDCs were sorted by CD11c expression and stimulated with bacteria, both KIM5-pLpxL-26°C and KIM5pLpxL Y. pestis-37° induced IL-12p40 production at levels higher than that induced by KIM5 Y. pestis-37° (Fig. 4B); this IL-12p40 was secreted primarily as IL-12(p40)2 (Fig. 4C). The increased IL-12p40 production elicited by KIM5pLpxL Y. pestis-37°, as compared with KIM5 Y. pestis-37°, was associated with the restored ability of DCs to migrate toward CCL19 (Fig. 4D). Thus, the temperature sensitivity of LpxP is implicated in the ability of Y. pestis to evade activation of IL-12p40 production and migration of DCs.


Figure 4
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  		FIGURE 4. Y. pestis stimulation of DC IL-12(p40)2 production is LPS lipid A- and TLR4-dependent. A, Yet40 BMDCs were stimulated with either KIM5 Y. pestis-26° or KIM5 Y. pestis-37° (top), or KIM5pLpxL Y. pestis-26° or KIM5pLpxL Y. pestis-37° (bottom). Cells were then gated based on size and expression of I-Ab, and histograms show the level of IL-12p40 (YFP) expression in CD11c+I-Ab+ cells as detected by flow cytometry. Dot plots show representative values for three independent experiments. B–D, B6 CD11c+ BMDCs were stimulated with KIM5 Y. pestis-26° ({blacksquare}), KIM5 Y. pestis-37°({square}), KIM5pLpxL Y. pestis-26° (•), or KIM5pLpxL Y. pestis-37° ({circ}). B, The level of IL-12p40 in the supernatant was determined by ELISA. Data points represent the mean and SD of duplicate values and show one experiment representative of three; for the difference between values induced by all experimental groups relative to Y. pestis-37°, *, p = 0.05, by Student’s t test. C, The nature of IL-12p40-containing proteins was determined as described for Fig. 1G in supernatants of DC stimulated by the indicated strains. Data represent one experiment representative of two. D, The CCL19-dependent chemotaxis of the stimulated DCs was determined as described for Fig. 2A. Data points represent the mean and SD of duplicate values and show one experiment representative of three; for the difference between values induced by all experimental groups relative to Y. pestis-37°, *, p = 0.05, **, p = 0.005 by Student’s t test. E, Both B6 (open symbols) and tlr4–/– (closed symbols) CD11c+ BMDCs were stimulated with KIM5 Y. pestis-26° ({square}, {blacksquare}), KIM5pLpxL Y. pestis-26° ({circ}, •) or KIM5pLpxL Y. pestis-37° ({triangleup}, {blacktriangleup}) and the level of IL-12p40 produced measured by ELISA. Because KIM5 Y. pestis-37° failed to induce IL-12p40 ({diamond}), it was not tested against tlr4–/–DC. Data points represent the mean and SD of duplicate values and show one experiment representative of two; for the difference between values induced by all experimental groups relative to B6 DCs stimulated by Y. pestis-37°, *, p = 0.05 by Student’s t test. F, The nature of IL-12p40-containing proteins in the supernatants from B6 and TLR4–/– CD11c+ BMDCs stimulated with 40 MOI KIM5 Y. pestis-26° was analyzed using nondenaturing Western blot analysis as for Fig. 1G. Data shown are one experiment representative of two.

 
The above result implicated LPS induced signaling as a factor in initiation of DC IL-12(p40)2 production. It has been repeatedly demonstrated that TLR4 is the pattern recognition receptor for Gram-negative bacterial LPS (34). Therefore, we generated BMDCs from B6.tlr4–/– mice and exposed CD11c+ cells to KIM5 Y. pestis-26°, KIM5 Y. pestis-37°, KIM5-pLpxL-26°, and KIM5-pLpxL-37°. In contrast to cells from wild-type control mice, cells from B6.tlr4–/– mice secreted very low levels of IL-12p40 in response to all stimuli (Fig. 4E). Thus, the ability of Y. pestis to induce IL-12p40 production in DCs requires TLR4. The induction of chemokine responsiveness seen for KIM5 Y. pestis-26°(Fig. 2, A and B) was not seen when TLR4-deficient DCs were stimulated with KIM5 Y. pestis-26° (data not shown).

Having determined that the principal IL-12p40-containing cytokine produced by DCs exposed to Y. pestis-26°C is IL-12(p40)2 and that IL-12p40 production is dependent on TLR4, we tested whether TLR4 was required for the change from IL-12p40 to IL-12(p40)2 production. Supernatants were collected from wild-type and TLR4-deficient CD11c+ BMDCs stimulated with KIM5 Y. pestis-26°C and were analyzed by nondenaturing Western blot analysis for IL-12p40-containing cytokines. Although stimulation of wild-type DCs with KIM5 Y. pestis-26°C results in the production of IL-12(p40)2, this cytokine does not appear in the supernatants of TLR4-deficient DCs, with IL-12p40 monomer being the only form of IL-12p40 produced (Fig. 4F). Thus, the ability of DC to produce IL-12(p40)2 and become responsive to chemokines following Y. pestis exposure is dependent on TLR4.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
DCs are pivotal in the initiation of immunity and pathogens have therefore developed mechanisms to alter DC function. Our data demonstrate that although Y. pestis grown at 26°C (i.e., a surrogate of flea vector temperature) is capable of activating DCs, it is able to evade activating DCs when growing at 37°C (i.e., akin to growth within the vertebrate host or when transmitted via the pulmonary route). We show that the ability of Y. pestis to initiate DC activation is determined by the lipid A structure and depends on the pattern recognition receptor TLR4. We further show that evasion of DC activation can be overcome if Y. pestis is modified to express hexa- rather than tetra-acylated lipid A, and this supports the hypothesis that modulation of the lipid A structure is a major virulence determinant (24).

Herein we have measured DC activation by the induction of IL-12p40 transcription and the initiation of responsiveness to homeostatic chemokines. We have previously shown that, upon exposure to bacteria and bacterial products, these two events are induced early and that chemokine responsiveness is dependent on the induction of IL-12p40 (18). We extend this result to Y. pestis and show here for the first time that Y. pestis is an inducer of IL-12(p40)2 and that this induction is required for bacterially induced responsiveness to chemokines. In the previous study, we characterized neither the nature of the IL-12p40 being released by the DCs nor the receptor pathway by which it was being induced. Our data show that the induction of IL-12p40 transcription that occurs in response to Y. pestis results in the release of IL-12(p40)2 in large excess relative to any other IL-12p40 containing cytokine and that this response is rapid. Further, our observation that TLR4 and lipid A structure are essential for production of IL-12(p40)2 provides the beginning of the molecular pathway by which this underappreciated cytokine is induced. Finally, the observation that it is the IL-12p40-transcribing DCs that migrate and that IL-12(p40)2 is enriched in the supernatant of DCs that have migrated provides support for the hypothesis that IL-12(p40)2 plays a role in bacterially induced migration of DCs (18).

The impact of lipid A modulation on virulence is clear as the hexa-acylated lipid A molecule is much more stimulatory than the tetra-acylated form (24). Further, forcing Y. pestis to maintain expression of the hexa-acylated lipid A at 37°C results in reduced virulence and efficient induction of protection against subsequent Y. pestis challenge (24). Our data demonstrate that the nature of the lipid A influences the ability of Y. pestis to activate DCs. Because the flea-borne bacteria need to initiate infection of the lymph nodes, the ability of the hexa-acylated lipid A to initiate DC activation and migration would allow efficient colonization of the lymph node. On the other hand, the temperature-sensitive induction of the tetra-acylated lipid A could allow the bacteria to remain and grow within the lymph node without stimulating immunity, thereby enabling development of very high bacteremia. In addition, the inability to induce DC migration when delivered by the aerosol route (i.e., bacteria grown at 37°C) may promote the establishment of disease in the lung. Although DCs represent a likely population to mediate transport of Y. pestis, it is also possible that other cell types such as neutrophils or monocytes could mediate transport of this acute inflammatory pathogen.

The regulation of transcription of IL-12p40-containing cytokines has been extensively studied, and the fact that LPS and other microbial lipoproteins can trigger IL-12(p40)2 production through TLR signaling pathways is a well-described phenomenon (35, 36). Although activation of a single TLR usually results in IL-12(p40)2 production, the presence of other pattern recognition receptor ligands or signals from activated T cells are required for IL-12p70 production (35). Our data demonstrate that TLR4 and hexa-acylated lipid A-dependent activation of DC (likely via direct ligation of TLR4 by hexa-acylated lipid A of Y. pestis), results in very potent production of IL-12(p40)2, and we correlate that expression with the migration of the DCs. These data provide further support to the hypothesis that IL-12(p40)2 plays an agonistic role in initiation of immunity to bacteria (37).

Understanding of IL-12(p40)2 biology is important in light of its newly described activities (37). Specifically, as the two subunits of IL-12(p40)2 are held together by a disulfide bond, it will be important to understand how TLR4 signaling directs the activity of the enzymes that catalyze disulfide bond formation. In eukaryotes, disulfide bonds occur in the endoplasmic reticulum under the direction of oxidoreductases (38). One such enzyme is protein disulfide isomerase which is expressed in DCs and which has been demonstrated to enhance IL-12p40 dimerization (39, 40). Whether protein disulfide isomerase activity is impacted by TLR4 and whether this impacts the level of IL-12p40 dimerization should be investigated.

In conclusion, our data demonstrate that the innate induction of IL-12(p40)2 by microbial challenge is required for DC migration, and we also describe a mechanism by which a pathogen minimizes such migration. We also provide a mechanistic insight into why lipid A of Y. pestis grown at vector temperature is more inflammatory than the lipid A of Y. pestis grown at vertebrate host temperature (24).


   Acknowledgments
 
We thank Celine Pujol, James Bliska, and Robert Brubaker for Y. pestis strains and Lawrence Kummer for technical assistance.


   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 Trudeau Institute Training Fellowship T32 AI49823 (to R.T.R.) as well as National Institutes of Health Grants R01-AI67723 (to A.M.C.), R01-A1057588 (to E.L.), R01-AI61577 (to S.T.S.), R01-AI26918 (to R.M.L.), and U54-AI057158-Lipkin, a New York Community Trust-Heiser Fund Fellowship and Career Development Award AI057158 (North East Biodefense Center-Lipkin) (to S.A.K.). Back

2 Current address: Division of Medicine, Allergy and Immunology, Children’s Hospital of Pittsburgh, Pittsburgh, PA 15213. Back

3 Address correspondence and reprint requests to Dr. Andrea M. Cooper, Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY 12983. E-mail address: acooper{at}trudeauinstitute.org Back

4 Abbreviations used in this paper: DC, dendritic cell; IL-12(p40)2, IL-12p40 homodimer; Y. pestis-26°, Y. pestis grown at 26°C; Y. pestis-37°, Y. pestis grown at 37°C; YFP, yellow-fluorescent protein; Yet40 mice, IL-12p40-IRES-YFP reporter mice; BMDC, bone marrow-derived DC; MOI, multiplicity of infection. Back

Received for publication June 16, 2008. Accepted for publication August 8, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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