of April 10, 2019.This information is current as

and Subsequent Cell Migration by Dendritic Cells2Induction of IL-12(p40)

Evades TLR4-dependentYersinia pestis

CooperLocksley, Egil Lien, Stephen T. Smiley and Andrea M. Richard T. Robinson, Shabaana A. Khader, Richard M.

http://www.jimmunol.org/content/181/8/5560doi: 10.4049/jimmunol.181.8.5560

2008; 181:5560-5567; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/181/8/5560.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2008 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Yersinia pestis Evades TLR4-dependent Induction ofIL-12(p40)2 by Dendritic Cells and Subsequent Cell Migration1

Richard T. Robinson,* Shabaana A. Khader,2* Richard M. Locksley,† Egil Lien,‡

Stephen T. Smiley,* and Andrea M. Cooper3*

At the temperature of its flea vector (�20–30°C), the causative agent of plague, Yersinia pestis, expresses a profile of genes distinctfrom 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 inducesmigration of DCs to the homeostatic chemokine CCL19, whereas Y. pestis-37° does not; migratory DCs are positive for IL-12p40transcription and secrete mostly IL-12(p40)2; DCs lacking IL-12p40 do not migrate. Expression of acyltransferase LpxL fromEscherichia coli in Y. pestis-37° results in the production of a hexa-acylated lipid A, also seen in Y. pestis-26°, rather thantetra-acylated lipid A normally seen in Y. pestis-37°. The LpxL-expressing Y. pestis-37° promotes DC IL-12(p40)2 production andinduction of DC migration. In addition, absence of TLR4 ablates production of IL-12(p40)2 in DC exposed to Y. pestis-26°. Thedata demonstrate the molecular pathway by which Y. pestis evades induction of early DC activation as measured by migration andIL-12(p40)2 production. The Journal of Immunology, 2008, 181: 5560–5567.

Y ersinia pestis, a Gram-negative facultative intracellularbacterium, is the causative agent of plague (1). Although itis primarily a rodent pathogen, Y. pestis can be transmitted

intradermally to humans through the bite of an infected flea (2). Itis believed that through this mode of transmission and also aerosoltransmission, Y. pestis was responsible for the Black Death in theMiddle Ages (3, 4). Plague is a global disease and poses an in-creased threat to world health due to the rise of both drug resis-tance and the ability to aerosolize this pathogen and create a bio-logical weapon (5–8). Understanding the biology of Y. pestis istherefore important, and recent work has resulted in the sequencingof a number of virulent Y. pestis strains (9) and definition of thecomplete Y. pestis virulence proteome (10). Currently, there is nosafe and efficacious vaccine against plague (11, 12), and rationaldesign of such a vaccine depends on knowledge of the factors thatimpact immunity to Y. pestis. In this paper, we investigate theinteraction of Y. pestis with dendritic cells (DC),4 the cells that arepivotal in the initiation of immune responses.

Although induction of humoral immunity has been the focus ofmost plague vaccine research, both cellular and humoral immunitycan mediate protection (12). A critical step in the generation ofboth cellular and humoral responses is the activation of DCs to astate that allows them to migrate to the lymphoid follicle, whereinthey promote acquired immune responses (13). This activation isinitiated as a result of the DCs being exposed to microbial com-ponents, and DC migration is mediated by the chemokines CCL19and CCL21, acting on their receptor, CCR7 (14–17). We recentlyreported that DCs deficient in IL-12p40 fail to migrate towardCCR7 ligands upon exposure to bacterial stimuli and that migra-tion is rescued by the addition of IL-12p40 homodimer (IL-12(p40)2) but not IL-12p70 or denatured IL-12(p40)2 (18). Cellsthat migrate after s.c. vaccination also express IL-12p40 alone,suggesting that expression of IL-12(p40)2 by DCs is a key com-ponent of the initiation of immunity (19). Herein we describe amechanism for induction of both IL-12(p40)2 production and che-mokine responsiveness in response to Y. pestis.

Because Y. pestis modulates its gene expression in a tempera-ture-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 che-mokine responsiveness in DCs. Whereas Y. pestis-26° is a potentinducer of IL-12(p40)2, Y. pestis-37°-stimulated DCs secrete onlylow levels of IL-12p40. Further, whereas Y. pestis-26° inducesresponsiveness of DCs to chemokine in an IL-12p40-dependentmanner, Y. pestis-37° does not. The ability of Y. pestis-37° toevade both IL-12(p40)2 induction and chemokine responsivenessis overcome when Y. pestis-37° is forced to express hexa-acylatedrather than only tetra-acylated lipid A. Finally, the induction ofIL-12(p40)2 secretion and chemokine responsiveness is dependenton TLR4. We have therefore demonstrated a molecular pathwayby which Y. pestis evades DC activation.

*Trudeau Institute, Saranac Lake, NY 12983; †Howard Hughes Medical Institute, De-partment of Medicine and Microbiology and Immunology, University of California, SanFrancisco, CA 94143; and ‡Division of Infectious Diseases and Immunology, Departmentof Medicine, University of Massachusetts Medical School, Worcester, MA 01655

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

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Trudeau Institute Training Fellowship T32 AI49823 (toR.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 CareerDevelopment Award AI057158 (North East Biodefense Center-Lipkin) (to S.A.K.).2 Current address: Division of Medicine, Allergy and Immunology, Children’s Hos-pital of Pittsburgh, Pittsburgh, PA 15213.3 Address correspondence and reprint requests to Dr. Andrea M. Cooper, TrudeauInstitute, 154 Algonquin Avenue, Saranac Lake, NY 12983. E-mail address: acooper@trudeauinstitute.org4 Abbreviations used in this paper: DC, dendritic cell; IL-12(p40)2, IL-12p40 ho-modimer; 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 reportermice; BMDC, bone marrow-derived DC; MOI, multiplicity of infection.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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Materials and MethodsMice

All mice were bred at the Trudeau Institute (Saranac, NY) and were treatedaccording to National Institutes of Health (Bethesda, MD) and TrudeauInstitute Animal Care and Use Committee guidelines. C57BL/6 (B6) andB6-il12b mice were originally purchased from The Jackson Laboratory.IL-12p40-IRES-YFP reporter mice (Yet40 mice) expressing a yellow-flu-orescent protein upon IL-12p40 transcription were a gift from Dr. RichardM. Locksley and have been described (19). Mice deficient in the TLR4gene (hereafter referred to as tlr4�/� mice; Ref. 23) were purchased fromOriental 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�pgm and pigmentation-positive Y. pestisstrain KIM10�caf1� (pCD1�, pMT�, pPCP�, F1�) were kindly providedby Celine Pujol and James B. Bliska (State University of New York, StonyBrook, NY). Y. pestis strain KIM5-pLpxL was created by transformingKIM5 with an pLpxL-containing plasmid as previously described (24). AllY. 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 ODunit � 5.8 � 108 CFU), washed with PBS, inactivated by heating at 60°Cfor 1 h, and frozen. Heat inactivation was performed to eliminate the abilityof Y. pestis grown at 26°C to alter its gene expression/surface phenotypeafter its addition to a 37°C culture.

Bone marrow-derived DC (BMDC) culture

DCs were generated from the bone marrow of C57BL/6, Yet40, andB6.tlr4�/� mice basically as described previously (25). Briefly, cultureswere harvested at day 6 and either sorted by CD11c expression usingmagnetic beads (Miltenyi Biotec; all protein detection experiments) or usedwithout sorting (chemotaxis assays or Yet40 YFP expression experiments).Generation of CD11c� immature DC (i.e., low expression of cell surfacemarkers, CD80, CD86, I-Ab, CD40) was similar in terms of numbers andfrequency between wild-type and B6.tlr4�/�-derived populations (data notshown).

In vitro Y. pestis exposure

BMDCs were resuspended in cDMEM at a concentration of 2.5 � 105/ml,and 2 ml were pipeted into individual wells of a 24-well plate (i.e., 5 � 105

total cells). Aliquots of Y. pestis (stock, 1 � 109/ml) were thawed andserially diluted in cDMEM; 2 ml were added to 2 ml of BMDCs to generate40 multiplicity of infection (MOI), 20 MOI, 10 MOI, and 5 MOI cultures.Medium alone (2 ml) was used for 0 MOI cultures. After the indicatedincubation period at 37°C, the supernatants were collected and used forELISA or Western blot analysis. Cells were centrifuged and washed twicewith FACs buffer or chemotaxis buffer, depending on the subsequent aimsof each individual experiment. Some cells received IL-12(p40)2 (R&DSystems) as previously described (18).

Flow cytometry

All Abs used for flow cytometric analysis were purchased from BD Pharm-ingen or eBiosciences. After exposure to varying MOIs of Y. pestis,BMDCs were washed with FACS buffer (2% FCS in PBS), Fc blocked, andstained with Abs that recognize CD11c (clone HL3) and I-Ab (clone AF6-120.1). YFP expression by Yet40 DCs was determined by gatingCD11c�I-Ab� cells; distinguishing YFP expression from autofluorescencewas performed using nontransgenic control BMDCs. For all surface mark-ers, positive staining was established using appropriate isotype controls.Data were acquired using a FACSCalibur (BD Biosciences) and analyzedwith FlowJo software (Tree Star).

Chemotaxis measurement

BMDCs were activated with varying MOIs of Y. pestis as indicated, andtheir ability to respond to the chemokine CCL19 (25 ng/ml; R&D Systems)was determined using the previously described in vitro transwell chemo-taxis 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 centrifu-gation in iCon concentrators (Pierce) with a molecular weight cutoff of9K. Nondenaturing sample buffer (Invitrogen) was added to each con-centrate and then loaded onto a 4 –16% gradient Bis-Tris gel. Electro-phoresis and transfer to a polyvinylidene difluoride membrane was per-formed 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 �g/mlCon 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 experimentalgroups was determined using Student’s t test. Means were considered dif-ferent if p was �0.05.

ResultsY. pestis-26°, but not Y. pestis-37°, induces IL-12(p40)2

production

Because Y. pestis-37° expresses a different transcriptional pattern thanY. pestis-26°, we compared the ability of these two preparations toactivate DCs. To do this, we exposed BMDC from Yet40 mice toboth Y. pestis-37° and Y. pestis-26° and assessed transcription of IL-12p40. BMDCs exposed to varying MOI of KIM5 Y. pestis-26°-ex-pressed YFP indicating a significant increase in IL-12p40-transcrip-tion among CD11c�I-Ab� cells compared with unstimulated controls(Fig. 1A). KIM5 Y. pestis-37°did not induce IL-12p40 transcriptionabove baseline (Fig. 1A). These results were dose dependent and ob-servable across multiple experiments as shown by the fold change infrequency of YFP expressing cells (Fig. 1B). IL-12p40 is a subunit ofthe cytokines IL-12p70 and IL-23 as well as being secreted as a ho-modimer, IL-12(p40)2. To assess which IL-12p40-dependent cyto-kines were produced by Y. pestis-exposed DCs, we sorted C57BL/6BMDC 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-de-pendent manner by Y. pestis-26°, only a small amount of IL-23 and noIL-12p70 were induced (Fig. 1C). The IL-12p40 response was muchstronger 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 toinduce significant IL-12p70 (Fig. 1E) both induced a small amount ofIL-23 (Fig. 1F).

Although the IL-12p40 response was much stronger follow-ing Y. pestis-26° stimulation, IL-12p40 could be induced by Y.pestis-37° (Fig. 1E). To determine the nature of the secretedIL-12p40 after Y. pestis stimulation, we concentrated the DCsupernatants, separated the proteins by size on a nondenaturinggel, and probed the subsequent blot with an anti-IL-12p40 mAb.IL-12(p40)2 was the dominant IL-12p40-containing cytokineproduced by DC exposed to KIM5 Y. pestis-26°; in contrast,DCs stimulated with KIM5 Y. pestis-37° secrete IL-12p40 inmonomeric form (Fig. 1Gii). The IL-23 and IL-12p70 (measur-able by ELISA) was below the level of detection by this method(Fig. 1Gi). Lysate from Con A-stimulated B6 splenocytes wasused to provide size markers for the IL-12p40-containing pro-teins 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-dependentchemotaxis of IL-12(p40)2-producing DCs

In addition to IL-12p40 production, DCs respond to bacterialexposure by becoming responsive to homeostatic chemokines(18). We therefore tested whether DC chemokine responsive-ness was differentially induced by exposure to Y. pestis-26° and

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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 MOIused, DCs stimulated with KIM5 Y. pestis-37° failed to migratetoward CCL19 (Fig. 2A). DCs that migrated across the trans-well were confirmed by flow cytometry to be mostly CD11c�

(data not shown). Y. pestis-26° therefore is able to stimulate DCchemokine responsiveness whereas Y. pestis-37° cannot. To ad-dress whether the migration was dependent on IL-12(p40)2, weused DCs from mice lacking il12b and therefore unable to generateIL-12p40 (18). DCs lacking il12b were unable to migrate to CCL19when stimulated with Y. pestis-26°, however, responsiveness could

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 MOIof 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 histogramsshow 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 (f) or Y. pestis-37°C (�) relative to unstimulated DCs (�YFP � [YFPexpressed in response to Y. pestis]/[YFP expressed in response to media alone]) was determined by flow cytometry for 3 experiments. Data pointsshow 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 proteinin the supernatant was determined by ELISA over time. Data points represent the mean � SD of duplicate readings and show one experimentrepresentative 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° for3 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 readingsand 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, byStudent’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 andIL-12p40�/� splenocytes were run simultaneously as size indicators and as a positive and negative control, respectively.

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be restored by the addition of IL-12(p40)2 but not denatured IL12(p40)2 (Fig. 2B).

To determine whether the Y. pestis-stimulated DCs that mi-grated to CCL19 also transcribed IL-12p40, we challenged Yet40DCs with the different bacterial strains and measured YFP inCD11c� cells that had migrated to the CCL19 in the lower cham-ber. The increased percentage of CD11c� YFP� cells in the lowerchamber confirmed that chemotaxis toward CCL19 resulted in anenrichment of IL-12p40-transcribing cells (Fig. 2C).

To determine the dominant form of secreted IL-12p40 amongmigratory DCs, we performed nondenaturing Western blot anal-ysis of the chemotaxis buffer that remained in the upper and

lower chambers of the transwell following the collection ofYet40 cells for the experiment shown in Fig. 2C. In the un-stimulated Yet40 DC cultures (0 MOI; Fig. 2D), IL-12p40 alonewas observed in both upper and lower chambers. However,stimulation with 20 MOI KIM5 Y. pestis-26° resulted in theappearance 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 concludefrom that the dominant form of secreted IL-12p40 associatedwith migratory DCs is IL-12(p40)2.

The increase in CD11c�YFP� cells was not due to an ability ofCCL19 to increase IL-12p40 transcription because coincubation of

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 werestimulated with either KIM5 Y. pestis-26° (f) or KIM Y. pestis-37° (�) at varying MOIs and the ability of the stimulated DCs to migrate towarda 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 betweenchemotaxis 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° (f, B6; F, B6.il12b�/�), KIM Y. pestis-37° (�, B6), KIM5 Y. pestis-26° plus IL-12(p40)2 (E,B6.il12b�/�) or KIM5 Y. pestis-26° plus denatured IL-12(p40)2 (�, B6.il12b�/�). The chemotaxis index was determined as for A. Data pointsrepresent the mean and SD of duplicate values and show one experiment representative of two; for the difference between chemotaxis index inducedby 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 werestimulated with KIM5 Y. pestis-26° and exposed to a CCL19 gradient. The frequency of CD11c� cells that were transcribing IL-12p40 (YFP�) wasdetermined by flow cytometry for the cells before they were exposed to CCL19 (o), for the cells remaining in the upper chamber (i.e., nonresponsive;�) and for cells that had migrated toward CCL19 (i.e., lower chamber; f). Bars represent the mean of duplicate values and show one experimentrepresentative 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 gradientfor 90 min. The nature of the IL-12p40-containing protein released into the supernatant during the 90-min incubation in the upper and lower chamberwas determined as described for Fig. 1G; data are from one experiment representative of two.

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Yet40 DCs with CCL19 did not significantly enhance YFP ex-pression (data not shown).

KIM5 Y. pestis-37° evasion of DC IL-12p40 productionoccurs with other strains of Y. pestis and is independent ofpCD1 and caf1

To determine whether the ability to evade DC activation was ageneral feature of Y. pestis, we compared the ability of KIM5 toinduce IL-12p40 transcription with that of strain CO92�pgm,which derives from a distinct Y. pestis biovar. We found thatCO92�pgm Y. pestis-37° induced a similar low level of IL-12p40transcription (YFP expression) in CD11c�I-Ab� cells as theKIM5 Y. pestis-37° (Fig. 3) and that this was significantly less thanthat seen for KIM5 Y. pestis-26° and CO92�pgm Y. pestis-26°(Fig. 3).

Several genes important to Y. pestis pathogenesis are known tobe transcriptionally up-regulated upon transition from 26°C to37°C. These include a type III secretion system and the F1 capsularprotein. The type III secretion system is encoded by the pCD1plasmid and injects host cells with effector proteins that blockphagocytosis and down-regulate proinflammatory signals (1, 22,27). The F1 protein, on the other hand, is encoded by the caf1 geneon pMT1 and forms a protective capsule around Y. pestis andsuppresses phagocytosis (1, 28–31). To test whether either pCD1or 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 ofCD11c�I-Ab�YFP� cells induced upon exposure was equally lowamong all strains grown at 37°C (Fig. 3, bottom) relative to thesame strains grown at 26°C. These data suggest that KIM5 Y.pestis-37° evasion of DC IL-12p40 production is independent ofpCD1 and caf1 and that this activity can be generalized to severalstrains of Y. pestis.

Y. pestis-37° evasion of DC IL-12p40 induction is overcome byexpression 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 atlower temperatures the acyl transferase LpxP enables the biosyn-thesis of hexa-acylated lipid A. Upon transition to 37°C, Y. pestisLpxP is presumably not produced and/or is inactivated and lipid Abecomes mostly tetra-acylated (24, 32, 33). Recently, a strain of Y.pestis (Y. pestis KIM5-pLpxL) expressing Escherichia coli acyl-transferase LpxL was generated. The enzyme activity of E. coliLpxL in KIM5 appears temperature insensitive; thus, the produc-tion of tetra-acylated lipid A of Y. pestis KIM5-pLpxL does notoccur upon growth at 37°C (24). To test whether LpxL expressionalters the ability of the Y. pestis KIM5 to evade DC activation, wegrew KIM5-pLpxL at 26°C and 37°C and examined the ability ofthe 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 atlevels 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 expressionand stimulated with bacteria, both KIM5-pLpxL-26°C andKIM5pLpxL Y. pestis-37° induced IL-12p40 production at levelshigher than that induced by KIM5 Y. pestis-37° (Fig. 4B); thisIL-12p40 was secreted primarily as IL-12(p40)2 (Fig. 4C). Theincreased IL-12p40 production elicited by KIM5pLpxL Y. pestis-37°, as compared with KIM5 Y. pestis-37°, was associated with therestored ability of DCs to migrate toward CCL19 (Fig. 4D). Thus,the temperature sensitivity of LpxP is implicated in the ability ofY. pestis to evade activation of IL-12p40 production and migrationof DCs.

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�pgm Y. pestis-26°, and CO92�pgm Y. pestis-37° (top), or the pCD1�caf1� strainKIM6 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 onsize and expression of I-Ab, and representative histograms show the level of IL-12p40 (YFP) expression in CD11c�I-Ab� cells as detected by flowcytometry. Dot plots show representative values for three independent experiments.

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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 witheither KIM5 Y. pestis-26° or KIM5 Y. pestis-37° (top), or KIM5pLpxL Y. pestis-26° or KIM5pLpxL Y. pestis-37° (bottom). Cells were then gatedbased 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 flowcytometry. Dot plots show representative values for three independent experiments. B–D, B6 CD11c� BMDCs were stimulated with KIM5 Y.pestis-26° (f), KIM5 Y. pestis-37°(�), KIM5pLpxL Y. pestis-26° (F), or KIM5pLpxL Y. pestis-37° (E). B, The level of IL-12p40 in the supernatantwas determined by ELISA. Data points represent the mean and SD of duplicate values and show one experiment representative of three; for thedifference 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 experimentrepresentative of two. D, The CCL19-dependent chemotaxis of the stimulated DCs was determined as described for Fig. 2A. Data points representthe mean and SD of duplicate values and show one experiment representative of three; for the difference between values induced by all experimentalgroups 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° (�, f), KIM5pLpxL Y. pestis-26° (E, F) or KIM5pLpxL Y. pestis-37° (‚, Œ) and the level ofIL-12p40 produced measured by ELISA. Because KIM5 Y. pestis-37° failed to induce IL-12p40 (�), it was not tested against tlr4�/�DC. Data pointsrepresent the mean and SD of duplicate values and show one experiment representative of two; for the difference between values induced by allexperimental 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 proteinsin the supernatants from B6 and TLR4�/� CD11c� BMDCs stimulated with 40 MOI KIM5 Y. pestis-26° was analyzed using nondenaturing Westernblot analysis as for Fig. 1G. Data shown are one experiment representative of two.

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The above result implicated LPS induced signaling as a factorin initiation of DC IL-12(p40)2 production. It has been repeat-edly demonstrated that TLR4 is the pattern recognition receptorfor Gram-negative bacterial LPS (34). Therefore, we generatedBMDCs from B6.tlr4�/� mice and exposed CD11c� cells toKIM5 Y. pestis-26°, KIM5 Y. pestis-37°, KIM5-pLpxL-26°, andKIM5-pLpxL-37°. In contrast to cells from wild-type controlmice, cells from B6.tlr4�/� mice secreted very low levels ofIL-12p40 in response to all stimuli (Fig. 4E). Thus, the abilityof Y. pestis to induce IL-12p40 production in DCs requiresTLR4. The induction of chemokine responsiveness seen forKIM5 Y. pestis-26°(Fig. 2, A and B) was not seen when TLR4-deficient DCs were stimulated with KIM5 Y. pestis-26° (datanot shown).

Having determined that the principal IL-12p40-containingcytokine 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 collectedfrom wild-type and TLR4-deficient CD11c� BMDCs stimu-lated with KIM5 Y. pestis-26°C and were analyzed by nonde-naturing Western blot analysis for IL-12p40-containing cyto-kines. Although stimulation of wild-type DCs with KIM5 Y.pestis-26°C results in the production of IL-12(p40)2, this cyto-kine does not appear in the supernatants of TLR4-deficient DCs,with IL-12p40 monomer being the only form of IL-12p40 pro-duced (Fig. 4F). Thus, the ability of DC to produce IL-12(p40)2

and become responsive to chemokines following Y. pestis ex-posure is dependent on TLR4.

DiscussionDCs are pivotal in the initiation of immunity and pathogens havetherefore developed mechanisms to alter DC function. Our datademonstrate that although Y. pestis grown at 26°C (i.e., a surrogateof flea vector temperature) is capable of activating DCs, it is ableto evade activating DCs when growing at 37°C (i.e., akin to growthwithin the vertebrate host or when transmitted via the pulmonaryroute). We show that the ability of Y. pestis to initiate DC activa-tion is determined by the lipid A structure and depends on thepattern recognition receptor TLR4. We further show that evasionof DC activation can be overcome if Y. pestis is modified to ex-press hexa- rather than tetra-acylated lipid A, and this supports thehypothesis that modulation of the lipid A structure is a major vir-ulence determinant (24).

Herein we have measured DC activation by the induction ofIL-12p40 transcription and the initiation of responsiveness tohomeostatic chemokines. We have previously shown that, uponexposure to bacteria and bacterial products, these two events areinduced early and that chemokine responsiveness is dependenton 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 inducerof IL-12(p40)2 and that this induction is required for bacteriallyinduced responsiveness to chemokines. In the previous study,we characterized neither the nature of the IL-12p40 being re-leased by the DCs nor the receptor pathway by which it wasbeing induced. Our data show that the induction of IL-12p40transcription that occurs in response to Y. pestis results in therelease of IL-12(p40)2 in large excess relative to any other IL-12p40 containing cytokine and that this response is rapid. Fur-ther, our observation that TLR4 and lipid A structure are es-sential for production of IL-12(p40)2 provides the beginning ofthe molecular pathway by which this underappreciated cytokineis induced. Finally, the observation that it is the IL-12p40-tran-scribing DCs that migrate and that IL-12(p40)2 is enriched in

the supernatant of DCs that have migrated provides support forthe hypothesis that IL-12(p40)2 plays a role in bacterially in-duced migration of DCs (18).

The impact of lipid A modulation on virulence is clear as thehexa-acylated lipid A molecule is much more stimulatory thanthe tetra-acylated form (24). Further, forcing Y. pestis to main-tain expression of the hexa-acylated lipid A at 37°C results inreduced virulence and efficient induction of protection againstsubsequent Y. pestis challenge (24). Our data demonstrate thatthe nature of the lipid A influences the ability of Y. pestis toactivate DCs. Because the flea-borne bacteria need to initiateinfection of the lymph nodes, the ability of the hexa-acylatedlipid A to initiate DC activation and migration would allowefficient colonization of the lymph node. On the other hand, thetemperature-sensitive induction of the tetra-acylated lipid Acould allow the bacteria to remain and grow within the lymphnode without stimulating immunity, thereby enabling develop-ment of very high bacteremia. In addition, the inability to in-duce DC migration when delivered by the aerosol route (i.e.,bacteria grown at 37°C) may promote the establishment of dis-ease in the lung. Although DCs represent a likely population tomediate transport of Y. pestis, it is also possible that other celltypes such as neutrophils or monocytes could mediate transportof this acute inflammatory pathogen.

The regulation of transcription of IL-12p40-containing cyto-kines has been extensively studied, and the fact that LPS andother microbial lipoproteins can trigger IL-12(p40)2 productionthrough TLR signaling pathways is a well-described phenom-enon (35, 36). Although activation of a single TLR usually re-sults in IL-12(p40)2 production, the presence of other patternrecognition receptor ligands or signals from activated T cellsare required for IL-12p70 production (35). Our data demon-strate that TLR4 and hexa-acylated lipid A-dependent activa-tion of DC (likely via direct ligation of TLR4 by hexa-acylatedlipid A of Y. pestis), results in very potent production of IL-12(p40)2, and we correlate that expression with the migration ofthe DCs. These data provide further support to the hypothesisthat IL-12(p40)2 plays an agonistic role in initiation of immu-nity to bacteria (37).

Understanding of IL-12(p40)2 biology is important in light of itsnewly described activities (37). Specifically, as the two subunits ofIL-12(p40)2 are held together by a disulfide bond, it will be im-portant to understand how TLR4 signaling directs the activity ofthe enzymes that catalyze disulfide bond formation. In eukaryotes,disulfide bonds occur in the endoplasmic reticulum under the di-rection of oxidoreductases (38). One such enzyme is protein di-sulfide isomerase which is expressed in DCs and which has beendemonstrated to enhance IL-12p40 dimerization (39, 40). Whetherprotein disulfide isomerase activity is impacted by TLR4 andwhether this impacts the level of IL-12p40 dimerization should beinvestigated.

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

AcknowledgmentsWe thank Celine Pujol, James Bliska, and Robert Brubaker for Y. pestisstrains and Lawrence Kummer for technical assistance.

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DisclosuresThe authors have no financial conflict of interest.

References1. Perry, R. D., and J. D. Fetherston. 1997. Yersinia pestis: etiologic agent of plague.

Clin. Microbiol. Rev. 10: 35–66.2. Goldman, L., E. Rockwell, and D. F. Richfield 3rd. 1952. Histopathological stud-

ies on cutaneous reactions to the bites of various arthropods. Am. J. Trop. Med.Hyg. 1: 514–525.

3. Gage, K. L., and M. Y. Kosoy. 2005. Natural history of plague: perspectives frommore than a century of research. Annu. Rev. Entomol. 50: 505–528.

4. Raoult, D., G. Aboudharam, E. Crubezy, G. Larrouy, B. Ludes, andM. Drancourt. 2000. Molecular identification by “suicide PCR” of Yersinia pestisas the agent of medieval black death. Proc. Natl. Acad. Sci. USA 97:12800–12803.

5. Welch, T., W. Fricke, P. McDermott, D. White, M. Rosso, D. Rasko,M. Mammel, M. Eppinger, M. Rosovitz, D. Wagner, et al. 2007. Multiple anti-microbial resistance in plague: an emerging public health risk. PLoS One 2: e309.

6. Inglesby, T. V., D. T. Dennis, D. A. Henderson, J. G. Bartlett, M. S. Ascher,E. Eitzen, A. D. Fine, A. M. Friedlander, J. Hauer, J. F. Koerner, et al. 2000.Plague as a biological weapon: medical and public health management: WorkingGroup on Civilian Biodefense. JAMA 283: 2281–2290.

7. Won, W. D., and H. Ross. 1966. Effect of diluent and relative humidity onapparent viability of airborne Pasteurella pestis. Appl. Microbiol. 14: 742–745.

8. Zilinskas, R. A. 2006. The anti-plague system and the Soviet biological warfareprogram. Crit. Rev. Microbiol. 32: 47–64.

9. Parkhill, J., B. W. Wren, N. R. Thomson, R. W. Titball, M. T. Holden,M. B. Prentice, M. Sebaihia, K. D. James, C. Churcher, K. L. Mungall, et al.2001. Genome sequence of Yersinia pestis, the causative agent of plague. Nature413: 523–527.

10. Chromy, B. A., M. W. Choi, G. A. Murphy, A. D. Gonzales, C. H. Corzett,B. C. Chang, J. P. Fitch, and S. L. McCutchen-Maloney. 2005. Proteomic char-acterization of Yersinia pestis virulence. J. Bacteriol. 187: 8172–8180.

11. Titball, R., and E. Williamson. 2004. Yersinia pestis (plague) vaccines. ExpertOpin. Biol. Ther. 4: 965–973.

12. Smiley, S. 2008. Current challenges in the development of vaccines for pneu-monic plague. Expert Rev. Vaccines 7: 209–221.

13. Randolph, G. J., J. Ochando, and S. Partida-Sanchez. 2008. Migration of den-dritic cell subsets and their precursors. Annu. Rev. Immunol. 26: 293–316.

14. Luther, S. A., H. L. Tang, P. L. Hyman, A. G. Farr, and J. G. Cyster. 2000.Coexpression of the chemokines ELC and SLC by T zone stromal cells anddeletion of the ELC gene in the plt/plt mouse. Proc. Natl. Acad. Sci. USA 97:12694–12699.

15. Beckmann, J. H., S. Yan, H. Luhrs, B. Heid, S. Skubich, R. Forster, andM. W. Hoffmann. 2004. Prolongation of allograft survival in ccr7-deficient mice.Transplantation 77: 1809–1814.

16. Martin-Fontecha, A., S. Sebastiani, U. E. Hopken, M. Uguccioni, M. Lipp,A. Lanzavecchia, and F. Sallusto. 2003. Regulation of dendritic cell migration tothe draining lymph node: impact on T lymphocyte traffic and priming. J. Exp.Med. 198: 615–621.

17. Ohl, L., M. Mohaupt, N. Czeloth, G. Hintzen, Z. Kiafard, J. Zwirner,T. Blankenstein, G. Henning, and R. Forster. 2004. CCR7 governs skin dendriticcell migration under inflammatory and steady-state conditions. Immunity 21:279–288.

18. Khader, S., S. Partida-Sanchez, G. Bell, D. Jelley-Gibbs, S. Swain, J. Pearl,N. Ghilardi, F. Desauvage, F. Lund, and A. Cooper. 2006. Interleukin 12p40 isrequired for dendritic cell migration and T cell priming after Mycobacteriumtuberculosis infection. J. Exp. Med. 203: 1805–1815.

19. Reinhardt, R., S. Hong, S. Kang, Z. Wang, and R. Locksley. 2006. Visualizationof IL-12/23p40 in vivo reveals immunostimulatory dendritic cell migrants thatpromote Th1 differentiation. J. Immunol. 177: 1618–1627.

20. Motin, V. L., A. M. Georgescu, J. P. Fitch, P. P. Gu, D. O. Nelson, S. L. Mabery,J. B. Garnham, B. A. Sokhansanj, L. L. Ott, M. A. Coleman, et al. 2004. Tem-

poral global changes in gene expression during temperature transition in Yersiniapestis. J. Bacteriol. 186: 6298–6305.

21. Zhang, P., M. Skurnik, S. S. Zhang, O. Schwartz, R. Kalyanasundaram,S. Bulgheresi, J. J. He, J. D. Klena, B. J. Hinnebusch, and T. Chen. 2008. HumanDC-SIGN (CD209) is a receptor for Yersinia pestis that promotes phagocytosisby dendritic cells. Infect. Immun. 76: 2070–2079.

22. Marketon, M. M., R. W. DePaolo, K. L. DeBord, B. Jabri, and O. Schneewind.2005. Plague bacteria target immune cells during infection. Science 309:1739–1741.

23. Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y. Takeda, K. Takeda,and S. Akira. 1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice arehyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene prod-uct. J. Immunol. 162: 3749–3752.

24. Montminy, S. W., N. Khan, S. McGrath, M. J. Walkowicz, F. Sharp, J. E. Conlon,K. Fukase, S. Kusumoto, C. Sweet, K. Miyake, et al. 2006. Virulence factors ofYersinia pestis are overcome by a strong lipopolysaccharide response. Nat. Im-munol. 7: 1066–1073.

25. Lutz, M. B., N. Kukutsch, A. Ogilvie, S. Rossner, F. Koch, N. Romani, andG. Schuler. 1999. An advanced culture method for generating large quantities ofhighly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223:77–92.

26. Partida-Sanchez, P., S. Goodrich, K. Kusser, N. Oppenheimer, T. D. Randall, andF. E. Lund. 2004. Regulation of dendritic cell trafficking by the ADP-ribosylcyclase CD38: impact on the development of humoral immunity. Immunity 20:279–291.

27. Orth, K., Z. Xu, M. B. Mudgett, Z. Q. Bao, L. E. Palmer, J. B. Bliska,W. F. Mangel, B. Staskawicz, and J. E. Dixon. 2000. Disruption of signaling byYersinia effector YopJ, a ubiquitin-like protein protease. Science 290:1594–1597.

28. Nakajima, R., and R. R. Brubaker. 1993. Association between virulence of Yer-sinia pestis and suppression of � interferon and tumor necrosis factor �. Infect.Immun. 61: 23–31.

29. Brubaker, R. 2003. Interleukin-10 and inhibition of innate immunity to Yersiniae:roles of Yops and LcrV (V antigen). Infect. Immun. 71: 3673–3681.

30. Sing, A., D. Rost, N. Tvardovskaia, A. Roggenkamp, A. Wiedemann,C. J. Kirschning, M. Aepfelbacher, and J. Heesemann. 2002. Yersinia V-antigenexploits Toll-like receptor 2 and CD14 for interleukin 10-mediated immunosup-pression. J. Exp. Med. 196: 1017–1024.

31. Viboud, G. I., and J. B. Bliska. 2005. Yersinia outer proteins: role in modulationof host cell signaling responses and pathogenesis. Annu. Rev. Microbiol. 59:69–89.

32. Rebeil, R., R. K. Ernst, C. O. Jarrett, K. N. Adams, S. I. Miller, andB. J. Hinnebusch. 2006. Characterization of late acyltransferase genes of Yersiniapestis and their role in temperature-dependent lipid A variation. J. Bacteriol. 188:1381–1388.

33. Kawahara, K., H. Tsukano, H. Watanabe, B. Lindner, and M. Matsuura. 2002.Modification of the structure and activity of lipid A in Yersinia pestis lipopoly-saccharide by growth temperature. Infect. Immun. 70: 4092–4098.

34. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev. Im-munol. 21: 335–376.

35. Trinchieri, G., and A. Sher. 2007. Cooperation of Toll-like receptor signals ininnate immune defence. Nat. Rev. Immunol. 7: 179–190.

36. Goriely, S., M. Neurath, and M. Goldman. 2008. How microorganisms tip thebalance between interleukin-12 family members. Nat. Rev. Immunol. 8: 81–86.

37. Cooper, A. M., and S. Khader. 2007. IL-12p40: an inherently agonistic cytokine.Trends Immunol. 28: 33–38.

38. Rietsch, A., and J. Beckwith. 1998. The genetics of disulfide bond metabolism.Annu. Rev. Genet. 32: 163–184.

39. Park, B., S. Lee, E. Kim, K. Cho, S. R. Riddell, S. Cho, and K. Ahn. 2006. Redoxregulation facilitates optimal peptide selection by MHC class I during antigenprocessing. Cell 127: 369–382.

40. Martens, E., I. Alloza, C. J. Scott, A. Billiau, and K. Vandenbroeck. 2000. Proteindisulfide isomerase-mediated cell-free assembly of recombinant interleukin-12p40 homodimers. Eur. J. Biochem. 267: 6679–6683.

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