effect of bacillus anthracis virulence factors on human dendritic cell activation

Effect of Bacillus anthracis virulence factors on human dendritic cell activation Andrew C. Hahn a , C. Rick Lyons a,b , Mary F. Lipscomb a,c * a Center for Infectious Disease and Immunity, University of New Mexico Health Science Center, Albuquerque, NM, USA b Department of Internal Medicine, University of New Mexico, Albuquerque, NM, USA c Department of Pathology, University of New Mexico, Albuquerque, NM, USA Received 3 April 2008; received in revised form 13 June 2008; accepted 24 June 2008 Summary Bacillus anthracis possesses three primary virulence factors: capsule, lethal toxin (LT), and edema toxin (ET). Dendritic cells (DCs) are critical to innate and acquired immunity and represent potential targets for these factors. We examined the ability of B. anthracis spores and bacilli to stimulate human monocyte-derived DC (MDDC), primary myeloid DC (mDC), and plasmacytoid DC (pDC) cytokine secretion. Exposure of MDDCs and mDCs to spores or vegetative bacilli of the genetically complete strain UT500 induced significantly increased cytokine secre- tion. Spores lacking genes required for capsule biosynthesis stimulated significantly higher cytokine secretion than UT500 spores from mDCs, but not MDDCs. In contrast, bacilli lacking capsule stimulated significantly higher cytokine secretion than UT500 bacilli in both MDDCs and mDCs. Spores or bacilli lacking both LT and ET stimulated significantly higher cytokine secretion than UT500 spores or bacilli, respectively, in both mDCs and MDDCs. pDCs exposed to spores or bacilli did not produce significant amounts of cytokines even when virulence factors were absent. In conclusion, B. anthracis employs toxins as well as capsule to inhibit human MDDC and mDC cytokine secretion, whereas human pDCs respond poorly even when capsule or both toxins are absent. © 2008 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. KEYWORDS Human myeloid DC; Human plasmacytoid DC; B. anthracis; B. anthracis toxins; B. anthracis capsule Introduction Bacillus anthracis, the Gram-positive, nonmotile, spore- forming bacterium that causes anthrax, possesses three pri- mary virulence factors: an extracellular poly-D-glutamate (D-PGA) capsule and the secreted toxins, lethal toxin (LT) and edema toxin (ET), that are induced by physiologic tem- perature, CO 2 , and bicarbonate concentrations found in mammalian hosts [1–3]. Capsule is produced by the enzymes CapA, CapB, and CapC encoded by the capBCAD operon lo- cated on virulence plasmid pXO2. LT and ET are binary toxins composed of enzymatic A-components lethal factor (LF) or edema factor (EF), respectively, sharing the B-component protective antigen (PA), which translocates LF and EF into the cytoplasm [4]. LF, EF, and PA are encoded by lef, cya, and pagA respectively, contained on virulence plasmid pXO1 [5–10]. The D-PGA capsule is critical to virulence in that deletion of the capBCAD operon from the pXO1 pXO2 strain UT500 * Corresponding author. Fax: (505) 272-8084. E-mail address: [email protected] (M.F. Lipscomb). Human Immunology (2008) 69, 552–561 0198-8859/$ -see front matter © 2008 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2008.06.012

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Human Immunology (2008) 69, 552–561


ffect of Bacillus anthracis virulence factors onuman dendritic cell activation

ndrew C. Hahna, C. Rick Lyonsa,b, Mary F. Lipscomba,c*

Center for Infectious Disease and Immunity, University of New Mexico Health Science Center, Albuquerque, NM, USADepartment of Internal Medicine, University of New Mexico, Albuquerque, NM, USADepartment of Pathology, University of New Mexico, Albuquerque, NM, USA

eceived 3 April 2008; received in revised form 13 June 2008; accepted 24 June 2008

Summary Bacillus anthracis possesses three primary virulence factors: capsule, lethal toxin(LT), and edema toxin (ET). Dendritic cells (DCs) are critical to innate and acquired immunityand represent potential targets for these factors. We examined the ability of B. anthracis sporesand bacilli to stimulate human monocyte-derived DC (MDDC), primary myeloid DC (mDC), andplasmacytoid DC (pDC) cytokine secretion. Exposure of MDDCs and mDCs to spores or vegetativebacilli of the genetically complete strain UT500 induced significantly increased cytokine secre-tion. Spores lacking genes required for capsule biosynthesis stimulated significantly highercytokine secretion than UT500 spores from mDCs, but not MDDCs. In contrast, bacilli lackingcapsule stimulated significantly higher cytokine secretion than UT500 bacilli in both MDDCs andmDCs. Spores or bacilli lacking both LT and ET stimulated significantly higher cytokine secretionthan UT500 spores or bacilli, respectively, in both mDCs and MDDCs. pDCs exposed to spores orbacilli did not produce significant amounts of cytokines even when virulence factors wereabsent. In conclusion, B. anthracis employs toxins as well as capsule to inhibit human MDDC andmDC cytokine secretion, whereas human pDCs respond poorly even when capsule or both toxinsare absent.© 2008 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. Allrights reserved.

KEYWORDSHuman myeloid DC;

Human plasmacytoid


B. anthracis;

B. anthracis toxins;

B. anthracis capsule



acillus anthracis, the Gram-positive, nonmotile, spore-orming bacterium that causes anthrax, possesses three pri-ary virulence factors: an extracellular poly-D-glutamate

D-PGA) capsule and the secreted toxins, lethal toxin (LT)nd edema toxin (ET), that are induced by physiologic tem-erature, CO2, and bicarbonate concentrations found in

* Corresponding author. Fax: (505) 272-8084.

oE-mail address: [email protected] (M.F. Lipscomb).

198-8859/$ -see front matter © 2008 American Society for Histocompatibioi:10.1016/j.humimm.2008.06.012

ammalian hosts [1–3]. Capsule is produced by the enzymesapA, CapB, and CapC encoded by the capBCAD operon lo-ated on virulence plasmid pXO2. LT and ET are binary toxinsomposed of enzymatic A-components lethal factor (LF) ordema factor (EF), respectively, sharing the B-componentrotective antigen (PA), which translocates LF and EF intohe cytoplasm [4]. LF, EF, and PA are encoded by lef, cya,nd pagA respectively, contained on virulence plasmid pXO15–10].

The D-PGA capsule is critical to virulence in that deletion

f the capBCAD operon from the pXO1� pXO2� strain UT500

lity and Immunogenetics. Published by Elsevier Inc. All rights reserved.













553B. anthracis virulence factors and human dendritic cells

esults in a 10,000-fold increase in LD50 [11]. The capsule,hich forms the outermost layer of bacilli, is a weak im-unogen [12] and inhibits phagocytosis by mouse macro-hages [13]. LF, a Zn2�-dependent protease, cleaves andnactivates several human mitogen-activated protein kinaseinases (MAPKKs) critical to innate and adaptive immuneesponses [14–17]. Purified LT has deleterious effects onacrophages and DCs, including induction of apoptosis and

nhibition of cytokine secretion [18–23]. EF, a calmodulin-ctivated adenylate cyclase, also inhibits innate immuneunctions, including phagocytosis by neutrophils and macro-hages, and cytokine secretion by macrophages and DCs [22–5].

Based on current understanding, anthrax disease progresseshrough several stages including spore deposition, spore phago-ytosis, spore germination during migration to lymph nodes,roliferation of bacilli in lymph nodes, and systemic dissemi-ation of bacilli [26–28]. The relatively short time period be-ween initial infection and death [29] suggests that immuneystem failure lies primarily in the innate response during earlyisease stages to detect bacteria and/or generate responses tonflammatory signals. Pathogens commonly disable innate im-une responses by inactivating DCs [30], which detect patho-

ens at environmental interfaces resulting in cytokine secre-ion and migration to draining lymph nodes. DC responses maye particularly important to anthrax as DCs extensively take upnd transport spores to lymph nodes in mouse studies [23,31].

Understanding interactions between B. anthracis and hu-an DCs is critical to extrapolating information from animalodels and facilitating development of new therapeutics.revious work on B. anthracis virulence factor effects on DCssed purified LT [21] or toxin-deficient mutants derived fromtrain Sterne 7702, which lacks plasmid pXO2 containing cap-CAD and possibly other genes contributing to virulence22,23,27,32]. Of these studies, some examined murine DCs22,23], and human DC studies used monocyte-derived DCsxclusively [21,27,32].

Here we assessed the impact of capsule, LT and ET onecretion by human DC subsets of cytokines important tonnate and adaptive immune responses following in vitroxposure to B. anthracis. We used a genetically completeXO1� pXO2� strain (UT500) and UT500-derived isogenic


B. anthracis Bacillus anthracisDC dendritic cellEF edema factorET edema toxinIFN interferonIL interleukinLF lethal factorLT lethal toxinmDC primary myeloid DCMDDC human monocyte–derived DCPA protective antigenpDC plasmacytoid DCTNF tumor necrosis factor

eletion mutant strains lacking the genes required for pro- S

uction of capsule, LF, EF, or both LF and EF. We examinedytokine secretion by three types of human peripherallood-derived DCs: primary mDCs and pDCs isolated fromeripheral blood, and monocyte-derived DCs (MDDCs). Be-ause the different stages of B. anthracis infection are char-cterized by distinct anatomical compartments and bacte-ial forms, we examined different subsets of DCs likelyssociated with these compartments, as well as both sporend vegetative forms.

ubjects and methods

. anthracis strains

he fully toxigenic capsule-forming strain UT500 (pXO1� pXO2�)as constructed previously by transducing plasmid pX02 from strain602 (Pasteur) into strain 7702 (Sterne) [33]. UT538 (�capBCADSpcr]), UT539 (�lef [Kanr]), UT540 (�cya [Kanr]), UT541 (�lef �cyaKanr, Spcr]) were derived previously from UT500 by targeted dele-ion of genes encoding capsule biosynthetic enzymes, LF, EF, oroth LF and EF respectively [11,34].

reparation of spores and vegetative bacilli

pores and vegetative bacilli were prepared as previously described34]. Spore stocks were confirmed by microscopy to contain morehan 95% spore forms.

Human MDDC preparation. MDDCs were prepared as describedreviously [35,36], with the following modifications. Peripherallood mononuclear cells isolated from anonymous human donoruffy coats (United Blood Services; Albuquerque, NM) by Ficoll-aque Plus centrifiguation (GE Healthcare, Uppsala, Sweden) werencubated at 4°C to induce cell aggregation, layered over FBS, re-ggregated at 4°C, washed twice in PBS containing 0.6 mmol/lthylenediaminetetraacetate (EDTA), and resuspended in PBS with% FBS, 2 mmol/l EDTA (Gibco; Grand Island, NY). T cells, NK cells,cells, and neutrophils were removed with antibody-linked mag-

etic beads recognizing CD3, CD16, CD19, or CD56 (Miltenyi Biotec,uburn, CA). Cell preparations were seeded onto 10-cm cultureishes at 2–3 � 106 cells/ml in Dendritic Cell Maturation MediumDCMM) (Sigma-Aldrich, St. Louis, MO) containing 2 mmol/l L-glu-amine, 100U/ml penicillin, 100 �g/ml streptomycin, 55 �mol/l-mercaptoethanol (2-ME), and incubated with interleukin (IL)–41000 IU/ml) and GM-CSF (50 ng/ml) (Invitrogen; Carlsbad, CA),ith cytokine replenishment at day 3, to yield monocyte-derivedCs at day 6 (MDDCs).

Human primary DC isolation. Peripheral blood mononuclear cellsere isolated by Ficoll-Paque centrifugation of buffy coats (Unitedlood Services; Albuquerque, NM) followed by antibody-linked mag-etic bead depletion of cells expressing CD3, CD14, CD16, CD19,D56, or Glycophorin A (Miltenyi Biotec; Auburn, CA). Cells labeledith anti-CD123-PE, anti-HLA-DR-PerCP, anti-CD11c-APC, and FITC-onjugated Lineage Markers (CD3, CD19, CD16, CD56, CD14) (BDharmingen; San Jose, CA) were processed by fluorescence-acti-ated cell sorting (FACS) with MoFlo™ High-Performance Cell SorterDako Cytomation; Ft. Collins, CO). Gating strategies were deter-ined using unstained and single-stained (compensation) controls.Cs, which were identified as Lin-, HLA-DR�, were categorized asDCs or pDCs by expression of CD11c or CD123, respectively. Byost-sort analysis, mDC preparations were 84–98% pure (averageurity 89%), with 0.2–0.6% contaminating pDCs. pDC preparationsere 90–99% pure (average purity 94%), with 0.3–0.6% contaminat-

ng mDCs. Cells were resuspended in DCMM with 2 mmol/l glutaminend 55 �mol/l 2-ME, and in the case of pDCs, 10 ng/ml IL-3 (R&D

ystems; Minneapolis, MN) was added.













554 A.C. Hahn et al.

. anthracis cultures with DCs

pores or bacilli were added to DCs at a multiplicity of infectionMOI) of 1 in 96-well round-bottom plates in DCMM containing 2mol/l glutamine, 55 �mol/l 2-ME. Spore experiments were per-

ormed with 50,000 MDDCs, 25,000 mDCs or 25,000 pDCs. Bacillixperiments were performed with 25,000 cells for each DC type.OIs were determined in each experiment by CFU measurement of

nocula. MOIs between 0.6 and 1.8 were considered usable, as cy-okine secretion was proportional to MOI in this range for bothpores and bacilli (data not shown). MOIs of virulence factor mu-ants did not differ from UT500 by more than 2-fold in any givenxperiment. Following inoculation, plates were centrifuged 5 min-tes at 500 rpm and incubated at 37°C with 5% CO2. Ciprofloxacinfinal concentration 1.0 �g/ml) was added 1 hour after spore inoc-lation or 3 hours after bacilli inoculation. Spore germination wasetermined by susceptibility to killing after incubation at 65°C for0 minutes, as measured by CFU count. At 24 hours post-inocula-ion, plates were centrifuged 10 minutes at 1000 rpm, and superna-ants stored at �80°C. Positive controls for MDDCs and mDCs con-isted of lipopolysaccharide (LPS) from Escherichia coli strain111:B4 (Sigma-Aldrich, St. Louis, MO) at 1 �g/ml, and influenzatrain A/PR/8/34 (H1N1) virus (Advanced Biotechnologies Incorpo-ated; Columbia, MD) at MOI of 10 for pDCs. Experiments wereonducted in an ABSL-3 facility.

ytokine quantitation

ytokine concentrations were measured using antibody-linkedeads recognizing 21 different cytokines (BioSource-Invitrogen;arlsbad, CA) and a Luminex 100 reader (Luminex Corporation, Aus-in, TX), and raw data were analyzed with the StatLIA statisticalnalysis program (Brendan Scientific, Carlsbad, CA). Statistical anal-sis of cytokine concentrations was performed with GraphPad PrismLa Jolla, CA).


etermination of DC-B. anthracis cultureonditions

nhalational anthrax is carried out by airborne spores thateposit in the lung and germinate in response to phagocyto-is as they are transported to draining lymph nodes [28].acilli predominate for the remainder of the infection andccount for proliferation, systemic dissemination, and toxinroduction. Because DCs may respond differently to sporesnd bacilli, we exposed DCs to each form separately. Sporesnd bacilli were generated from B. anthracis strain UT500, aenetically complete pXO1� pXO2� strain that producesapsule, LT and ET [33], as well as from UT500-derived iso-enic virulence factor deletion mutants lacking the capsuleiosynthesis operon, LF, EF, or both LF and EF. Preliminarytudies determined that UT500 spores or bacilli inoculatedt an MOI of 1 stimulated cytokine secretion in MDDCs, andhat higher MOIs did not significantly affect cytokine levelsdata not shown).

Spores encountered by DCs are rapidly taken up and ger-inate. In exposing DCs to spores we aimed to expose DCs toathogen-associated molecular patterns (PAMPs) and otherotential immunomodulatory factors contained in dormantpores or expressed during germination, while limiting ex-osure to bacilli and bacillary stage-specific processes in-

luding replication. MDDCs and mDCs were incubated with e

T500 and mutant spores for 1 hour. Over 95% of spores ofach strain initiated germination within 20 minutes afterddition to DC cultures as determined by susceptibility toilling by incubation at 65°C for 30 minutes (data nothown). Previous studies showed that at one hour post-in-ection, human MDDCs and mouse bone marrow–derived DCsontain endocytosed spores [22,27]. Therefore, the antibi-tic ciprofloxacin was added one hour after spore inocula-ion with each DC type, resulting in complete killing of bac-eria as determined by plating for CFU (data not shown). Forach strain, numbers of total bacteria recovered from MDDCr mDC cultures did not change markedly over the 1 hourncubation period, typically increasing between 1.0-fold and.7-fold (Figures 1A and 1C). Addition of ciprofloxacin at 1our postinfection resulted in MDDC, mDC and pDC viabili-ies at 24 hours postinfection similar to respective unex-osed controls, regardless of which bacterial strain wasresent in the culture (Supplemental Figure 1).

In studying responses of DCs exposed to bacilli, our goalsere to (1) use bacilli grown under conditions to induceirulence factor expression, including a fully developed cap-ule in appropriate strains, prior to initiation of contact withCs; (2) allow sufficient time for appropriate strains to ex-ress and secrete toxins during in vitro cultures with DCs;nd (3) avoid significant losses in DC viability that wouldomplicate interpretation of data. Pilot studies determinedhat addition of ciprofloxacin 3 hours postinfection allowedhese conditions to be met. In culture medium without DCs,ll five strains proliferated at similar rates (data not shown).hen co-cultured with MDDCs, although bacterial numbers

f each strain increased over the 3-hour incubation period,capsular capBCAD � bacilli proliferated at a lower ratehan UT500 or any of the toxin mutant strains (Figure 1B). Inontrast, when co-cultured with mDCs, capBCAD�, UT500,nd toxin mutant bacilli proliferated at similar rates (FigureD). These findings suggest that MDDCs, but not mDCs, pos-ess B. anthracis growth–limiting functions that are inhibitedy capsule but unaffected by either toxin. MDDCs and mDCsncubated with bacilli showed slight viability decreases at 24ours postinoculation relative to unexposed controls, butecreases were similar whether incubated with UT500 or anyf the mutant strains (Supplemental Figure 1). pDCs showedarger viability decreases relative to unexposed controlshan did MDDCs or mDCs, but viabilities were similar whetherncubated with UT500 or mutant strains.

uman MDDC, mDC and pDC cytokine secretionostexposure to spores and bacilli of B. anthracisarent strain UT500

T500 spores—DC subsets. We first determined for each DCype whether UT500 spores stimulate cytokine secretion.PS was used as a positive control for MDDCs and mDCs, andnfluenza A virus for pDCs. In MDDCs and mDCs, UT500 sporestimulated large increases in secretion of proinflammatoryytokines including IL-12p40, tumor necrosis factor (TNF)–�,nd IL-6 (Figures 2A and 2B), as well as MIP-1�, MIP-1�, IL-8,L-10, GM-CSF, RANTES, and GRO-� (data not shown), rang-ng from one to two orders of magnitude higher than unex-osed controls. No increases in IL-1�, IL-1�, G-CSF, IFN-�, or

otaxin secretion were observed (data not shown). In pDCs,










F(seE htly o

555B. anthracis virulence factors and human dendritic cells

T500 spores failed to stimulate elevated cytokine secre-ion, in contrast to influenza A virus which stimulated largemounts of IFN-� and TNF-� (Figure 2C).

T500 bacilli—DC subsets. In MDDCs and mDCs, UT500 ba-illi, like UT500 spores, stimulated large increases in secre-ion of IL-12p40, TNF-�, and IL-6, ranging from one to tworders of magnitude higher than unexposed controls (FiguresA and 3B). UT500 bacilli failed to stimulate pDC cytokineecretion (Figure 3C).

ole of capsule in MDDC, mDC, and pDC cytokineecretion after exposure to spores and bacilli

pores—DC subsets. We next asked whether capsule forma-ion impacts cytokine secretion by DCs exposed to spores.apBCAD� spores, which are genetically incapable of form-ng capsule, stimulated several-fold higher IL-12p40, IL-6nd TNF-� secretion than UT500 spores in mDCs, but not inDDCs (Figures 4A and 4C; data not shown). Nevertheless, inoth MDDCs and mDCs, capBCAD� spores stimulated signif-cantly higher cytokine secretion than unexposed controlsdata not shown). Like UT500 spores, capBCAD� spores failedo stimulate IFN-� or IL-6 secretion by pDCs (Figure 4E).

acilli—DC subsets. capBCAD� bacilli stimulated several-

igure 1. Bacterial colony-forming units (CFU) in spore andMDDCs) and primary myeloid dendritic cells (mDCs). Bacteria wpores (A, C) or bacilli (B, D), and addition of ciprofloxacin, forxperiments, 3 hours for bacilli experiments). Results shown arerror bars indicate standard error of mean. Data points are slig

old higher IL-12p40, IL-6, and TNF-� secretion than UT500 l

acilli not only from mDCs but also from MDDCs (Figure 4Bnd 4D; data not shown). capBCAD� bacilli failed to stimu-ate pDC secretion of any cytokines, including TNF-� (FigureF; data not shown).

ole of toxins in MDDC, mDC, and pDC cytokineecretion after exposure to spores and bacilli

pores—DC subsets. In MDDCs and mDCs, LF-/EF-deficientpores stimulated small, but statistically significant, in-reases in IL-12p40 and IL-6 secretion relative to UT500pores (Figures 5A and 5C). In contrast, single-toxin mutantpores deficient in only LF or only EF stimulated similar lev-ls of cytokine secretion as UT500 spores in both MDDCs andDCs, suggesting that the two toxins function redundantly

o inhibit cytokine secretion. Again, pDCs showed no cyto-ine response to spores deficient in either or both toxinsFigure 5E).

acilli—DC subsets. In MDDCs and mDCs, LF-/EF-deficientacilli stimulated several-fold increases in IL-12p40, IL-6 andNF-� secretion relative to UT500 bacilli, although in MDDCshe increase in IL-6 secretion was not statistically significantFigures 5B and 5D; data not shown). EF-deficient bacillitimulated similar levels of IL-12p40 and IL-6 as UT500 bacillin MDDCs and mDCs. LF-deficient bacilli stimulated similar

lli experiments with human monocyte-derived dendritic cellsuantified by plating for CFU at timepoints between addition of(A, B) and mDC (C, D) cultures (1 hour postinfection for spore

ages of two (spore) or three (bacilli) independent experiments.ffset along x-axis to aid viewing.

baciere qMDDCaver

evels of IL-12p40 and IL-6 as UT500 bacilli in MDDCs, but






Fwpofcc 1.

556 A.C. Hahn et al.

ignificantly higher levels in mDCs. Interestingly, in mDCs,F-deficient bacilli stimulated a similar amount of IL-6 se-retion as LF-/EF-deficient bacilli, but less than half themount of IL-12p40. This result suggests that in mDCs, LFcts independently of EF to inhibit IL-6 secretion, but com-ines with EF to inhibit IL-12p40 secretion. pDCs showedtatistically significant increases in IFN-� and IL-6 secretionn response to LF-/EF- bacilli as compared with UT500 bacilliFigure 5F), but IL-6 quantities were negligible comparedith MDDCs and mDCs exposed to LF-/EF- bacilli, and IFN-�uantities were only slightly above the limit of detection.


n human beings, B. anthracis causes three forms of anthraxisease according to the portal of entry—namely, subcuta-eous, gastrointestinal, and inhalational. Inhalational an-hrax, in particular, is associated with a high mortality rate,nd is the form most relevant to biologic warfare. In thisorm, B. anthracis proliferates to high titers within severalays of spore deposition in the lungs, indicating the failuref innate immunity. Studies point toward inhibition of mul-iple innate immune functions of several cell types by thehree primary B. anthracis virulence factors—namely, cap-ule, LT, and ET. Among innate immune cells are DCs, whichecrete cytokines in response to pathogen-associated molec-lar patterns (PAMPs) [30]. In the mouse model, lung DCsave been implicated as the primary cell to take up andarry deposited spores to draining lymph nodes [31]. Opti-

igure 2. Human DC cytokine secretion after exposure to spoere measured in cell culture supernatants 24 hours after sporrimary pDCs. As positive controls, Escherichia coli LPS was addef 10 to pDC cultures. Graphs depict mean concentrations fromrom a different individual human donor. Error bars indicateoncentration differences between DCs exposed to UT500 sporealculated by the one-tailed paired t-test; *p � 0.05, **p � 0.0

al DC responses require a sequence of events frequently p

argeted by viral and bacterial virulence factors. This se-uence includes initiation of contact with pathogens, phago-ytosis, phagolysosome formation and degradation of theathogen, and activation of signaling pathways leading toytokine secretion and cell maturation [30,37,38]. Weought to extend previous work into the effect of B. anthra-is virulence factors on DCs [19,21,22,27,32] by addressingeveral issues relevant to human anthrax infection: (1) doesfully virulent, pXO1� pXO2� B. anthracis strain stimulateuman DC cytokine secretion; (2) do human DCs respondifferently to spores than to bacilli; (3) do the primary B.nthracis virulence factors, in particular capsule, inhibit DCesponses; and (4) do cytokine responses vary between DCubtypes?

Two prominent DC categories include resident DCs re-ruited from the bloodstream to undiseased tissue duringhe course of normal homeostasis to act as sentinels, andnflammatory DCs recruited to diseased tissue as monocyteshat subsequently undergo cytokine-induced differentiation39,40]. Therefore, primary mDCs and pDCs, which are DCsirectly purified from peripheral blood under steady-stateonditions, may be better surrogates for resident human DCshan are MDDCs, which by virtue of cytokine-induced differ-ntiation may closely resemble inflammatory DCs [39]. Be-ause both resident and inflammatory DCs are likely to beelevant to B. anthracis pathogenesis, we examined re-ponses of surrogates for both types.

Each form of B. anthracis performs specific functions in

f B. anthracis strain UT500 (capBCAD�, LT�, ET�). Cytokinesosure at an MOI of 1 for (A) MDDCs, (B) primary mDCs, and (C)1 �g/ml to MDDC and mDC cultures, and influenza virus at a MOI(MDDC and pDC) or six (mDC) independent experiments, eachdard error of the mean. Statistical significance of cytokineS or influenza, versus the respective unexposed DC type, were

res oe expd atfivestan

s, LP

athogenesis. A relatively small number of spores cause in-








557B. anthracis virulence factors and human dendritic cells

ection and germinate shortly thereafter, while bacilli pro-iferate, produce capsule and toxins, and eventually dissem-nate systemically during the remainder of the disease.herefore, the innate immune response to bacilli, especiallyuring early stages of bacillary proliferation in the lymphode, may be as or more important than the response topores. Previous studies examined DCs exposed to spores21–23,27,32], but bacilli and spores bear several obvioustructural and compositional differences [41] and hence maylicit distinct DC responses. In addition, virulence factorsould affect responses to spores or bacilli differently de-ending on how each form stimulates DC signaling pathways.herefore, we assessed DCs responses to spores and bacilli ineparate experiments.

We found that spores from the fully virulent pXO1�XO2� strain UT500 stimulated cytokine secretion in MDDCsnd mDCs, consistent with previous findings using the pXO2-eficient Sterne 7702 strain [22,27,32]. We conclude thatxposure to B. anthracis spores rapidly activates signalingathways leading to cytokine secretion in human mDCs andDDCs. Previous work showing that killed spores activateurine splenocyte cytokine secretion through multipleyD88-dependent pathways [42] raises the possibility that

ive spores activate analogous pathways in human mDCs andDDCs, but relevant pathogen related receptors (PRRs) andAMPs await identification.

We found that UT500 bacilli also stimulated cytokine se-retion by both mDCs and MDDCs. Although we cannot ruleut the possibility that DCs were stimulated by bacterial

igure 3. Cytokine secretion by human DCs after exposureytokines were measured in cell culture supernatants 24 hours af 1. As positive controls, LPS was added at 1 �g/ml to MDDC anraphs for each DC type depict mean concentrations from at leaach from a different individual human donor. Error bars indiconcentration differences between UT500 bacilli-exposed DCs,ne-tailed paired t-test; *p � 0.05, **p � 0.01.

egradation products resulting from ciprofloxacin killing, i

his result suggests that presence of capsule alone does notrevent mDC or MDDC responses. Previous work implicatesLR2 [43] and multiple MyD88-dependent pathways [42,43]n the response to killed bacilli, suggesting standard Gram-ositive PAMPs such as PGN, lipoteichoic acid (LTA), or pu-ative B. anthracis lipoproteins [44] as candidate ligands.

ole of capsule in regulating DC cytokine secretion. Al-hough capsule is associated primarily with bacilli, the quan-ities of capsule present in dormant spores or produced dur-ng germination have not been well-characterized andemain a potential factor in DC responses. We found thatapBCAD� spores stimulated increased cytokine secretionelative to UT500 spores in mDCs, but not in MDDCs. Thisifference between MDDC and mDC responses suggests thatither MDDCs are more resistant to capsular effects, or thatDDCs allow less capsule production during co-incubation

han do mDCs, possibly because of greater or more rapidDDC phagocytic and/or killing ability as a result of in vitroaturation. One implication of these findings is that capsuleroduction may begin at the outset of infection to inhibit theesponse of lung-resident DCs to inhaled spores.

Our findings showed that capBCAD� bacilli stimulatedncreased cytokine secretion in both MDDCs and mDCs rela-ive to UT500 bacilli. In fact, capsule impacted cytokineecretion in MDDCs and mDCs to a similar degree as theombined effect of LF and EF, in keeping with the impor-ance of capsule to overall virulence in animal model studies11]. How capsule inhibits MDDC and mDC cytokine secretion

cilli from B. anthracis strain UT500 (capBCAD�, LT�, ET�).exposure of (A) MDDCs, (B) mDCs, and (C) pDCs to bacilli at MOIDC cultures, and influenza virus at a MOI of 10 to pDC cultures.x (MDDC), five (mDC) or three (pDC) independent experiments,tandard error of the mean. Statistical significance of cytokineus the respective unexposed DC type, were calculated by the

to bafterd mst si

ate svers

s unclear but could occur through a variety of mechanisms.



Fcsamv the


558 A.C. Hahn et al.

s the outermost layer of bacilli, capsule is positioned tohysically block host PRRs from internally located bacterialAMPs. Furthermore, capsule has been shown to inhibithagocytosis by neutrophils and macrophages [13,45] andould act similarly on MDDCs and mDCs. Reduction in DChagocytosis could result in diminished cytokine secretion

igure 4. Cytokine secretion by human DCs after exposure tapBCAD operon required for capsule formation. Cytokines werpores or bacilli at MOI of 1 for (A, B) MDDCs, (C, D) mDCs, and (Et least three independent experiments, each from a differentean. Statistical significance of cytokine concentration differe

ersus UT500 spores or bacilli, respectively, were calculated by

igure 5. Cytokine secretion by human DCs after exposure to sf genes required for production of Lethal Toxin (LT-), Edema Toulture supernatants 24 hours after exposure to spores or bacillor each DC type depict mean concentrations from at least threonor. Error bars indicate standard error of the mean. Statisticxposed to toxin-mutant spores or bacilli, versus UT500 spores

-test; *p � 0.05, **p � 0.01.

y decreasing the surface area of DC-pathogen contact, pre-enting bacterial digestion and concomitant PAMP release,r allowing increased LT and ET production by promotingacterial survival and proliferation. Indeed, we observedhat encapsulated bacilli strains, regardless of toxin geneeletions, showed higher proliferation rates than capBCAD�

res or bacilli from a UT500-derived mutant strain lacking theasured in cell culture supernatants 24 hours after exposure toDCs. Graphs for each DC type depict mean concentrations fromvidual human donor. Error bars indicate standard error of thebetween DCs exposed to capBCAD� mutant spores or bacilli,one-tailed, paired t-test; *p � 0.05, **p � 0.01.

s or bacilli of UT500-derived mutant strains containing deletionsET-), or both toxins (LT-/ET-). Cytokines were measured in cellOI of 1 for (A, B) MDDCs, (C, D) mDCs, and (E, F) pDCs. Graphs

ependent experiments, each from a different individual humangnificance of cytokine concentration differences between DCsacilli, respectively, were calculated by the one-tailed paired

o spoe me, F) pindi


porexin (

i at Me indal sior b











559B. anthracis virulence factors and human dendritic cells

acilli during incubation with MDDCs. However, the inhibi-ory effect of capsule on cytokine secretion is unlikely to beediated solely by increased toxin production as a result of

reater bacterial proliferation, because in mDC cultures,apBCAD� bacilli stimulated markedly higher cytokine se-retion than UT500 bacilli but proliferated to a similar de-ree as UT500 bacilli. Clearly, our results warrant furthertudies into mechanisms of the capsule’s effect on humanCs.

ole of toxins in regulating DC cytokine secretion. InDDCs and mDCs, LF-/EF-deficient spores stimulated smallut statistically significant increases in IL-12p40 and IL-6ecretion relative to UT500 spores, indicating that both tox-ns were secreted in sufficient quantities prior to ciprofloxa-in killing to have an appreciable impact on DCs. Indeed,romoters of genes encoding PA, LF, and EF are active soonfter spore germination in murine alveolar macrophages26]. In contrast, single-toxin mutant spores deficient in ei-her LF or EF stimulated similar levels of IL-12p40, IL-6, andNF-� secretion by MDDCs and mDCs as did UT500 spores,ndicating that at least in this experimental setting the ef-ects of LF and EF on cytokine secretion are redundant and ofimilar magnitude. This result is consistent with previouseports showing purified LT and ET are each capable of in-ibiting cytokine secretion [21,22,25].

LF-/EF-deficient bacilli, like LF-/EF-deficient spores,timulated greater cytokine secretion than UT500 bacilli inoth MDDCs and mDCs. Notably, differences in elicited cy-okine secretion between the LF-/EF-deficient strain andT500 were greater in bacilli experiments than in sporexperiments. We attribute this difference to the longer co-ncubation period of DCs with bacilli than with spores prioro addition of antibiotic, and the fact that vegetative bacillire already in a toxin-producing state while spores musterminate before beginning toxin production. Hence weould predict that longer incubation times of spores withCs prior to addition of ciprofloxacin would lead to largerifferences in elicited cytokine secretion between LF-/EF-pores and UT500 spores.

In MDDCs, single-toxin mutant bacilli lacking either LF orF stimulated similar levels of IL-12p40, IL-6, and TNF-�ecretion as UT500 bacilli, indicating that the two toxinsave redundant effects of similar magnitude. Like MDDCs,DCs showed no increase in TNF-� secretion in response to

ingle mutant LF-deficient or EF-deficient bacilli as com-ared with UT500 bacilli. However, mDCs secreted signifi-antly higher quantities of IL-12p40 and IL-6 in response toF-deficient bacilli than to UT500 bacilli, but similar quan-ities in response to EF-deficient bacilli as to UT500 bacilli,ndicating a larger role for LF than EF in inhibiting secretionf these cytokines by mDCs.

Our finding that LF and EF each inhibit MDDC and mDCytokine secretion agrees with a previous study using mouseone marrow-derived DCs exposed to spores from toxin mu-ants derived from the pXO1� pXO2- Sterne 7702 strain [22].he previous study differed slightly from ours in finding thatingle toxin-deficient mutants stimulated significant eleva-ions in cytokine secretion as compared with the referencetrain, whereas our spore exposure experiments did not.dditionally, the previous study found that EF, but not LF,

nhibited IL-12 secretion, whereas our MDDC and mDC bacilli D

xposure experiments found that both LF and EF inhibit IL-2p40 secretion. These differences could result from differ-nces between mouse and human cells, tissue of DC origin,ffects of other genes on pXO2 (which is absent from theterne strain), or antibiotic properties. Gentamicin, used inhe previous study, penetrates cells less readily than cipro-oxacin [46], which we used to simulate effects in humansollowing recommended anthrax treatment guidelines. Gen-amicin’s reduced cellular penetration may allow a longerime period for bacterial survival and toxin production, thusagnifying the effects of each toxin.We found that pDC cytokine secretion was minimal in

esponse to spores and bacilli even when capsule or bothoxins were absent. This lack of responsiveness is consistentith pDC expression of TLR7 and TLR9, which detect single-

tranded RNA and CpG DNA motifs respectively, to the ex-lusion of other TLRs [47,48]. In addition, pDCs respond toLR7 and TLR9 agonists with large amounts of IFN-� secre-ion, a response more appropriate for viral than for bacterialnfection [49]. Therefore, we predict that direct interactionetween human pDCs and B. anthracis has minimal impactn the innate immune response during early stages of infec-ion.

n vivo significance. In these studies, antibiotic was addedo prevent bacterial overgrowth and allow assessment ofarly DC responses. In the absence of antibiotic, in vivo toxinoncentrations will likely exceed those present in our exper-ments as vegetative bacilli continue toxin production andhe effective MOI rises as a result of bacterial replication.iven the concentration-dependent effects of each toxin

21,22], we expect that increases in in vivo incubation timesnd bacterial numbers would reduce DC cytokine secretiono levels below those observed in our experiments becausef direct inhibition of cytokine secretion and/or DC apopto-is [19] or lysis [50]. Other in vivo factors potentially rele-ant to pathogenesis include the contribution of connectiveissue together with blood and lymph flow to distribution ofacilli and toxins.

Taking these considerations into account, our experi-ents suggest that resident lung DCs respond to spore up-

ake with a burst of cytokine secretion as they migrate andnter lymph nodes. If the spore burden is small, the resultingost innate immune response may be adequate to clear in-ection. However a sufficiently large spore burden may gen-rate enough LT, ET, and capsule to inhibit cytokine secre-ion and establish an environment favorable to bacterialurvival and replication. As bacterial numbers and incuba-ion times increase, elevated concentrations of LT and ETould inhibit functions of inflammatory DCs and other im-une cells recruited from the bloodstream. Systemic toxin

oncentrations would eventually increase and allow bacillio disseminate and seed target organs.

Our studies raise several questions. LT and ET have beenhown to disrupt MAPK and cAMP pathways, respectively,ut it is not known whether they act upon a common down-tream signaling factor controlling cytokine secretion or onistinct factors, nor is it known whether they affect secre-ory pathways. Although we used cytokine secretion as aeadout for DC activation, future studies should also assessffects of LT, ET, and capsule on other processes integral to

C activation such as antigen presentation.



























560 A.C. Hahn et al.

In summary, we found that both spores and bacilli from aenetically complete pXO1� pXO2� B. anthracis straintimulated secretion of proinflammatory cytokines in humanDDCs and mDCs. LF and EF exerted inhibitory effects on

pore- and bacilli-induced cytokine secretion that were re-undant in MDDCs, but LF had a markedly stronger effecthan EF in mDCs. Our findings are consistent with a model inhich infecting B. anthracis spores produce capsule and tox-

ns soon after germination to compromise the ability of res-dent DCs to initiate innate immune functions that wouldtherwise help eliminate spores. Bacilli formed subse-uently in lymph nodes continue to produce capsule andoxins in order to inhibit immune cell recruitment and inac-ivate any cells that manage to be recruited.


e thank Gwyneth Olson, Cheryl Miller, Elizabeth McKown,harlene Hensler, and the UNM HSC Flow Cytometry Facilityor providing DCs, and Lorena Diehl for performing Luminexeasurements. Support was provided by NIH grants NIAID19 AI57234 (CRL, AH, MFL), NIAID PO1 AI56295 (CRL, AH,FL), and P50-HL56384 T32 (MFL).

ppendix. Supplementary data

Supplementary data associated with this article can beound, in the online version, at doi:10.1016/j.humimm.008.06.012.


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