Exposure to Bacillus anthracis Capsule Results in Suppression ofHuman Monocyte-Derived Dendritic Cells

Tanya M. Jelacic,a Donald J. Chabot,a Joel A. Bozue,a Steven A. Tobery,a Michael W. West,a Krishna Moody,c De Yang,b

Joost J. Oppenheim,b Arthur M. Friedlandera

United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USAa; Laboratory of Molecular Immunoregulation, Frederick NationalLaboratory for Cancer Research, Frederick, Maryland, USAb; Department of Microbiology, Boston University, School of Medicine, Boston, Massachusetts, USAc

The antiphagocytic capsule of Bacillus anthracis is a major virulence factor. We hypothesized that it may also mediate virulencethrough inhibition of the host’s immune responses. During an infection, the capsule exists attached to the bacterial surface butalso free in the host tissues. We sought to examine the impact of free capsule by assessing its effects on human monocytes andimmature dendritic cells (iDCs). Human monocytes were differentiated into iDCs by interleukin-4 (IL-4) and granulocyte-mac-rophage colony-stimulating factor (GM-CSF) over 7 days in the presence of capsule derived from wild-type encapsulated B. an-thracis Ames (WT) or a control preparation from an isogenic B. anthracis Ames strain that produces only 2% of the capsule ofthe WT (capA mutant). WT capsule consistently induced release of IL-8 and IL-6 while the capA mutant control preparation elic-ited either no response or only a minimal release of IL-8. iDCs that were differentiated in the presence of WT capsule had in-creased side scatter (SSC), a measure of cellular complexity, when assessed by flow cytometry. iDCs differentiated in the presenceof WT capsule also matured less well in response to subsequent B. anthracis peptidoglycan (Ba PGN) exposure, with reducedupregulation of the chemokine receptor CCR7, reduced CCR7-dependent chemotaxis, and reduced release of certain cytokines.Exposure of naive differentiated control iDCs to WT capsule did not alter cell surface marker expression but did elicit IL-8.These results indicate that free capsule may contribute to the pathogenesis of anthrax by suppressing the responses of immunecells and interfering with the maturation of iDCs.

Bacillus anthracis is the causative agent of anthrax and an im-portant biothreat agent. The virulence of B. anthracis derives

primarily from the products of two plasmids, pX01, which carriesthe genes for lethal toxin and edema toxin (1), and pX02, whichcarries the genes required for synthesizing capsule (2, 3). B. an-thracis capsule covers the outer surface of the bacilli and protectsthem from phagocytosis by immune cells (4–6). While mostbacterial capsules are polysaccharides, B. anthracis capsule is ahomopolymer composed entirely of D-glutamic acid residues con-nected by � linkages (7). These unusual attributes make B. anthra-cis capsule both relatively resistant to degradation by mammalianproteases and a poorly immunogenic thymus-independent type 2antigen (8, 9).

In in vitro broth culture and during infection, B. anthracis ba-cilli release capsule fragments. These free capsule fragments canaccumulate to significant concentrations in the blood of mori-bund animals (7). Serum concentrations of B. anthracis capsulegreater than 500 �g/ml have been reported in mice (10), and con-centrations greater than 1 mg/ml have been reported in rhesusmacaques (11). Concentrations in local lesions could potentiallybe as high or even higher. Capsule free from the bacillus has beendemonstrated to accumulate in animal tissues (12, 13). Early ex-periments with capsule recovered from the plasma and exudatesof moribund guinea pigs showed that purified capsule could en-hance the virulence of spores when coinjected with them (14).More recently, coinjection of purified capsule with an attenuatedmutant of B. anthracis restored its virulence (15). We have alsoobserved that free capsule can inhibit the antimicrobial activity ofhuman defensins (D. K. O’Brien and A. M. Friedlander, unpub-lished data). Given this evidence that free capsule as well as bacil-lus-bound capsule contributes to virulence, we hypothesized thatthe shed capsule might contribute to pathogenesis by interfering

with innate immune responses as has been observed with the an-thrax toxins (16). Therefore, we sought to examine the effect offree capsule on human monocytes and immature dendritic cells(iDCs). Capsule released by B. anthracis into the medium duringin vitro growth was harvested from broth culture supernatants ofwild-type encapsulated B. anthracis Ames (WT). Human mono-cytes were differentiated into iDCs in the presence of the purifiedfree capsule over the course of 7 days. Cytokine levels were mea-sured on days 3 and 7 to assess the nature of the response overtime. We found that WT capsule consistently elicited interleu-kin-8 (IL-8) and IL-6 on days 3 and 7. The resulting iDCs had analtered phenotype showing increased side scatter (SSC) when theywere assessed by flow cytometry. Furthermore, maturation ofthese iDCs in response to B. anthracis peptidoglycan (Ba PGN)was impaired as indicated by reduced CCR7 expression, reducedchemotaxis toward the CCR7 ligands CCL19 and CCL21, andreduced cytokine secretion. These results indicate that capsuleshed from B. anthracis acts on cells of the human innate immune

Received 31 March 2014 Accepted 14 May 2014

Published ahead of print 2 June 2014

Editor: S. R. Blanke

Address correspondence to Tanya M. Jelacic, tanya.obreiter@us.army.mil, orArthur M. Friedlander, arthur.friedlander@us.army.mil.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.01857-14.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.01857-14

The authors have paid a fee to allow immediate free access to this article.

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system, inhibiting the function of DCs, and thus may play a role inthe pathogenesis of anthrax.

MATERIALS AND METHODSB. anthracis strains. B. anthracis Ames was from the USAMRIID collec-tion. An isogenic capsule-reduced strain with inactivated capA (capA mu-tant) was derived from B. anthracis Ames as follows. A 1.24-kb DNAfragment containing the capA gene was amplified from the B. anthracisAmes genome. The capA gene was inactivated by the insertion of thenonpolar kanamycin fragment (17) into the BamHI site located 700 bpdownstream of the ATG site and cloned into the Escherichia coli/Bacillussubtilis shuttle vector pEO-3 (18, 19). The resulting plasmid, designatedpKLM2, was passaged through the dam dcm strain of E. coli GM2683. Toconstruct the isogenic capA mutant strain of B. anthracis, pKLM2 was firsttransformed into B. anthracis Ames by electroporation and selected bygrowth in the presence of erythromycin at 30°C. Next, the plasmid con-taining the insertionally inactivated capA gene was integrated into pXO2by selection and growth at 42°C in the presence of kanamycin (18). Toresolve the plasmid from pX02, mutants were selected as previously de-scribed (20) and confirmed by PCR analysis.

Capsule preparation. Capsule was harvested from broth culture su-pernatants of the WT and capA mutant B. anthracis strains as follows. Asingle colony was used to inoculate brain heart infusion (BHI) broth.After overnight growth on a shaker at 37°C, the culture was diluted 1:1,000in BHI broth containing 0.8% (wt/vol) sodium bicarbonate in a flask witha filter cap. Broth cultures were incubated at 37°C for 20 to 24 h in a shakerincubator with an atmosphere containing 20% carbon dioxide, and thenthe bacterial cells were pelleted at 3,000 � g for 30 min. Supernatants werefiltered (0.2 �m), brought to a final concentration of 1 mM magnesiumchloride and 2.5 U/ml Benzonase (EMD Chemicals, Gibbstown, NJ), andincubated overnight at 4°C. Trichloroacetic acid was then added to 10%(vol/vol). After 1 h on ice, mixtures were centrifuged as before, and thesupernatant was neutralized with 10% (wt/vol) sodium acetate powder.Three volumes of 95% ethanol was added to precipitate the capsule over-night at �20°C. After centrifugation as before, the capsule-containingpellets were washed three times with 70% ethanol. The pellets from WTand capA mutant cultures were dried overnight and then dissolved inequivalent volumes of endotoxin-free 100 mM sodium phosphate buffer.Equivalent cultures of WT and capA mutant B. anthracis were grown andharvested in parallel. The preparation from the capsule-reduced capAmutant strain was used as a control for any material that copurified duringthe purification process. The capsule preparations were shown to be freeof DNA after being run on agarose gels and stained with ethidium bro-mide and to be free of protein after being run on SDS-PAGE gels andstained with Coomassie blue. All capsule preparations were tested forendotoxin contamination using a Limulus amebocyte lysate (LAL) end-point chromogenic assay (Lonza, Walkersville, MD) before use in cellculture.

Capsule quantitation was based upon the WT preparation. The prep-aration from the capA mutant strain, which was cultured and purified inparallel, was resuspended in an equivalent volume. The capA mutantstrain produces 2% of the amount of capsule produced by the WT strain asdetermined by amino acid analysis (data not shown). The relativeamounts of capsule in the two strains were also analyzed by agarose gelelectrophoresis as previously described (21).

Peptidoglycan testing. The WT and capA mutant capsule prepara-tions were tested for peptidoglycan by Immunetics, Inc. (Boston, MA),using the BacTx assay.

Ba PGN. Ba PGN was purified similarly to the method of Candela andFouet (22). B. anthracis Ames was grown overnight on Trypticase soy agarat 37°C. A single colony was used to inoculate BHI broth for overnightgrowth in a 37°C shaker incubator. Ten microliters was inoculated into100 ml nutrient broth with yeast extract (8 g nutrient broth, 3 g yeastextract per liter) without added bicarbonate or carbon dioxide. Afterovernight growth in a 37°C shaker incubator, bacteria were centrifuged at

2,500 � g for 30 min. The pellet was resuspended in 20 ml of TNS (50 mMTris, pH 7.4, 150 mM NaCl, 1% SDS) and put in a 100°C water bath for 10min. The boiled bacteria were pelleted, resuspended, and boiled a secondtime. After the second round of boiling, the bacteria were pelleted andthen resuspended in 50 mM Tris, pH 7.4. The suspension was sonicated,frozen overnight at �70°C, and ultracentrifuged at 40,000 � g for 20 min.The pellet was resuspended in TNS, boiled, and ultracentrifuged again.The resulting pellet was resuspended in 10 ml 50 mM Tris, pH 7.4, with200 �g proteinase K and incubated overnight at 4°C. One milliliter of 10%SDS was added, and the sample was boiled and washed four times withsterile water for injection. The sample was then dried in a hood and re-suspended in sterile water. Amino acid analysis results for Ba PGN (Al-phalyse Inc., Palo Alto, CA) were very similar to those reported by Langeret al. (23). PGN purified from B. anthracis Ames did not have more glu-tamic acid than did PGN purified from B. anthracis �ANR (pX01�

pX02�), which lacks the capsule, indicating that there is no significantamount of capsule associated with the purified Ba PGN (data not shown).All Ba PGN preparations were tested for endotoxin contamination usingan LAL endpoint chromogenic assay as described above before use in cellculture.

Monocyte-DC cultures. Human monocytes were purified by Percolldensity gradient centrifugation from leukopacks obtained from the De-partment of Transfusion Medicine (Clinical Center, National Institutes ofHealth, Bethesda, MD). Purified monocytes were washed three times withsterile Dulbecco’s phosphate-buffered saline (Gibco, Grand Island, NY)and differentiated into iDCs by cultivation at 37°C in 5% CO2 in thefollowing medium for 7 days: RPMI 1640 (Mediatech, Manassas, VA)supplemented with 2 mM glutamine (Gibco), 25 mM HEPES (Gibco),10% fetal bovine serum (FBS) (HyClone, Logan, UT), 50 �M �-mercap-toethanol, 50 ng/ml IL-4 (Peprotech, Rocky Hill, NJ), and 50 ng/ml gran-ulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech).Experimental cultures included up to 1,000 �g/ml of B. anthracis WTcapsule in 100 mM sodium phosphate buffer. Control cells were culturedwithout capsule in the presence of an equivalent volume of 100 mM so-dium phosphate buffer or in differentiation medium alone. Ten nano-grams of lipopolysaccharide per milliliter (from E. coli 055:B5; Sigma, St.Louis, MO) diluted in 100 mM sodium phosphate buffer was used as apositive control in the 7-day differentiation experiments. On days 3 and 5,50% of the medium was replaced with fresh differentiation medium sup-plemented with the same concentration of test material in order to main-tain the same conditions throughout the experiment. On day 7 when themonocytes had differentiated into iDCs, either the experiment was con-cluded or 50% of the medium was replaced with fresh medium plus 1�g/ml Ba PGN or 100 ng/ml LPS to induce maturation of the iDCs tomature DCs (mDCs). For experiments with naive differentiated iDCs,cells were cultured in differentiation medium alone for 7 days with feedson days 3 and 5, stimulation on day 7, and harvest of medium samples onday 9.

Cytokine measurement. Duplicate wells of medium samples were as-sayed for human IL-1�, IL-6, IL-8, IL-10, IL-12p70, IL-2, gamma inter-feron (IFN-�), GM-CSF, and tumor necrosis factor alpha (TNF-�) usinghuman proinflammatory 9-plex platform ultrasensitive plates (MesoScale Discovery, Gaithersburg, MD). The plates were analyzed using aSector Imager 2400 (Meso Scale Discovery) according to the manufactur-er’s protocol. Data are shown as the mean pg/ml standard error of themean (SEM) or as fold increases in cytokines in cells exposed to capsulecompared to untreated control cells. IL-23 was measured using the hu-man IL-23 enzyme-linked immunosorbent assay (ELISA) Ready SET Go!kit (eBioscience, San Diego, CA) as mean pg/ml SEM according to themanufacturer’s protocol. Statistical significance of the data presented asmean pg/ml SEM was determined by two-tailed t tests comparing themeans for the treated samples to those of the untreated control (GraphPadSoftware, La Jolla, CA). A P value of 0.05 was considered significant.

Flow cytometry. After differentiation of monocytes to iDCs or matu-ration of iDCs to mDCs, the cells were stained with antibodies specific for

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human major histocompatibility complex class I (MHC I), MHC II,CD83, or CCR7 or with isotype control antibodies purchased from BDPharmingen (San Jose, CA). Flow cytometry experiments were run in aFACSCalibur cytometer (BD Biosciences, Billerica, MA), and the datawere analyzed using Cell Quest Pro software (BD Biosciences). Statisticalsignificance of the data was determined by two-tailed t tests comparingaverage mean fluorescence intensities (MFIs) for the capsule-treated sam-ples to those for the controls (GraphPad Software). Statistical significanceof differences in SSC between capsule-treated cells and controls was sim-ilarly determined by two-tailed t tests. A P value of 0.05 was consideredsignificant.

Chemotactic activity assay. After maturation of iDCs to mDCs, cellswere pelleted and resuspended in binding medium (RPMI 1640 supple-mented with 1% bovine serum albumin) at 5 � 105 live cells/ml. Cellswere added to the inner chambers of transwells with 5-�m-pore polycar-bonate membranes (Corning, Corning, NY). Binding medium alone orsupplemented with 10 ng/ml recombinant human CCL19 or recombinanthuman CCL21 (Peprotech, Rocky Hill, NJ) was added to the outer cham-ber. Cells were incubated in the transwells for 4.5 h at 37°C and 5% CO2.The transwells were then aspirated and transferred to fresh wells contain-ing cell detachment solution (Cell Biolabs, Inc., San Diego, CA) and in-cubated for 30 min at 37°C and 5% CO2. The transwells were then dis-carded. Duplicate samples of the combined medium and cell detachmentsolution containing the migrated cells for each cell treatment were trans-ferred to a black-walled 96-well plate (Corning) and lysed with lysis buffer(Cell Biolabs, Inc.) supplemented with CyQuant GR dye (Life Technolo-gies, Eugene, OR). After 20 min of incubation at room temperature, flu-orescence was read at 480 nm/520 nm in a SpectraMax M5 fluorescenceplate reader (Molecular Devices, Sunnyvale, CA). Chemotactic index wascalculated by dividing fluorescence units observed in the presence of theligands by fluorescence units observed in the absence of ligand. P valueswere determined by comparing the means of the chemotactic indices forthe mDCs of all four donors matured with or without WT capsule in atwo-tailed t test. A P value of 0.05 was considered significant.

RESULTSB. anthracis capsule elicits a distinct cytokine response fromhuman monocytes over the course of differentiation into iDCs.We first sought to determine whether or not B. anthracis capsulecould elicit a cytokine response from differentiating humanmonocytes. Human monocytes were cultured in medium supple-mented with human IL-4 and GM-CSF to promote their differen-tiation into iDCs over the course of 7 days. Capsule prepared fromthe WT strain of B. anthracis at concentrations of 250 to 1,000�g/ml or equivalent amounts of the preparation from the capAmutant strain were included in the differentiation medium. ThecapA mutant strain produced only 2% of the amount of capsule ofthe WT strain as determined by amino acid analysis (data notshown), and the relative amounts of capsule in the two prepara-tions are shown in Fig. 1. Cells cultured with equivalent volumesof the sodium phosphate control buffer or 10 ng/ml LPS served asnegative and positive controls, respectively. Medium samples col-lected on days 3 and 7 were assayed for human IL-8, IL-6, IL-10,IL-1�, IL-12p70, TNF-�, IL-2, and IFN-� to assess responses ofthe cells to the capsule over time. The results for a representativedonor are shown in Fig. 2. The WT capsule consistently elicitedstatistically significant IL-8 and IL-6 responses on day 3 (Fig. 2A,C, and D) in all six donors tested at 500 �g/ml (P range, 0.01 to0.0001 for IL-8 and for IL-6). In contrast, an equivalent amountof the capA mutant preparation, which contained all the potentialcopurifying material derived from the broth culture and harvestprocess, elicited only very low levels of IL-8 (Fig. 2B and C) fromthe six donors tested at 500 �g/ml (P range, 0.05 to 0.0001).

The capA mutant preparation also elicited very low levels of IL-6(Fig. 2B and D) from five out of six donors tested at 500 �g/ml (Prange, 0.05 to 0.001), including the one shown in Fig. 2. Threeout of six donors, including the one shown in Fig. 2, also exhibitedsignificant release of IL-1� in response to 500 �g/ml WT capsuleon day 3 (Fig. 2A and E, P range, 0.01 to 0.001), but only onetreated with an equivalent amount of the capA mutant prepara-tion did (P 0.05). Four out of six donors exhibited significantrelease of TNF-� in response to 500 �g/ml WT capsule on day 3(Fig. 2A and F; P range, 0.01 to 0.001), while only three of sixtreated with an equivalent amount of the capA mutant prepara-tion did (Fig. 2B and F; P range, 0.05 to 0.01). The cytokinerelease elicited by WT capsule was in all cases dramatically greaterthan that observed with the capA mutant preparation as illustratedby the donor shown in Fig. 2 (A versus B). Similar cytokine pro-files were observed on day 7 (data not shown). The WT capsuleelicited a dose-dependent cytokine response pattern that was dis-tinctly different from that elicited by LPS, which typically eliciteda combination of IL-1�, IL-10, and TNF-� in addition to IL-8 andIL-6 (Fig. 2G). Furthermore, LPS elicited larger amounts of IL-6than IL-8, while the reverse was observed with WT capsule. WTcapsule, even at the highest dose of 1,000 �g/ml, elicited fewercytokines and in smaller amounts than did LPS at 10 ng/ml (com-pare Fig. 2A and G).

FIG 1 Capsule expression is greatly reduced in the capA mutant B. anthracisstrain. Capsule was purified from broth culture supernatants as described inMaterials and Methods. Fifty micrograms of capsule from WT (lane 1) andcapA mutant (lane 2) per lane was electrophoresed on a 1% agarose gel, and thegel was stained with methylene blue.

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The cytokine responses to the WT capsule are not due to anybroth culture or harvest-derived components or contaminantssuch as endotoxin because the capA mutant preparation, whichwas subjected to the same culture and purification conditions inparallel, did not elicit comparable cytokine responses and bothpreparations have similarly low levels of endotoxin (0.25 endo-toxin units [EU]/ml). In addition, the cytokine pattern observedwith LPS is distinctly different from that seen with WT capsule asnoted above. Because capsule is covalently attached to the PGNlayer of the bacillus (22, 24), and because PGN has been reportedin the literature to be a potent inducer of cytokine release (23,25–28), PGN in the capsule preparation was also a possible stim-ulator of the cytokine responses. However, the WT capsule andcapA mutant preparations were found to contain only 0.05% and0.11% PGN, respectively. Thus, the cytokine release induced bythe WT capsule is not due to PGN, as it contained half the amountof PGN present in the relatively inactive capA mutant preparation.

Exposure to B. anthracis capsule during differentiationyields iDCs with a slightly altered phenotype. We hypothesizedthat since continuous exposure to capsule during differentiationelicited cytokine responses from the monocytes, it might affect thephenotype of the resulting iDCs. Capsule prepared from the WTstrain of B. anthracis at concentrations of 125 to 500 �g/ml orequivalent amounts of the preparation from the capA mutantstrain were included in the differentiation medium. On day 7, thephenotype of the differentiated iDCs was analyzed by flow cytom-etry. Naive control iDCs differentiated in the absence of capsuleexpressed high levels of MHC I and II but very little CD83 orCCR7 as shown with the donor in Fig. 3. Cells differentiated in thepresence of 500 �g/ml WT capsule did not show any significantchanges in cell surface marker expression compared to the naive

control cells (P � 0.05 for all markers, n � 6 donors, Fig. 3B), nordid the cells differentiated in the presence of an equivalent amountof the capA mutant preparation (P � 0.05 for all markers, n � 6donors, Fig. 3C). However, there was a dose-dependent increasein SSC in the cells exposed to WT capsule (Fig. 3D). This effect wasobserved in five out of six donors (P 0.01 for 500 �g/ml) in-cluding the one shown. The capA mutant preparation also in-creased the SSC over that of the untreated control (P 0.05 for the500-�g/ml equivalent), but without the dose dependence ob-served with WT capsule (data not shown).

Differentiation in the presence of capsule results in DCs withreduced CCR7 expression and reduced CCR7-dependent che-motaxis upon maturation with Ba PGN. Since iDCs differenti-ated in the presence of WT capsule did not have an entirely normalphenotype as evidenced by the increase in SSC, we hypothesizedthat they might not convert to an mDC phenotype in response toPGN as do normal iDCs when stimulated with a maturation factor(29). To test this, monocytes differentiated into iDCs in the pres-ence or absence of WT capsule (500 �g/ml) or an equivalentamount of the capA mutant preparation were subsequently stim-ulated with a maturing dose of Ba PGN (1 �g/ml) on day 7. Con-trol naive iDCs were maintained in differentiation medium alonewithout Ba PGN in order to retain the immature phenotype (Fig.4A). On day 9, medium samples were collected for cytokine anal-ysis (see below) and the cells were stained with fluorescently la-beled antibodies specific for human MHC I and II, plus CD83 andCCR7, cell surface markers that are associated with mDCs (29,30), and analyzed by flow cytometry. Day 9 naive control iDCsexpress high levels of MHC I and II and low levels of CD83 andCCR7 (Fig. 4A). Ba PGN induced maturation of naive controliDCs that were not exposed to capsule preparations, as indicated

FIG 2 B. anthracis capsule induces cytokine responses from differentiating human monocytes. (A to F) Differentiating human monocytes were exposed to 250to 1,000 �g/ml of WT capsule (A, C, D, E, and F) or an equivalent amount of capA mutant preparation (B, C, D, E, and F) for 7 days. Medium samples collectedon day 3 were analyzed for cytokines as described in Materials and Methods. (A and B) Data are presented as the fold increase over the basal cytokine levelsreleased by unstimulated control cells cultured in parallel. Data for IL-8 (C), IL-6 (D), IL-1� (E), and TNF-� (F) from day 3 are also presented as mean pg/ml SEM (n � 2). Statistical significance of the IL-8, IL-6, IL-1�, and TNF-� responses compared to the unstimulated control was determined by t test (�, P 0.05;��, P 0.01; ���, P 0.001; ����, P 0.0001). (G) The comparable fold increase in cytokine response of differentiating human monocytes exposed to 10 ng/mlLPS is shown after 3 days of culture. Data from a representative donor are shown.

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by the increased cell surface expression of MHC I, MHC II, CD83,and CCR7 (Fig. 4B). Increased expression of these cell surfacemarkers was still noted in cells that had been differentiated in thepresence of the WT capsule, but CCR7 upregulation was inhibited(Fig. 4C). This occurred in all five donors tested with WT capsule(P 0.05, n � 5 donors), including the one shown in Fig. 4, whilethe capA mutant preparation showed no statistically significantinhibition of CCR7 in the same group of donors (Fig. 4D, P �0.05, n � 5 donors). When a similar experiment was conductedusing LPS to induce maturation instead of Ba PGN, increasedexpression of MHC I, MHC II, CD83, and CCR7 was similarlyobserved while CCR7 upregulation was also significantly inhib-ited in cells exposed to WT capsule during differentiation (P 0.05, n � 5 donors; see Fig. S1C in the supplemental material), butnot in cells exposed to an equivalent amount of the capA mutantpreparation (P � 0.05, n � 5 donors; see Fig. S1D).

CCR7 expression by mDCs allows them both to migrate to andto enter lymph nodes (31). Since CCR7 expression was reduced in

cells exposed to WT capsule that were matured with Ba PGN, wesought to determine whether or not CCR7-dependent chemotaxiswas also reduced by testing the cells in a chemotactic activity assayusing the CCR7 ligands CCL19 and CCL21 (31) as chemoattrac-tants. Human monocytes were differentiated into iDCs in thepresence of 500 �g/ml capsule or an equivalent amount of thecapA mutant preparation. Control cells were differentiated inthe absence of capsule. One microgram of Ba PGN per ml wasadded on day 7 to capsule-differentiated, capA mutant-differenti-ated, and naive control cells to induce maturation. Additionalcontrol cells were maintained unstimulated to preserve the iDCphenotype. On day 9, the cells were assessed for CCR7-dependentchemotaxis in a chemotactic activity assay using CCL19 or CCL21as the chemoattractive ligand. Control iDCs matured with BaPGN demonstrated significant chemotaxis toward both CCL19and CCL21 compared to the unstimulated control cells (P 0.001, n � 4 donors, Fig. 4E and F). Capsule-differentiated, BaPGN-matured cells showed a significant decrease in chemotaxis

FIG 3 Exposure of monocytes to B. anthracis capsule during differentiation alters the phenotype of the resulting iDCs. Differentiating human monocytes wereexposed to 125 to 500 �g/ml WT capsule or equivalent amounts of capA mutant preparation for 7 days. On day 7, the cells were assessed for MHC I, MHC II,CD83, CCR7, and SSC by flow cytometry. (A) The filled histograms represent the isotype control staining, and the black-outlined histograms represent themarker staining for the naive control iDCs. (B and C) The filled histograms represent the marker staining for the naive control iDCs. The black-outlinedhistograms represent the marker staining for the cells differentiated in the presence of 500 �g/ml WT capsule (B) or capA mutant preparation (C). P values weredetermined by comparing the means of the MFIs for the WT capsule- and capA mutant-treated cells for all donors tested to the mean MFI for the naive controliDCs (P � 0.05 for all markers, n � 6 donors). (D) The filled histograms represent the SSC for the naive control iDCs. The black-outlined histograms representthe SSC for the cells differentiated in the presence of WT capsule. Statistical significance of changes in SSC compared to the naive control was determined by t test(P 0.01, for 5 of 6 donors). Data from a single donor are shown.

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toward both CCL19 and CCL21 compared to that of Ba PGN-matured control cells (P 0.05, n � 4 donors, Fig. 4E and F),while the capA mutant-differentiated, Ba PGN-matured cells didnot (P � 0.05, n � 4 donors, Fig. 4E and F), consistent with thelack of statistically significant CCR7 expression inhibition in thesecells (Fig. 4D). These data indicate that not only is CCR7 expres-sion decreased in monocytes differentiated in capsule prior tomaturation induction by Ba PGN but CCR7-dependent che-motaxis toward CCL19 and CCL21 is decreased as well.

Differentiation in the presence of capsule results in DCs withreduced cytokine release upon maturation with Ba PGN. In ad-dition to inducing increased cell surface marker expression byiDCs, Ba PGN also induces cytokine release. Thus, we next deter-mined if differentiation in the presence of capsule would alter thisresponse of iDCs to Ba PGN. Monocytes differentiated into iDCsin the presence or absence of WT capsule (500 �g/ml) or an equiv-alent amount of the capA mutant preparation were subsequentlystimulated with a maturing dose of Ba PGN (1 �g/ml) on day 7and medium was collected on day 9 as described above. Ba PGNstimulated the release of all of the cytokines tested except for IL-2.Exposure to WT capsule during differentiation resulted in de-creased release of IL-6 (Fig. 5A), IL-10 (Fig. 5B), IL-1� (Fig. 5C),TNF-� (Fig. 5D), IFN-� (Fig. 5E), and IL-23 (Fig. 5F) upon stim-ulation with Ba PGN. IL-6 release was inhibited by WT capsuleonly (P 0.05) in three out of five donors, including the oneshown in Fig. 5. IL-10 was inhibited by WT capsule (P 0.05) infour out of five donors, with the capA mutant preparation havinga similar effect in one donor. IL-1� release was inhibited by WTcapsule in three out of five donors while the capA mutant prepa-ration inhibited IL-1� release in only one donor. TNF-� release

was inhibited by WT capsule (P 0.05) in four out of five donors,with the capA mutant preparation having a similar effect in onlyone donor. IFN-� release was inhibited (P 0.05) in all five do-nors receiving WT capsule, with the capA mutant preparationhaving a similar effect in four donors, including the one shown.IL-23 was significantly inhibited in cells exposed to the capsule inthree out of four donors (P 0.001, Fig. 5F). IL-23 was alsosignificantly inhibited in cells exposed to the capA mutant prepa-ration in three out of four donors (P 0.01). The inhibition ofIL-23 was greater in cells exposed to the WT capsule in two out ofthree donors, including the one shown in Fig. 5.

In a similar experiment, we also determined if differentiationin the presence of capsule would alter the cytokine response ofiDCs to maturation with LPS. Human monocytes differentiatedinto iDCs in the presence or absence of WT capsule (500 �g/ml) oran equivalent amount of the capA mutant preparation were sub-sequently stimulated with a maturing dose of LPS (100 ng/ml) onday 7, and medium was analyzed for cytokines on day 9. IL-10release was significantly inhibited (P 0.05) in WT capsule-dif-ferentiated cells compared to the naive differentiated control infive out of five donors tested, compared to four out of five donorsfor the capA mutant preparation. In three of the five donors, theinhibition was greater for the WT capsule (P 0.05), while in onedonor it was greater for the capA mutant preparation (data notshown). IL-12p70 release was significantly inhibited (P 0.05)compared to the naive control in three out of five donors by boththe WT capsule and the capA mutant preparations but was onlysignificantly greater with WT in one donor (data not shown).There was no significant inhibition by WT capsule of LPS-stimu-lated release of any other cytokines in the majority of donors.

FIG 4 Differentiation in the presence of B. anthracis capsule results in DCs with reduced CCR7 expression and reduced CCR7-dependent chemotaxis uponmaturation with Ba PGN. Human monocytes were differentiated into iDCs in the presence of 500 �g/ml WT capsule or an equivalent amount of capA mutantpreparation for 7 days. Control cells were differentiated in parallel in the absence of capsule. On day 7, the cells were stimulated with 1 �g/ml Ba PGN to inducethe mDC phenotype. Additional control cells were maintained in differentiation medium alone to preserve the iDC phenotype. On day 9, half of the cells wereassessed for MHC I, MHC II, CD83, and CCR7 expression by flow cytometry (A, B, C, and D). (A) The filled histograms represent the isotype control staining,and the black-outlined histograms represent the marker staining for the naive control iDCs. (B) The filled histograms represent the unstimulated naive controliDCs. The black-outlined histograms represent control iDCs after stimulation with 1 �g/ml Ba PGN to induce the mDC phenotype. (C and D) The filledhistograms represent the mDC control. The black-outlined histograms represent Ba PGN-stimulated cells that were exposed to 500 �g/ml WT capsule (C) or tocapA mutant preparation (D). P values were determined by comparing the means of the MFI for the WT capsule- and capA mutant-treated cells for all donorstested to the mean MFI for the Ba PGN-matured controls (P 0.05 only for CCR7 for WT capsule, n � 5 donors). Histograms and MFIs shown are from a singledonor. (E and F) The other half of the cells was subjected to a migration assay using CCL19 (E) or CCL21 (F) as the chemoattractive ligand, and the chemotacticindex was calculated as described in Materials and Methods. Data shown are the means of the chemotactic indices for all the donors tested (n � 4). Statisticalsignificance was determined by t test (�, P 0.05, n � 4 donors).

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Thus, while there were differences among the donors, the WTcapsule significantly inhibited upregulation of CCR7 in responseto Ba PGN or LPS while the capA mutant preparation had nosignificant effect on CCR7. Effects on the cytokine release in re-sponse to a maturing dose of Ba PGN were more variable, withWT capsule showing more significant inhibition for most cyto-kines tested. However, the similarities between WT capsule andthe capA mutant preparation with respect to IFN-� release in re-sponse to Ba PGN and IL-10 and IL-12p70 release in response toLPS suggest that these particular inhibitory effects may be due notto capsule but to other undefined components of the preparationsthat remain to be identified.

Simultaneous exposure to B. anthracis capsule and Ba PGNdoes not inhibit the responses of naive iDCs. The decreased re-sponsiveness to Ba PGN by the iDCs that had been differentiatedin the presence of capsule could be mediated by capsule interfer-ing directly with Ba PGN or by a change in the cells that occurredover time. In order to test whether or not capsule could interferewith the response to Ba PGN directly, naive iDCs differentiated inthe absence of capsule were exposed to capsule plus a maturingdose of Ba PGN (1 �g/ml) simultaneously. Human monocyteswere differentiated into iDCs over the course of 7 days. On day 7,the naive iDCs were exposed to capsule from the WT (500 �g/ml)or an equivalent amount of the capA mutant preparation plus 1�g/ml Ba PGN simultaneously. Control iDCs were exposed to 1�g/ml Ba PGN alone or were maintained in differentiation me-dium alone to retain the immature phenotype. On day 9, medium

samples were collected and the cells were analyzed for surfaceexpression of MHC I, MHC II, CD83, and CCR7 as describedabove. Ba PGN induced upregulation of MHC I, MHC II, CD83,and CCR7 when introduced alone or with WT capsule (data notshown). Indeed, upregulation of these cell surface markers wasalmost entirely unaffected in cells from four of four donors ex-posed to capsule plus Ba PGN. This is in direct contrast to the cellsexposed to WT capsule during differentiation (Fig. 4) and suggeststhat capsule does not directly inhibit the interaction of Ba PGNwith cells and that more prolonged preexposure to capsule medi-ates changes in the cells that make them less responsive. Consis-tent with the absence of changes in cell surface marker expressionafter simultaneous exposure to Ba PGN and WT capsule, therewere no significant changes in the cytokine response profile to BaPGN given together with WT capsule compared to Ba PGN alone(data not shown). Similar results were obtained when LPS wasused to mature the cells instead of Ba PGN (data not shown).

B. anthracis capsule is not a maturation factor for humaniDCs. Since capsule reproducibly elicits a cytokine response fromdifferentiating human monocytes, we next determined whether itwould also elicit a cytokine response from naive differentiatedhuman iDCs and possibly alter expression of cell surface markersas well. Human monocytes were differentiated over 7 days intoiDCs, after which they were exposed to capsule derived from WT(125 to 500 �g/ml) or an equivalent amount of the capA mutantpreparation. Control cells either were maintained in differentia-tion medium in order to retain their immature phenotype or were

FIG 5 Exposure of monocytes to B. anthracis capsule during differentiation alters the resulting iDCs’ cytokine response to Ba PGN. Human monocytes weredifferentiated into iDCs in the presence of 500 �g/ml capsule or capA mutant preparation for 7 days. Control cells were differentiated in parallel in the absenceof capsule. On day 7, the treated and naive control cells were stimulated with 1 �g/ml Ba PGN. Additional control cells were maintained unstimulated. On day9, the media were analyzed for cytokines. Several donors exhibited decreased release of IL-6 (A), IL-10 (B), IL-1� (C), TNF-� (D), IFN-� (E), and IL-23 (F) uponstimulation with Ba PGN (�, P 0.05; ��, P 0.01; ���, P 0.001; n � 2). P values for the responses to the two preparations (WT and capA mutant) weredetermined by comparing the mean pg/ml of each cytokine detected for the treated cells to the mean pg/ml of each cytokine detected for the control cells for eachof four or five donors in a two-tailed t test. Data shown are from a representative donor.

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stimulated with 100 ng/ml LPS to induce maturation. On day 9,medium samples were collected and the cells were analyzed forsurface expression of human MHC I, MHC II, CD83, and CCR7by flow cytometry. Naive control iDCs expressed high levels ofMHCs and very low levels of CD83 and CCR7 (Fig. 6A). Exposureto 100 ng/ml LPS induced upregulation of MHC I, MHC II, CD83,and CCR7 as expected (Fig. 6B), while exposure to up to 500�g/ml of the WT capsule did not significantly affect cell surfaceexpression of MHC I, MHC II, CD83, and CCR7 (P � 0.05, n � 3donors, Fig. 6C). Similarly, no significant effect on cell surfacemarkers was observed with the capA mutant preparation (P �0.05, n � 3 donors, Fig. 6D). Despite the lack of changes in cellsurface marker expression, there were cytokine responses to theWT capsule and capA mutant preparations. Results from a repre-sentative donor are shown in Fig. 6E to G. As with the differenti-ating monocytes, 500 �g/ml WT capsule induced significant re-lease of IL-8 in all donors tested (P range, 0.01 to 0.0001; n �

3). In contrast to the differentiating monocytes, WT capsule in-duced significant (P 0.01) release of IL-6 in two out of threedonors tested (including the one shown in Fig. 6), rather than inall donors. Also in contrast to the differentiating monocytes, low-level, but statistically significant (P range, 0.05 to 0.001),TNF-� release was observed with WT capsule in all donors tested.Similar to the differentiating monocytes, the capA mutant prepa-ration elicited statistically significant (P 0.01) IL-8 only from alldonors. The magnitude of the IL-8 response to WT capsule wassignificantly greater than that to the equivalent amount of capAmutant preparation (P 0.01). Low, but statistically significant,IL-6 and TNF-� release in response to the equivalent amount ofcapA mutant preparation (P 0.01 for IL-6 and 0.05 forTNF-�) was observed in two out of three donors, including theone shown in Fig. 6. However, these responses were much smallerthan those to WT capsule (P 0.05 for IL-6, P 0.01 for TNF-�).Thus, the cytokine response of the iDCs to anthrax capsule was

FIG 6 Exposure of naive iDCs to B. anthracis capsule does not induce an mDC phenotype but does elicit cytokines. Human monocytes were differentiated intoiDCs over the course of 7 days. On day 7, the iDCs were exposed to 500 �g/ml WT capsule or an equivalent amount of capA mutant preparation. On day 9, thecells were assessed for MHC I, MHC II, CD83, and CCR7 expression by flow cytometry (A, B, C, and D) and the media were analyzed for cytokines (E, F, and G).(A) The filled histograms represent the isotype control staining, and the black-outlined histograms represent the marker staining for the untreated naive controliDCs. (B) The filled histograms represent the unstimulated naive control iDCs, and the black-outlined histograms represent control iDCs that were stimulatedwith 100 ng/ml LPS to induce the mDC phenotype. (C and D) The filled histograms represent the unstimulated naive control iDCs. The black-outlinedhistograms represent cells that were exposed to 500 �g/ml WT capsule (C) or to capA mutant preparation (D). P values were determined by comparing the meansof the MFI for the WT capsule and capA mutant-treated cells for all donors tested to the mean MFI for the unstimulated naive control iDCs (P � 0.05 for allmarkers, n � 3 donors). (E, F, and G) Data are presented as mean pg/ml SEM (n � 2) for IL-8 (E), IL-6 (F), and TNF-� (G). Statistical significance of the IL-8,IL-6, and TNF-� responses compared to the unstimulated iDC control was determined by t test (�, P 0.05; ��, P 0.01; ���, P 0.001). Data from a singledonor are shown.

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similar, though not identical, to the response of the differentiatingmonocytes.

DISCUSSION

Capsule is one of the chief virulence factors for B. anthracis. Themechanism by which capsule enhances virulence is presumed tobe mainly by the prevention of ingestion and killing of the bacteriaby phagocytic cells. However, vegetative bacilli shed largeamounts of capsule during their growth, and previous studieshave indicated that the free capsule may also contribute to viru-lence (14, 15). We hypothesized that free capsule might contributeto pathogenesis by interfering with innate immune responses.Therefore, in this study we examined the effects of free capsule oncells of the human innate immune system. We purified capsuleshed naturally by vegetative bacilli during broth culture to acquirefree capsule that would be similar to the free capsule present in theinfected host. To control for possible contaminants or compo-nents arising from the broth culture and purification procedure,we prepared capsule material from a fully virulent WT B. anthracisstrain and from an isogenic capsule-reduced capA mutant strainthat produces only 2% of the capsule of the WT. When these twopreparations were applied to differentiating human monocytes,the WT capsule preparation consistently elicited both IL-8 andIL-6 from all donors, while the capA mutant preparation elicitedonly very low levels of IL-8 from all donors and very low levels ofIL-6 from some donors. Cytokine responses to WT capsule wereconsistently significantly greater and showed better dose depen-dence than did cytokine responses to the capA mutant prepara-tion. Although B. anthracis capsule consistently elicited IL-8 andIL-6, it is a weak inducer of cytokines in differentiating monocytesand iDCs compared to LPS. We could detect a cytokine responseat capsule concentrations as low as 125 �g/ml, but 500 �g/ml ormore was required for robust responses. Even at concentrations of1,000 �g/ml, the cytokines released were only a fraction of thosereleased in response to 10 ng/ml of LPS. Although the concentra-tions of capsule used in this study were high, they were not so highas to be physiologically irrelevant, considering that serum concen-trations greater than 500 �g/ml have been reported in mice (10)and concentrations greater than 1 mg/ml have been reported inrhesus macaques (11) with anthrax.

Various controls suggested that the cytokine responses were infact due to the capsule in the WT preparation. The capA mutantpreparation, which is greatly reduced in capsule and prepared inparallel in a manner identical to that of the WT capsule, producednearly no cytokine release from the differentiating monocytes.Both preparations were practically free of LPS (0.25 EU/ml),and furthermore, the pattern of cytokines induced by capsule wasdistinctly different from that seen with LPS. PGN, a relatively po-tent cytokine inducer (22, 24–27), has been shown to be releasedby vegetative B. anthracis bacilli during culture (25). While verylow levels of PGN were found in the preparations, this was notlikely to be the source of cytokine release since the inactive capAmutant preparation contained twice as much PGN as did the ac-tive WT capsule. The trace amounts of capsule, PGN, and possiblyother B. anthracis cell wall components in the capA mutant prep-aration may be responsible for the very low IL-8 and IL-6 re-sponses observed.

These results are in contrast to those in a recent report describ-ing release of IL-1� by human monocyte-derived dendritic cells inresponse to 100 �g/ml B. anthracis capsule (32). In that report,

Cho et al. did not see the increase in IL-6 that we observed, andthey did not test for IL-8. The capsule purification protocol thatthey used differs substantially from ours. Furthermore, they donot mention testing for contamination by LPS. Also, they do notindicate the amount of PGN present in their capsule preparation.Unlike B. anthracis capsule, Ba PGN, as noted above, induces arobust cytokine response and, in our study, maturation of humaniDCs. Either LPS contamination or PGN might account for theIL-1� observed by Cho et al.

The consistency and specificity of the cytokine responses toWT capsule suggest that it may bind to a pattern recognition re-ceptor. This idea is supported by a recent report suggesting that B.anthracis capsule mediates TNF-� release through TLR2, based onthe differential response of macrophages from strains of hamsterswith or without functional TLR2 (33). It is possible that humanTLR2 also functions as a receptor for B. anthracis capsule. Exper-iments are under way to explore this hypothesis.

Examination of the iDCs differentiated in the presence of B.anthracis capsule by flow cytometry revealed a subtle change in thephenotype. SSC increased in response to WT capsule in a dose-dependent manner. This change could reflect the presence of vac-uoles filled with undegraded capsule, or an increase in dendrites,or a combination of the two. Increases in SSC were observed withthe capA mutant preparation as well, but these were small by com-parison and did not show dose dependence. No significantchanges in MHC I, MHC II, CD83, or CCR7 expression comparedto the naive control were observed in the capsule-differentiatediDCs. The combination of the cytokine response and the increasein SSC indicated that the presence of B. anthracis capsule duringdifferentiation altered the resulting iDCs. The relevance of thesechanges was demonstrated by the impaired responses of the cap-sule-differentiated iDCs to maturation induced by Ba PGN, asindicated by consistently smaller increases in cell surface expres-sion of CCR7 and by the concomitant reduction in CCR7-depen-dent chemotaxis toward CCL19 and CCL21. It is possible that thiseffect was due to the IL-6 released by the differentiating mono-cytes in response to the capsule. Human monocytes differentiatedinto iDCs in the presence of as little as 5 ng/ml IL-6 have also beenshown to be impaired in upregulation of CCR7 in response to amaturing dose of LPS (34). In addition to inhibition of CCR7upregulation, release of IL-6, IL-10, IL-1�, TNF-�, IFN-�, andIL-23 upon maturation with Ba PGN was inhibited in the majorityof donors whose cells were exposed to capsule. Surprisingly, re-lease of IFN-� upon maturation with Ba PGN was also inhibited inthe majority of donors whose cells were exposed to the capA mu-tant preparation. We also noted that the capA mutant preparationas well as the capsule inhibited LPS induction of IL-10 release. Thisindicates that the effect on Ba PGN-stimulated IFN-� release andLPS-stimulated IL-10 release is likely due to another unidentifiedcomponent present in both of the preparations. This other com-ponent was not as effective as capsule in suppressing DC re-sponses, as it did not mediate statistically significant inhibition ofCCR7 expression and function like the capsule did. Its primaryeffect in Ba PGN-matured cells was inhibition of IFN-� release.One donor treated with the capA mutant preparation showed in-hibition of IL-10, IL-1�, and TNF-� in response to Ba PGN aswell. While that one individual was remarkably sensitive to theunidentified component, the majority of donors were affectedmore by the capsule.

Not only was upregulation of CCR7 expression upon matura-

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tion with Ba PGN significantly impaired in the capsule-differen-tiated DCs, but chemotaxis toward the CCR7 ligands CCL19 andCCL21 was also significantly impaired. The impairment of CCR7that we observed could have an impact on the development of anadaptive immune response. CCR7 expression by mature DCs al-lows them both to migrate to and to enter lymph nodes (31).Furthermore, signaling through CCR7 inhibits apoptosis of ma-ture DCs, thereby extending their life span and increasing theprobability of T cell activation (35, 36). Therefore, impaired CCR7expression and the consequently impaired CCR7-dependent mi-gration due to capsule exposure could contribute to pathogenesisby potentially reducing activation of T cells and impairing thedevelopment of an adaptive immune response. This could haveconsequences for pathogenesis, as the successful induction of aTh1 immune response has been shown to be protective in miceagainst a nontoxigenic, encapsulated strain of B. anthracis (37).

Less consistent than the reduction in CCR7 expression andCCR7-dependent chemotaxis, but still significant, there were alsoreductions in the release of IL-10 and TNF-� in four out of fivedonors, IL-6 and IL-1� in three out of five donors, and IL-23 inthree out of four donors. Inhibition of IL-23 was also noted withboth capsule and the capA mutant preparation but was greaterwith capsule in two of three subjects. Although inhibition ofIFN-� release cannot be ascribed solely to capsule, inhibition wasmore consistently observed with capsule than with the capA mu-tant preparation. These inhibitory effects were due to changes thatoccurred in the cells over time rather than to direct interference ofcapsule with the Ba PGN, since capsule added together with BaPGN to naive differentiated iDCs had no effect. Impaired releaseof these cytokines could have consequences in the context of an-thrax. Although IL-10 usually acts as an anti-inflammatory cyto-kine, it has also been shown to be a potent stimulator of NK cells(38, 39) that can increase their killing capacity and release of per-forin and granzyme B (37). NK cells have recently been shown tocontribute to suppressing bacteremia during B. anthracis infectionin mice (40, 41). IL-6 can support survival of the naive T cell pool(42) and can drive differentiation of Th17 cells (43). TNF-� andIL-1� also drive differentiation of Th17 cells from naive CD4 Tcells (44), while IL-23 supports Th17 survival and activity (45).The Th17 lineage contributes to immunity against both extracel-lular and intracellular bacterial infection as well as fungal patho-gens by inducing production of antimicrobial peptides by epithe-lial cells and by recruiting and activating phagocytes (44, 46).Simultaneous inhibition of IL-6, IL-1�, TNF-�, and IL-23 couldthus blunt the development of a Th17 response that could con-tribute to impaired resistance to infection.

Statistically significant reductions in the expression of CCR7upon maturation, in chemotaxis toward the CCR7 ligands CCL19and CCL21, and inhibition of some cytokines as indicated abovewere observed only in cells exposed to capsule, indicating thatthese effects are mediated by the capsule. Inhibition of the Th1-promoting and macrophage-activating (36) cytokine IFN-� in re-sponse to Ba PGN was almost as effectively mediated by the capAmutant control preparation, indicating that there is likely anotheractive component present in both preparations. However, thisother component lacked the breadth of effect that capsule had.Between the impairment of CCR7 expression and CCR7-depen-dent chemotaxis and the inhibition of the release of Th17-pro-moting cytokines by capsule, there may be interference with adap-tive immune responses and thus resistance to anthrax.

WT capsule elicited a distinct cytokine response from differen-tiating human monocytes, including IL-8, IL-6, and TNF-�, whilethe capA mutant preparation had a much lesser effect, elicitingonly minimal amounts of IL-8 and IL-6 consistently. No remark-able changes in cell surface markers were noted when naive iDCswere exposed to the capsule preparations. Similarly, the mono-cytes differentiated in the presence of capsule showed only subtlephenotypic changes until they were subsequently exposed to BaPGN or LPS. The naive iDCs that were exposed to capsule for 48 hretained the naive phenotype but were not subsequently chal-lenged with a maturation stimulus such as Ba PGN or LPS. Per-haps, this shorter exposure to capsule might also have impairedthe subsequent responses of the DCs to Ba PGN. The minimumamount of time that capsule needs to be present to impair the cells’sensitivity to inflammatory stimuli will require further study.

Capsule is known to enhance the virulence of B. anthracis byprotecting the vegetative bacilli from phagocytosis by immunecells (4–6). This poorly immunogenic outermost layer may alsoshield more reactogenic bacterial factors on intact bacilli, such asPGN, from being sensed by pattern recognition receptors on hostcells. In support of this hypothesis, it has been shown that humanprimary myeloid DCs and human monocyte-derived DCs releaseless IL-12p40, IL-6, and TNF-� when they are cocultured with anencapsulated strain of B. anthracis than when they are coculturedwith an unencapsulated strain (47). Our results indicate that thecapsule may not need to be bound to the bacilli in order to protectthem from the immune system. In this study, we demonstrate theability of shed capsule to induce only a very modest cytokine re-sponse from differentiating monocytes compared to more robuststimuli like LPS and PGN and to inhibit the responsiveness andmaturation of iDCs differentiated in capsule to a subsequent in-flammatory stimulus. These findings might result in impairmentof DC function in vivo. It is thus possible that while bacillus-bound capsule protects vegetative bacilli from phagocytosis, theshed capsule also contributes to the virulence of B. anthracis byblunting the responses of innate and adaptive immune cells tomore inflammatory stimuli from the spore or bacillus, such asPGN, thus allowing the infection to become established.

ACKNOWLEDGMENTS

We thank Neil X. Krueger of Immunetics, Inc., for conducting the PGNanalysis. We also thank Anna Trivett for purifying the monocytes.

The opinions, interpretations, conclusions, and recommendations arethose of the authors and not necessarily endorsed by the US Army.

The work was supported by the Defense Threat Reduction Agencygrant CBM.VAXBT.03.10.RD.015.

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Erratum for Jelacic et al., Exposure to Bacillus anthracis CapsuleResults in Suppression of Human Monocyte-Derived Dendritic Cells

Tanya M. Jelacic,a Donald J. Chabot,a Joel A. Bozue,a Steven A. Tobery,a Michael W. West,a Krishna Moody,c De Yang,b

Joost J. Oppenheim,b Arthur M. Friedlandera

United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USAa; Laboratory of Molecular Immunoregulation, Frederick NationalLaboratory for Cancer Research, Frederick, Maryland, USAb; Department of Microbiology, Boston University, School of Medicine, Boston, Massachusetts, USAc

Volume 82, no. 8, p. 3405–3416, 2014. Page 3405, column 1, line 3: “pX01” should read “pXO1.”

Page 3405, column 1, line 4: “pX02” should read “pXO2.”

Page 3406, column 1, line 17: “pX02” should read “pXO2.”

Page 3406, column 2, lines 15 and 16: “pX01� pX02�” should read “pXO1� pXO2�.”

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.02665-14

ERRATUM

December 2014 Volume 82 Number 12 Infection and Immunity p. 5347 iai.asm.org 5347