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Atherogenic PotentialPeriodontitis Influences DC Differentiation,Dendritic Cells (DCs) in Chronic Microbial Carriage State of Peripheral Blood

Genco, David L. Brown and Christopher W. CutlerSabino, Amir E. Zeituni, Ying Gu, Adam Bear, Caroline A. Julio Carrion, Elizabeth Scisci, Brodie Miles, Gregory J.

http://www.jimmunol.org/content/189/6/3178doi: 10.4049/jimmunol.1201053August 2012;

2012; 189:3178-3187; Prepublished online 13J Immunol 

MaterialSupplementary

3.DC1http://www.jimmunol.org/content/suppl/2012/08/13/jimmunol.120105

Referenceshttp://www.jimmunol.org/content/189/6/3178.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2012 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

Microbial Carriage State of Peripheral Blood Dendritic Cells(DCs) in Chronic Periodontitis Influences DC Differentiation,Atherogenic Potential

Julio Carrion,* Elizabeth Scisci,* Brodie Miles,† Gregory J. Sabino,* Amir E. Zeituni,*

Ying Gu,* Adam Bear,* Caroline A. Genco,‡,x David L. Brown,{ and

Christopher W. Cutler‖

The low-grade oral infection chronic periodontitis (CP) has been implicated in coronary artery disease risk, but the mechanisms

are unclear. In this study, a pathophysiological role for blood dendritic cells (DCs) in systemic dissemination of oral mucosal

pathogens to atherosclerotic plaques was investigated in humans. The frequency and microbiome of CD192BDCA-1+DC-SIGN+

blood myeloid DCs (mDCs) were analyzed in CP subjects with or without existing acute coronary syndrome and in healthy

controls. FACS analysis revealed a significant increase in blood mDCs in the following order: healthy controls < CP < acute

coronary syndrome/CP. Analysis of the blood mDC microbiome by 16S rDNA sequencing showed Porphyromonas gingivalis and

other species, including (cultivable) Burkholderia cepacia. The mDC carriage rate with P. gingivalis correlated with oral carriage

rate and with serologic exposure to P. gingivalis in CP subjects. Intervention (local debridement) to elicit a bacteremia increased

the mDC carriage rate and frequency in vivo. In vitro studies established that P. gingivalis enhanced by 28% the differentiation of

monocytes into immature mDCs; moreover, mDCs secreted high levels of matrix metalloproteinase-9 and upregulated C1q, heat

shock protein 60, heat shock protein 70, CCR2, and CXCL16 transcripts in response to P. gingivalis in a fimbriae-dependent

manner. Moreover, the survival of the anaerobe P. gingivalis under aerobic conditions was enhanced when within mDCs. Immu-

nofluorescence analysis of oral mucosa and atherosclerotic plaques demonstrate infiltration with mDCs, colocalized with P.

gingivalis. Our results suggest a role for blood mDCs in harboring and disseminating pathogens from oral mucosa to atheroscle-

rosis plaques, which may provide key signals for mDC differentiation and atherogenic conversion. The Journal of Immunology,

2012, 189: 3178–3187.

Coronary artery disease (CAD) and its thrombotic com-plications are currently the most deadly and disablingcardiovascular diseases in affluent countries. CAD is the

leading cause of mortality in the United States, and it is believedthat CAD will continue to spread globally unless improvedmethods are developed to identify at-risk individuals (reviewed inRef. 1) and institute preventive measures.

The most commonly recognized risk factors for CAD includediabetes, smoking, hypertension, and hyperlipidemia (2). Chronic

low-grade infections with bacterial species Chlamydia pneumonia,

Helicobacter pylori, Porphyromonas gingivalis, and others are

also suspected of conferring increased CAD risk (3, 4); however,

the mechanisms are not clear.Chronic periodontitis (CP) is a low-grade infection identified as

a risk factor for CAD (4– 6) and other systemic diseases (7). The

oral submucosa in CP is a niche for growth of oral Gram-negative

anaerobes such as P. gingivalis. P. gingivalis is uniquely able to

infect myeloid dendritic cells (mDCs) and reprogram them to in-

duce an immunosuppressive T effector response (8–10). P. gingi-

valis has been identified in bacteremias (11, 12) and atherosclerotic

plaques in humans (13); moreover, it accelerates atherosclerosis in

ApoE2/2 mice in a manner that is dependent on expression of

fimbrial adhesins (4).Invasion of the arterial vessel walls by inflammatory cells is in-

dispensible to CADdevelopment. Infiltrating cells includemonocytes/

macrophages (14, 15), lymphocytes, neutrophils, and mDCs (16, 17).

An emerging body of literature supports a pivotal role for mDCs in

CAD development in humans (18) and mice (19, 20), as reviewed

previously (21). However, the predominant sources of mDCs in

atherosclerotic plaques and the factors that trigger their activation,

infiltration, and differentiation remain elusive.Circulating dendritic cells (DCs) called blood DCs and their

progenitors are likely sources of infiltrating DCs in CAD (22). In

humans, blood DC subsets include CD123+CD303+ plasmacytoid

DCs, CD192CD1c+ (BDCA-1) mDCs, and a minor subset of

CD141+ mDCs (23). Blood DCs are derived from bone marrow

*School of Dental Medicine, Stony Brook University, Stony Brook, NY 11794;†Department of Molecular Genetics and Microbiology, Center for Infectious Dis-eases, Stony Brook University, Stony Brook, NY 11794; ‡Section of Infectious Dis-eases, Department of Medicine, Boston University School of Medicine, Boston, MA02118; xDepartment of Microbiology, Boston University School of Medicine, Boston,MA 02118; {Department of Cardiology, School of Medicine, Stony Brook Univer-sity, Stony Brook, NY 11794; and ‖College of Dental Medicine, Georgia HealthSciences University, Augusta, GA 30912

Received for publication April 11, 2012. Accepted for publication July 7, 2012.

This work was supported by U.S. Public Health Service grants from NationalInstitutes of Health/National Institute of Dental and Craniofacial Research (K23DE018187 to J.C., R01 DE014328 and R21 DE020916 to C.W.C., F31 DE020014to A.E.Z., and F30 DE021649-01 to E.S.).

Address correspondence and reprint requests to Dr. Christopher W. Cutler, Depart-ment of Periodontics, College of Dental Medicine, Georgia Health Sciences Uni-versity, 1120 15th Street, Augusta, GA 30912-1220. E-mail address: chcutler@georgiahealth.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: ACS, acute coronary syndrome; CAD, coronaryartery disease; CP, chronic periodontitis; CTL, healthy control; DC, dendritic cell;gDNA, genomic DNA; HSP, heat shock protein; mDC, myeloid DC; MMP, matrixmetalloproteinase; moDC, monocyte-derived dendritic cell; MOI, multiplicity ofinfection; PMN, polymorphonuclear leukocyte; qRT-PCR, quantitative real-timePCR; S&RP, scaling and root planing.

Copyright� 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00

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progenitors, monocytes, and, ostensibly, DC-SIGN+ tissue DCsthat have reverse transmigrated into circulation after capture ofmicrobial Ags (24, 25). Previous work has documented mDCsactively infiltrating the oral submucosa in CP (26, 27) and rupture-prone atherosclerotic plaques (28). However, the role of bloodmDCs in clearance of bacteremias and dissemination to distantsites such as atherosclerotic plaques is undocumented in humans.In the current study, we show that blood mDCs of humans with CP

harbor microbes identified in oral mucosa and atherosclerotic pla-ques. mDCs provide thesemicrobeswith a protective niche andmodeof transport. The microbe in turn stimulates differentiation of mDCsfrom monocytes and converts mDCs into an atherogenic phenotype.

Materials and MethodsStudy population

The Committee on Research Involving Human Subjects at Stony BrookUniversity approved all protocols involving human subjects. Informedconsent was obtained from all subjects before commencement of the study.The cohort of subjects with CP consisted of 40 subjects with moderate tosevere CP as determined by the presence of .20 teeth, of which at least 8exhibited the following: probing depth .4 mm, attachment loss .3 mm,bleeding on probing, and alveolar bone crest .3 mm from cemento–

enamel junction. Demographic data and clinical parameters of the studysubjects are shown in Table I. Exclusion criteria included the following:steroidal anti-inflammatory agents, smoking, periodontal treatment withinthe past 6 mo, pregnancy, diabetes, heart disease, or cancer. After the initialexamination, all CP patients were subjected to scaling and root planing(S&RP; local debridement of the root surfaces and pockets) under localanesthesia, and the blood mDC response was evaluated at 24 h. A subset ofCP subjects included those with acute coronary syndrome (ACS) (n = 15),diagnosed as reported (29) and shown in Table I. ACS subjects without CPcould not be identified. Healthy controls (CTL) consisted of 25 age- andgender-matched subjects and nonsmokers without CP, who had no historyof ACS, diabetes, cancer, or other reported systemic disease. Healthycontrols were not subjected to S&RP because there is no clinical need andit can be detrimental to clinical attachment levels.

Blood mDC isolation

PBMCs were isolated from 30 ml whole blood, and a nucleated cell sus-pension was prepared using Ficoll-Paque Plus density gradient centrifu-gation (GE Healthcare). mDCs were isolated by positive immunoselection, asdescribed previously (30). Briefly, PBMCs labeled with BDCA-1 (CD1c) PE+

(BDCA-1; Miltenyi Biotec; catalog 130-090-508), CD209 allophycocyanin+

(BD Biosciences; catalog 551545), and CD19 FITC+ (BD Biosciences;catalog 557697) were FACS sorted (FACSAria; BD Biosciences) and thensorted again to remove CD19+ (B2) cells. These procedures routinelyyielded mDC (CD1c+CD209+CD192) preparations of .95% purity.

Table I. Clinical description, demographics, serum lipids, and cytokines

Patient Cohorts

Control (n = 25) CP (n = 25) ACS/CP (n = 15)

Clinical description Nonsmokers Moderate to severe CP Troponin + unstable anginaWithout CP .20 teeth ECG evidence of ischemiaNo ACS, diabetes, cancer, orother reported systemic disease

$8 with probing depth . 4 mmALOSS . 3 mm, BOP, alveolarbone crest . 3 mm from cemento–enamel junction, nondiabetic,nonsmoker

1-, 2-, or 3-vessel CAD, history of MIModerate to severe CPExcluded previous coronary bypass,untreated or incomplete treatmentof CAD, life expectancy of ,2 y

Age (y)

Median (range) 51 (38–63) 52 (31–72) 65 (32–89)

Gender

Males 12 11 10Females 13 14 5

Self-Reported Race or Ethnic Group (%)

White 12 (48) 16 (64) 11 (63)Black 9 (36) 2 (8) 0 (0)Asian 0 (0) 2 (8) 0 (0)Hispanic 4 (16) 4 (16) 4 (27)Arab 0 (0) 1 (4) 0 (0)

Serum Lipids (mg/dl)

Triglycerides 102 6 8.3 155 6 16a 146 6 17Total cholesterol 209 6 8 215 6 9 170 6 11LDL 119 6 7 134 6 8 96 6 9.1HDL 70 6 4 50 6 3a 42 6 11CHOL/HDL ratio 3.2 6 0.2 4.6 6 0.3a 4.2 6 0.9VLDL 20.3 6 1.6 31 6 3a 27 6 4

Serum Cytokines

FLT-3L (pg/ml) 65 6 24 66 6 18 137 6 27b

TNFr-I (pg/ml) 1750 6 564 1850 6 867 3520 6 2100TNFr-II (pg/ml) 2969 6 537 3239 6 247 4982 6 647b

hsCRP (ng/ml) 2293 6 1046 2797 6 525 2851 6 413

Serum IgG Titer against P. gingivalis DPG-3

Anti-PgIgG 1.06 6 0.06 2.69 6 0.19a n.d.

aSignificantly elevated versus CTL (p , 0.05, Student t test).bSignificantly elevated versus CP and CTL (p , 0.05, Student t test).ALOSS, Attachment loss; BOP, bleeding on probing; CHOL, cholesterol; ECG, electrocardiogram; HDL, high density lipoprotein; LDL, low density lipoprotein; MI,

myocardial infarction; n.d., not determined; Pg, P. gingivalis; VLDL, very low density lipoprotein.

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Generation of monocyte-derived DCs

To serve as in vitro model of DC infectivity and P. gingivalis survival,monocyte-derived dendritic cells (MoDCs) were generated, as we havedescribed previously (9, 27, 31). Briefly, monocytes were isolated frommononuclear cell fractions of the peripheral blood of healthy controls andseeded in the presence of GM-CSF (100 ng/ml; PeproTech catalog 300-03) and IL-4 (25 ng/ml; R&D Systems; catalog 204-IL-010) at a concen-tration of 1–2 3 105 cells/ml for 6–8 d, after which flow cytometry wasperformed to confirm the immature DC phenotype (CD142CD832CD1a+

CD1c+DC-SIGN+; all Abs from BD Biosciences, except CD1c [MiltenyiBiotec]).

In vitro infection and mDC differentiation model

After PBMC isolation and plastic adherence, monocytes were scraped offplates and collected. The total number of monocytes was counted usingAccuri’s C6 Flow Cytometer System, and baseline levels of CD14, CD1c,DC-SIGN, CD86, and CD83 expression were obtained. Monocytes werethen divided equally into 6-well plates and treated either with GM-CSFand IL-4 alone, wild-type Pg381 alone, GM-CSF and IL-4 plus wild-typePg381, or RPMI 1640 alone. Bacterial multiplicities of infection (MOIs)chosen were 0.1, 0.5, and 1. Each experimental condition was performed intriplicate. Cells for each condition were collected on subsequent days 1, 2,and 3 for analysis of MoDCs present and changes in receptor expression.Cells were gated on scattergram plots based on size characteristics for bothmonocytes and MoDCs. Flow samples were collected based on totalnumber of events rather than volume, so differentiation of monocytes intoDCs is represented as MoDCs per microliter of sample. Triplicates wereaveraged and assembled onto a line graph to show the differentiation ofmonocytes to MoDCs with and without growth factors. Intensity profilesof CD14, DC-SIGN, CD1c, and CD83 expression were compared withbaseline monocyte levels to show downregulation of CD14, upregulationof DC-SIGN and CD1c, and immature state of MoDCs (CD832CCR72)for each condition.

Flow cytometric phenotyping

mDCs were labeled with combinations of PE, FITC, allophycocyanin, andPerCP mouse anti–human lineage Ab (CD14, CD19, CD11b/Mac-1, CD1c[BDCA-1; Miltenyi Biotec], CD80, CD83, CD86, HLA-DR, CD209 [DC-SIGN], CD1a, or isotype controls [all from BD Biosciences, San Jose,CA]; and goat anti-mouse IgG [H+L] [Invitrogen]). Analysis was per-formed with a FACSCalibur flow cytometer (BD Biosciences). Markerexpression was analyzed as the percentage of positive cells in the relevantpopulation defined by forward scatter and side scatter characteristics.Expression levels were evaluated by assessing mean fluorescence intensityindices calculated by relating the mean fluorescence intensity noted withthe relevant mAb to that of the isotype control mAb for samples labeled inparallel and acquired using the same setting.

Blood mDC frequency change after treatment

Blood PBMCs were obtained from all study subjects at baseline and 24 hafter a single intensive bout of S&RP, as reported (32). This treatment haspreviously been shown to result in acute, short-term bacteremia (33, 34), aswell as systemic inflammation (32). The mDCs were labeled with fluo-rescent conjugated Abs (as described above), CD192CD1c+DC2SIGN+

sorted (FACSAria; BD Biosciences), and analyzed (FACS Diva v.6.1.3), asdescribed above.

Quantitative RT-PCR and DNA sequencing

To detect and quantitate P. gingivalis, genomic DNA (gDNA) and totalRNA were isolated from subgingival plaque and mDCs with the RNeasyMicro Kit (Qiagen), according to the manufacturer’s instructions, butwith a slight modification. Briefly, gDNA Eliminator spin columns werenot used during the isolation protocol. This was done to collect, in ad-dition to total RNA, gDNA for P. gingivalis 16S rDNA detection in themDCs. cDNA was synthesized using STR1 Enhanced Avian First StrandSynthesis Kit (Sigma-Aldrich, St. Louis, MO; catalog STR1-1KT). ThecDNA template was standardized to a concentration of 0.1 mg/ml usinga NanoDrop 3300 Fluorospectrometer (Thermo Scientitific; catalogND3300) with Quant-iT dsDNA HS Assay Kit (Invitrogen; catalogQ32851). Quantitative real-time PCR (qRT-PCR) was used to detect thepresence of P. gingivalis (16S rDNA) in MoDCs, mDCs, and dental plaquesamples. The 16S rDNA consisted of a forward Pg-specific primer (59-TGT AGA TGA CTG ATG GTG AAA ACC-39) and a universal reverseprimer (C11R) (59-ACG TCA TCC CCA CCT TCC TC-39) sequence, aspreviously described (35). The universal reverse primer was also used to

detect non-P. gingivalis amplification products, which were then subjectedto genomic blast sequencing. PCRs were performed using QuantiTectSYBR Green PCR Kit (Qiagen; catalog 204145) on a iCycler ThermalCycler (Bio-Rad). The thermal cycling conditions were an initial incu-bation step of 15 min at 95˚C to activate HotStartTaq DNA polymerase.This step prevents the formation of misprimed products and primerdimers during reaction setup and the first denaturation step, leading tohigh PCR specificity and accurate quantification. This initial incubationstep was followed by 45–50 cycles at 94˚C for 15 s, 54˚C for 30 s, and72˚C for 30 s. P. gingivalis 16S rDNA primer was a gift of S. Walker(State University of New York, Stony Brook, NY). As a quantitativestandard for qRT-PCR analysis of mDCs, MoDCs were spiked in vitrowith P. gingivalis 381 at a range of MOI and CFU/ml. This yieldeda linear regression curve consisting of log10 CFU/ml versus amplificationcycles (Supplemental Fig. 1C). The infection status of mDC from theblood was then expressed as estimated CFU/ml and MOI relative to ourMoDC standard.

To confirm the specificity of the PCR-amplified product using theP. gingivalis 16S rDNA primer (or the non-P. gingivalis–specific PCR-amplified products), the resultant amplicons were sequenced. Briefly,the amplified product was run in a 1% agarose gel electrophoresis (Bio-Rad; catalog 162-0102) along with a 0.1- to 10.0-kb DNA ladder (NewEngland BioLabs; catalog N3200S). The resultant band was extractedand purified from the agarose gel using QIAEX II Gel Extraction Kit(Qiagen; catalog 20051), according to the manufacturer’s instructions.The DNA template (3.0 ng) was combined with 3.2 pmol 16S rDNAprimer and sequenced using a 3730 DNA Analyzer (Applied Bio-systems). The sequenced product was then aligned against all human,prokaryotic, and eukaryotic known database genomes using GenomicBlastSequence (BLASTN 2.2.24+) (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi).

Intracellular survival

Wild-type Pg381 was used to infect MoDCs, or polymorphonuclear leu-kocytes (PMNs) at a multiplicity of infection of 100. Uptake of the bacteriaby human cells was confirmed by observing complete internalization ofCFSE-stained P. gingivalis via epifluorescence microscopy as soon as60 min after inoculation. Cells were then washed twice in PBS and resus-pended in culture medium for continued incubation. At each time point,cells were resuspended in sterile water on ice for 20 min to initiate celllysis. Remaining bacteria released from within the cells was resuspendedin PBS and streaked on anaerobic 5% blood agar plates in triplicate underanaerobic conditions (10% H2, 5% CO2 in nitrogen) at a 1:10 dilution. Plateswere incubated in anaerobic conditions at 35˚C for 14 d, after which colonieswere numerated and surviving cell-forming U/ml were determined.

Derivation of mAb to DC invasin, mfa-1 minor fimbriae

mAb 89.15 against the DC invasin, mfa-1 (minor fimbriae), was generatedby the Cell Culture/Hybridoma Facility at Stony Brook University. Briefly,three female 6- to 8-wk-old BALB/c mice (Charles River) were immunizedi.p. with three 50 mg doses of native minor fimbriae (Mfa-1) in Sigmaadjuvant (Sigma-Aldrich) at 2-wk intervals, following which sera wasdrawn and tested by ELISA for the presence of Ag-specific Abs. Themouse selected for splenectomy had a titer of.1:1000 to the protein. Priorto fusion, the mouse was boosted i.p. with 1 mg Mfa-1 in PBS (LifeTechnologies-Invitrogen, Carlsbad, CA). Four days following the booster,the mouse was sacrificed, and the spleen cells were isolated aseptically andfused with mouse myeloma cell line Sp2/0 (American Type Culture Col-lection), as described (36). Clones were screened by ELISA against nativeminor fimbriae. Clones were then further screened using a whole bacteriaELISA against MFI, which expresses only the major fimbriae (Pg min2/maj+), and DPG3, which expresses only the minor fimbriae (Pg min+/maj2). Clone 89 was determined to be positive both by native minorfimbria ELISA and whole bacteria ELISA, and thus was selected forsubcloning by limiting dilution. Subclone 89.15 was selected by ELISA forfurther study. mAb 89.15 was determined to be of the IgG1 isotype havinga k L chain, by use of the IsoStrip Mouse mAb Isotyping Kit (RocheApplied Science, Indianapolis, IN). Abs from this subclone are referred toas AEZaMfa1.

Immunofluorescence staining of oral mucosal tissues andcoronary artery tissues

Oral mucosal tissues from the human gingiva were collected from untreatedCP patients using a biopsy technique previously reported (37). Immediatelyafter collection, the tissues were rinsed with sterile saline to remove tracesof blood, embedded in Tissue Tek OCT compound (Sakura), snap frozen in

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liquid nitrogen, and sectioned into 7-mM–thick sections using a cryostat(Leica CM1850). Coronary artery tissues were obtained from humanpostmortem patients with atherosclerosis and CP (gift of E. Kozarov,Columbia University, New York) and cryosectioned, as described above.For immunofluorescent staining, sections were fixed in acetone for 5 min at220˚C, rehydrated in PBS lacking Ca2+ and Mg2+ (PBS2), blocked with5% BSA (Sigma-Aldrich) in PBS2 along with anti-human FcR block re-agent (Miltenyi Biotec, Auburn, CA) for 1 h, and washed. Sections wereincubated for 30 min at room temperature with conjugated primary Absdiluted in PBS2 and washed before mounting. All sections were mountedwith VectaShield mounting medium containing DAPI (Vector Laborato-ries, Burlingame, CA). To identify DC-SIGN within arterial plaques andgingival tissue sections, FITC-conjugated (BioLegend, San Diego, CA) orRPE-conjugated mouse anti-human CD209 (AbD Serotec, Raleigh, NC)was used. FITC-conjugated mouse anti-human CD1c was used (BD Bio-sciences) to identify CD1c. DyLight microscale Ab-labeling kit (ThermoScientific, Rockford, IL) was used to conjugate Alexa Fluor 594 to mfa-1Ab AEZaMfa1, according to manufacturer’s protocol. Controls includedisotype-matched Abs and preimmune Abs. Images were acquired witha Zeiss LSM 510 META NLO Two-Photon Laser Scanning ConfocalMicroscope System coupled with image processor software for imageprocessing. In addition, sections were stained with H&E stain to examinecell and tissue morphology. The H&E images were analyzed by image-enhanced light microscopy (Nikon E600).

In situ association of mDCs with P. gingivalis byimmunofluorescence

mDCs from blood of CP and ACS/CP patients were analyzed by immu-nofluorescence cytometry to determine infection with P. gingivalis. Briefly,FACS-sorted CD192CD1c+DC-SIGN+ mDCs were permeabilized andfixed in Shandon Cytospin Collection Fluid (Thermo Scientific; catalog6768315). The cytological specimen was deposited on Ultrastick slides(Thermo Scientific; catalog 3039) and cytocentrifuged (Shandon Cytospin4; Thermo Scientific). After blocking human FcR, as described above,mDCs were probed with PE-AEZaMfa1, and Vectashield mounting mediawith DAPI (VWR; catalog 101098-044) was added to the specimens; thenslides were analyzed by conventional epifluorescence (Nikon E600) andby confocal microscopy, as above.

Quantitation of serum DC poietins FLT3 ligand, soluble TNFRI, soluble TNF RII

Serum analysis of DC poietin levels was conducted with colorimetricsandwich ELISA using respective Quantikine immunoassays, as follows:human Flt-3 ligand (Quantikine DFK00), soluble TNF RI (QuantikineDRT100), and soluble TNF RII (Quantikine DRT200) (R&D Systems).Briefly, standard dilutions and recommended serum sample dilutions wereincubated in precoated microplates containing a specific mAb. Unbounddebris was washed, and an enzyme-linked polyclonal Ab specific for thetarget molecule was added. Unbound Ab was removed, and a substratesolution was added to give a proportionate color change relative to theamount of DC poietin in the well. The substrate reaction was stopped, andmicroplates were read immediately at 450 nm on a microplate reader witha correction wavelength of 540 nm (EMax; Molecular Devices).

Determination of human anti-P. gingivalis IgG titers

Serum levels of anti-P. gingivalis IgG Abs were determined by ELISA, aspreviously described (38). In brief, 96-well ELISA plates were coatedwith P. gingivalis strain DPG-3 (1 3 107 cells/well), followed by 1-hincubation at 37˚C. The wells were blocked for 2 h at 37˚C with 2% BSA(Sigma-Aldrich; catalog A3912) in PBS-Tween 20 (Sigma-Aldrich;catalog P1379). Appropriate dilution of serum samples as determinedby checkerboard titrations using pooled sera was added, and the plateswere incubated for 2 h at 37˚C. HRP-conjugated goat anti-human IgG(H+L) Abs (Promega, Madison, WI; catalog W4031) were added andincubated for 1 h at 37˚C. The 3,39,5,59-tetramethylbenzidine liquidsubstrate for ELISA (Sigma-Aldrich; catalog T4444) was used as asubstrate. The reaction was stopped by the addition of 3 N hydrochloricacid (LabChem, Pittsburgh, PA; catalog LC15360-2), and the OD wereread using an E-max microplate reader (Molecular Devices, Palo Alto,CA) at 450 nm.

Statistical analysis

Summary statistics were computed for human peripheral blood mDC counts(numbers and percentages), number of PBMCs, serum IgG titers, serum DCpoietins, and lipid profiles in humans, who were grouped according todisease status. D’Agostino-Pearson normality test at p , 0.05 was per-

formed to confirm Gaussian distribution, after which differences inmeans 6 SD of healthy versus CP or ACS/CP and CP versus ACS/CPsubjects were analyzed by Student t test at p , 0.05 (GraphPad Prizm 5;GraphPad Software, La Jolla, CA). The incidence of P. gingivalis insubgingival plaque and blood mDCs of CP patients was analyzed by x2 andFisher’s exact test, and Spearman rank test (data shown) with significancewas assessed at p , 0.05. In vitro assays of DC differentiation, DC ex-pression of matrix metalloproteinase (MMP)-9, and fold changes in ath-erogenic marker mRNA were performed in triplicate and repeated aminimum of three times. Data were analyzed either by repeated measuresANOVA or Kruskal–Wallis test at p , 0.05.

ResultsBlood mDC frequency increases with chronic periodontitis andACS

The number and percentages (Fig. 1) of blood mDCs were ana-lyzed in three groups, as follows: healthy controls, CP subjects atincreased coronary artery disease risk due to lipid profile andserologic exposure to the atherogenic pathogen P. gingivalis(Table I) (39), and 100% coronary artery disease risk due toexisting ACS, as well as CP. No ACS patients could be foundwithout CP. The results show significant increases in blood mDCsin the following order: CTL , CP , ACS/CP. This was not at-tributable to an increase in total PBMCs, which were actuallydecreased in CP and ACS/CP (Fig. 1B3). Due to previous corre-lation between blood DC frequency and serum levels of FLT-3Land soluble TNF receptors (40, 41), we analyzed these cytokinesin the same patient sera. The results show no difference in FLT-3L,TNFr1, or TNFr2 in CP versus CTL subjects. However, a signifi-cant increase was noted in all three cytokines in ACS/CP, relativeto CP and CTL (p , 0.05, Student t test) (Fig. 1C). As infectiousseropositivity has been correlated with cardiovascular diseasemortality (42), we analyzed the correlation between serum Abtiters against P. gingivalis DPG-3, a strain that solely expresses theDC adhesin, mfa-1 fimbriae (9), and blood mDC numbers. Theresults demonstrate a linear relationship between serum Ab titersto P. gingivalis DPG-3 and blood mDC frequency in CTL versusCP (r2 = 0.56, p , 0.0001) (Supplemental Fig. 1A). Note that seraand subgingival plaque from the ACS/CP cohort were unavailablefor analysis in this study, but previous studies have documented inACS subjects the presence of serum IgG Abs to P. gingivalis andP. gingivalis DNA in saliva (43).

Blood mDCs contain a microbiome, as well as viablepathogens and intact P. gingivalis

To determine whether the blood mDCs contain microbes that couldhelp explain their responsiveness in vivo, blood mDCs were iso-lated from CP and ACS/CP subjects and analyzed by 16S rDNAsequencing, immunofluorescence, and viable cultures. The 197-bpamplified product specific for P. gingivalis is shown in repre-sentative mDCs of CP and ACS/CP subjects, relative to in vitrocontrols (Supplemental Fig. 1B). Overall, 72% of CP subjectswho were orally colonized by P. gingivalis (Supplemental Table I)were also positive for P. gingivalis 16S rDNAwithin blood mDCs,whereas no CTL mDCs yielded the amplified product. The cor-relation between oral colonization with P. gingivalis and mDCinfection rate in CP subjects was positive and significant (Spear-man r = 0.5192, p = 0.0078). Although dental plaque was un-available from ACS/CP patients for analysis, 37.5% of their mDCswere positive for P. gingivalis 16S rDNA. To estimate the level ofP. gingivalis infection of blood mDCs, MoDCs were pulsed witha range of known MOIs and CFUs of P. gingivalis and 16S rDNAused to generate linear regression models (Supplemental Fig. 1C,Supplemental Table I). Based on these models, we calculatedestimated CFUs of P. gingivalis in blood mDCs from CP and

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ACS/CP patients, which were equal to 132,623 estimated CFUs(6121,484 SE), whereas estimated MOIs were all ,1. Otherpotential species identified and sequenced from 16S rDNA ofmDCs included H. pylori, Pseudomonas spp., Moraxella catar-rhalis, Klebsiella pneumonia, Salmonella enterica, and others.Live bacteria recovered on blood agar from mDCs includedBurkholderia cepacia from two ACS/CP patients (SupplementalTable I). Although viable P. gingivalis were not cultivable fromblood mDCs, probably due to their dormant state (44), we dididentify intact P. gingivalis at low MOI within mDCs of CPsubject by immunofluorescence-confocal microscopy (Fig. 2A).The specificity of our mAb for P. gingivalis mfa-1 (AEZaMfa1)was first established, as shown (Supplemental Fig. 1D). To con-firm the ability of P. gingivalis to infect CD1c+ DC-SIGN+ bloodmDCs, we pulsed ex vivo isolated mDCs from a CTL subject withCFSE-labeled P. gingivalis in vitro, which were then counter-labeled with DC-SIGN. The results show intact P. gingivalis-CFSE within DC-SIGN+ blood mDCs (Fig. 2B).

Induction of short-term bacteremia increases blood mDCcarriage state and frequency

To determine whether elevated peripheral blood DC counts cor-related with the presence of blood-borne infection, CP patientswere subjected to mechanical debridement (i.e., S&RP), thestandard therapy for CP. This treatment is well documented todrive oral bacteria such as P. gingivalis and others into the blood-stream (11, 32, 33). mDCs were isolated 24 h later and analyzedfor P. gingivalis 16S rDNA content and blood mDC frequency.The results (Fig. 2C) indicate a significant increase in P. gingivaliscontent of blood mDCs after S&RP. Accompanying this was a∼25% increase in blood mDC frequency (Fig. 2D). This response

was statistically significant when comparing pre- and posttreat-ment in all the CP subjects (p , 0.05, paired Student t test) (Fig.2E). There was no increase in total PBMCs after therapy, nor werelevels of DC poetins FLT-3L, TNFr1, or TNFr2 significantly al-tered by therapy (data not shown).

Increased mDC frequency involves de novo mDCdifferentiation in response to infection

A previous in vitro study indicated that differentiation of DCs frommonocytes can be enhanced by Ab ligation of DC-SIGN (45). Wereasoned that a similar ligation of DC-SIGN by P. gingivalis mfa-1, as reported (10), may enhance differentiation from monocytes.Therefore, monocytes were cultured with the growth factors GM-CSF/IL-4 and P. gingivalis added at low MOIs in select wells. Theresults confirmed that GM-CSF/IL-4 alone promotes differentia-tion of monocytes into immature CD1c+ DC-SIGN+ mDCs, afteran apparent 1-d delay. These were indeed immature mDCs, as theywere negative for CD83 and CCR7 (data not shown). Moreover,mDC differentiation was enhanced ∼28% by P. gingivalis at aMOI of 0.1, but was retarded by MOI of 0.5 and 1 in a dose-dependent manner (Fig. 3A). Interestingly, P. gingivalis alone at0.1 MOI induced differentiation of CD1c+ DC-SIGN+ mDCs inthe absence of GM-CSF/IL-4 (Fig. 3B), and this continued for 3 d.When the MOI dose was increased to 0.5, the effect was dimin-ished, whereas a MOI of 1 caused no enhancement of MoDCdifferentiation relative to controls.

mDCs provide a protective niche for P. gingivalis

Viable P. gingivalis (44) and C. pneumonia (46) have been re-covered with some difficulty from atherosclerotic plaques. Todetermine whether the obligate anaerobe P. gingivalis survives

FIGURE 1. Increased blood mDC

frequency in chronic periodontitis

and ACS. (A) Representative scatter-

grams from flow cytometry analysis

of CD1c+ (BDCA-1) (x-axis) DC-

SIGN+ (y-axis) blood mDCs at base-

line in healthy control (CTL) subjects

(n = 25), chronic periodontitis (CP)

subjects (n = 25), and subjects with

ACS and CP (ACS + CP) (n = 15).

(B) Mean numbers (1) and percen-

tages (2) of blood mDCs (mDCs/

20,000 PBMCs) and total numbers of

PBMCs/ml (3) in CP, ACS/CP rela-

tive to CTL. **p , 0.05, Student t

test. (C) Serum ELISA levels of FLT-

3L and soluble TNFR-1 and TNFR-2

(ng/ml) in all three patient groups

performed in triplicate for each pa-

tient sample. *Significant difference

in ACS + CP groups relative to CP

and CTL (p , 0.05, Student t test).

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within mDCs under aerobic conditions, in vitro culture studieswere performed. Our results (Fig. 3C) show that P. gingivalissurvives for 24 h within mDCs, whereas it dies rapidly in theabsence of mDCs. Pretreatment of mDCs with cytochalasin Dreduces the protective effect of mDCs 10-fold at 6 and 24 h. Wefurther show professional phagocyte PMNs kill P. gingivalis rapidly.

Conversion of mDCs to atherogenic phenotype by DC-invasiveP. gingivalis

MMPs are involved in extracellular matrix destruction, plaquerupture, and myocardial infarction (47). MMP-9 is particularlyimportant in mDC migration (48). We therefore analyzed secre-tion of MMP-9 by mDCs pulsed with P. gingivalis strains thatexpress the DC adhesin mfa-1 and gain access into mDCs (i.e.,Pg381, PgDPG-3) or not (PgMFI). The results support mfa-1–dependent secretion of MMP-9 by mDCs (Fig. 3D). Other indi-cators of risk of plaque rupture, including C1q (49), heat shockprotein (HSP) 60/HSP70 (reviewed in Ref. 50), CCR2 (51), andCXCL16 (52, 53), were analyzed at the mRNA level (Fig. 3E).Analysis of these transcripts indicates that C1q, HSP60, HSP70,CCR2, and CXCL16 are upregulated in response to fimbriatedPg381, relative to untreated control. Moreover, relative to thefimbriaeless mutant Pg MFB, Pg381 upregulates C1q, CXCL16,and HSP70 on mDCs.

mDCs infected by P. gingivalis infiltrate oral submucosa andatherosclerotic plaques in situ

Previous quantitative immunofluorescence histomorphometry es-tablished that the diseased oral mucosa (26, 54) and atheroscleroticplaques (16) are infiltrated with increasing numbers of DC-SIGN+

mDCs. In view of the current data and our previous in vitro workestablishing mfa-1 fimbriae as a DC-SIGN (CD209) ligand (9,10), we opted to ascertain whether mfa-1 could be identifiedwithin DC-SIGN+ and CD1c+ mDCs in oral mucosa. The resultsindicate that P. gingivalis mfa-1 colocalizes with DC-SIGN+

mDCs in oral submucosa (Fig. 4). Identical staining of healthycontrol (oral) tissue revealed trace mfa-1 and some DC-SIGNpositivity, but no mfa-1/DC-SIGN colocalization was detected(data not shown). We then probed postmortem coronary arterybiopsies from patients with CAD and CP using the same Abs (Fig.5). A representative sample is shown. Note erosion and vascularinflammation of the intimal subendothelial layer (box) (Fig. 5A).Evident are CD1c+ (Fig. 5B) and DC-SIGN+ (Fig. 5C) mDCs, aswell as colocalization of P. gingivalis mfa-1 with its receptor DC-SIGN (Fig. 5D).

DiscussionIn this study, we provide evidence in humans for infection of mDCsas a significant route for pathogen dissemination to atheroscleroticplaques. The blood DCs respond to the low-grade infection CP, aswell as the acute bacteremia elicited by treatment of CP. DCs werefirst identified in the human aortic intima in 1995 and were thoughtto be important in the development of atherosclerotic lesions (18).Soon after, DCs were shown to contact T cells and to overexpress,in early lesions, HSP70, an indicator of physiologic stress (55).HSP70 is also expressed by intracellular bacterial species (56),including P. gingivalis (57), and, as we show in this work, bymDCs infected with P. gingivalis. Subsequent studies of athero-sclerotic plaques showed a prodigious infiltrate of DC-SIGN+

immature mDCs (58), as we observed in oral mucosa of CPsubjects (54). The immature state of these mDCs is consistent witha reported role for DC-SIGN ligation in inhibiting TLR-mediatedDC maturation (9, 59) and in promoting differentiation of im-mature DCs from progenitors (45). The present work isolatedCD1c+DC-SIGN+ mDCs from the blood of patients at increasedrisk for CAD and those with existing CAD and showed that theyharbor DNA of P. gingivalis and other human pathogens. Theinfection of blood mDCs was linked to increased blood mDCfrequency and to increased relative risk of CAD. The blood mDCswere immature despite containing whole intact P. gingivalis. Thisis consistent with inefficient maturation of mDCs by P. gingivalis

FIGURE 2. P. gingivalis carriage state and frequency of blood mDCs,

before and after induced bacteremia. (A) Image series from scanning laser

confocal microscopy (1–7, z-stack series of 1-mm slices) of CD1c+

(BDCA-1+) blood mDCs from oral carriage-positive CP patient, per-

meabilized and cytocentrifuged on slides and probed with AEZaMfa1-PE,

followed by Vectashield mounting media. P. gingivalis (red) in mDC is

shown by white arrow. (B) Epifluorescence deconvolution image analysis

of blood mDCs from a healthy donor pulsed in vitro with CFSE-labeled P.

gingivalis 381 (green) at a MOI of 1 for 3 h. MDCs were counterstained

with DAPI (blue) and RPE-conjugated DC-SIGN (red). (C) Significant

increase in P. gingivalis 16S rDNA content of blood mDCs from CP

subjects (n = 6) 24 h after local debridement (S&RP). MDCs were ana-

lyzed for 16S rDNA of P. gingivalis, quantified as in Supplemental Fig. 1C

(*p = 0.02, paired t test). (D) Representative scattergrams from flow

cytometry analysis of CD1c+ (BDCA-1+) and CD209 (DC-SIGN+) blood

mDCs before (0 h) and 24 h after local debridement (S&RP) of CP

patients, as described in Materials and Methods. (E) Significant increase in

mean number (1) and percentages (2) of blood mDCs 24 h after S&RP (per

30,000 PBMCs) (**p , 0.05, paired t test).

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due to ligation of DC-SIGN by mfa-1 (9). The initial site of mDCinfection by P. gingivalis is most likely the oral submucosa. In-troduction of P. gingivalis into the oral mucosa in mice resultsin rapid (30-min) bacteremia (personal communication fromC. Genco), but the role of DCs in this bacteremia is not yet clear.Trafficking of mDCs through tissues is aided, in particular, byMMP-9 (48, 60), a biomarker of increased CAD risk and a me-diator of plaque instability (61). We show that human mDCsstimulated by P. gingivalis release high levels of MMP-9, and that

this is dependent on expression by P. gingivalis of the DC-invasinmfa-1. Increased mobilization of Langerhans cells, which expressCD1c (55), and dermal DCs, which express DC-SIGN, is observedin the oral submucosa in CP, and these DCs accumulate in closeproximity to the vasculature (27, 54). Hypoxic conditions, as inthe subgingival pockets where P. gingvalis resides (62), promotethe transmigratory activity of DCs through endothelium (63).Overall, the hypoxic microenvironment and local infection with P.gingivalis may be a potent driving force for reverse transmigration

FIGURE 3. P. gingivalis induces mDC differentiation, survives within mDCs, and induces atherogenic mDC phenotype. (A) Pre-DC monocytes from

healthy controls were cultured in triplicate with growth factors GM-CSF/IL-4+/2 P. gingivalis 381 at MOIs of 0.1, 0.5, and 1. The mean number of cells

appearing in the MoDC gate (CD1c+DC-SIGN+) per ml 6 SE is shown. Phenotype of immature MoDCs was further confirmed by evidence of down-

regulation of CD14 and low to no expression of CD83 and CCR7 (data not shown). *Significant difference between control and growth factors (GF) and

MOI of 0.1 + GF (ANOVA, p, 0.05). Data are representative of results of assay repeated three separate times. (B) Performed as in (A), but without growth

factors. Significant differences were noted in mean number of MoDCs per ml in MOI 0.1-GF versus MOI 0.5-GF at 2 and 3 d, and in MOI 0.1-GF, MOI 0.5-

GF versus control, and MOI 1-GF at 2 d (ANOVA, p , 0.05). (C) Wild-type Pg381 was incubated in mDC buffer alone (filled circle) or with mDCs (filled

square), or with mDCs pretreated with cytochalasin D (filled triangle), or control PMNs (inverted filled triangle) at a multiplicity of infection of 100 for 0, 6,

and 24 h. Internalization of CFSE-labeled P. gingivalis was confirmed by epifluorescence microscopy. Cells were lysed, and viable bacteria was recovered

on enriched anaerobic 5% blood agar plates in triplicate under anaerobic conditions (10% H2, 5% CO2 in nitrogen) at a 1:10 dilution at 35˚C for 14 d, after

which log10 CFU/ml was determined. Experiment was repeated three separate times, and data are representative of consistent results. (D) MoDCs were

pulsed with wild-type Pg 381, its mfa-1 minor fimbriae-deficient strain (MFI) or fimA major fimbriae-deficient strain (DPG-3), or no Pg (DC ctl) for 18 h at

a MOI of 1:25. Secretion of MMP-9 in pg/ml was assessed by ELISA. The data are the mean6 SD of triplicate assays. (E) MoDCs were pulsed in triplicate

with wild-type Pg381, its fimbriaeless mutant MFB, or no Pg (CTL) at a 25:1 MOI for 3 h, and uptake of CFSE-labeled Pg was monitored by FACS analysis

(data not shown). qRT-PCR (Bio-Rad) was used to determine expression levels, normalized to b-actin, and expressed as fold changes in mRNA. PCR

primers were designed using PRIMER3 Software (77). For relative quantification of transcript expression, cycle threshold values were obtained for each

gene and data were analyzed using the Excel (Microsoft) macro GENEX v1.10 (Gene Expression analysis for iCycle iQ Real-time PCR Detection System,

v1.10, 2004; Bio-Rad Laboratories).

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of tissue mDCs into the blood to atherosclerotic plaques (8, 25,64).Our findings also shed light on the enigmatic functions of blood

DCs. It has been speculated that blood DCs translocate antigenicmaterial from its point of origin to remote target tissues (25). Ourresults suggest that clearance of bacteremias may be a patho-physiological function of blood DCs. Bacteremia is a well-

described phenomenon in patients with CP (33), and is inducedby eating an apple (12), tooth brushing, flossing, or undergoingS&RP (65). We demonstrate that both the chronic condition CPand acute bacteremia increase the P. gingivalis content of bloodmDCs and result in an increase in blood mDC frequency. Otherinfectious conditions that alter DC frequency include tuberculosis(66), malaria (67), and filariasis (68). We initially suspected thatDC poietin levels were altering blood mDC frequency in CP, asreported in a number of diseases, including CAD in humans (40,41, 66, 69–74). Whereas elevated DC poietins were found in seraof our ACS/CP cohort, all had been on long-term cardiovascularcare, including lipid-lowering drugs. These are known to raisemDC levels (73). Moreover, 100% of our ACS cohort had CP;none could be identified without it. It should be noted that oursystemically healthy CP cohort were not on such drugs and DCpoietin levels were unchanged in CP. We therefore surmised thatthe bacteremia itself was the impetus for increasing mDC fre-quency. We thus implemented an in vitro infection assay to as-certain the mechanism. We show that indeed DC differentiationwas enhanced by P. gingivalis at low MOIs, but retarded at higherMOIs. We surmise that this differential response may be reflectiveof a balance between two driving forces. At low MOIs, the DC-SIGN ligand P. gingivalis mfa-1 (10) positively influences DCdifferentiation, as reported for anti–DC-SIGN Abs (45). Thisinvolves activation of a unique signaling pathway, the DC-SIGNsignalosome, which is counterregulatory to TLR signaling (59). Incontrast, infection by P. gingivalis at higher MOIs has a negativeinfluence on DC differentiation due to enhanced apoptosis (75).Although we have no evidence yet that this effect is specific for P.gingivalis, previous studies using several other species suggesta predominantly negative influence of bacterial infection on DCdifferentiation (76).Collectively, these data indicate that mDCs in tissues and blood

harbor pathogens of direct relevance to coronary artery disease andother human diseases. MDCs protect the pathogen, whereas theinfection promotes further mDC differentiation and converts mDCsto an atherogenic phenotype. We have thus identified an importantpathophysiological mechanism that links chronic low-grade in-fections to an increased risk of cardiovascular disease and otherdiseases.

AcknowledgmentsWe give special thanks to Ruth Tenzler for study coordination and Emil

Kozarov for postmortem biopsies.

DisclosuresThe authors have no financial conflicts of interest.

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