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Cell Response Strains Elicits a Distinct Effector Tgingivalis

PorphyromonasCells by Minor Fimbriated Targeting of DC-SIGN on Human Dendritic

W. CutlerAmir E. Zeituni, Ravi Jotwani, Julio Carrion and Christopher

http://www.jimmunol.org/content/183/9/5694doi: 10.4049/jimmunol.0901030October 2009;

2009; 183:5694-5704; Prepublished online 14J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Targeting of DC-SIGN on Human Dendritic Cells by MinorFimbriated Porphyromonas gingivalis Strains Elicits a DistinctEffector T Cell Response1

Amir E. Zeituni,2† Ravi Jotwani,2* Julio Carrion,* and Christopher W. Cutler3*

The oral mucosal pathogen Porphyromonas gingivalis expresses at least two adhesins: the 67-kDa mfa-1 (minor) fimbriae and the41-kDa fimA (major) fimbriae. In periodontal disease, P. gingivalis associates in situ with dermal dendritic cells (DCs), many ofwhich express DC-SIGN (DC-specific ICAM-3 grabbing nonintegrin; CD209). The cellular receptors present on DCs that areinvolved in the uptake of minor/major fimbriated P. gingivalis, along with the effector immune response induced, are presentlyunclear. In this study, stably transfected human DC-SIGN�/� Raji cell lines and monocyte-derived DCs (MoDCs) were pulsedwith whole, live, wild-type Pg381 or isogenic major (DPG-3)-, minor (MFI)-, or double fimbriae (MFB)-deficient mutant P.gingivalis strains. The influence of blocking Abs, carbohydrates, full-length glycosylated HIV-1 gp120 envelope protein, andcytochalasin D on the uptake of strains and on the immune responses was determined in vitro. We show that the binding of minorfimbriated P. gingivalis strains to Raji cells and MoDCs is dependent on DC-SIGN, whereas the double fimbriae mutant strain doesnot bind. Binding to DC-SIGN on MoDCs is followed by the internalization of P. gingivalis into DC-SIGN-rich intracellularcompartments, and MoDCs secrete low levels of inflammatory cytokines and remain relatively immature. Blocking DC-SIGN withHIV-1 gp120 prevents the uptake of minor fimbriated strains and deregulates the expression of inflammatory cytokines. Moreover,MoDCs promote a Th2 or Th1 effector response, depending on whether they are pulsed with minor or major fimbriated P.gingivalis strains, respectively, suggesting distinct immunomodulatory roles for the two adhesins of P. gingivalis. The Journal ofImmunology, 2009, 183: 5694–5704.

T he C-type lectin dendritic cell (DC)4-specific ICAM-3grabbing nonintegrin (DC-SIGN) is a pattern recognitionreceptor (PRR) and adhesion molecule expressed by den-

dritic cells (DCs) and by certain types of macrophages (1). It isused to endocytose microbial Ags in the periphery, bind toICAM-2 on endothelial cells (2) and mediate immune clusteringwith ICAM-3� T cells in the lymph nodes (1, 3, 4). It is alsoexpressed on blood DCs (5), in pathological conditions such asrheumatoid arthritis (6), and in rupture-prone atheroscleroticplaques (7, 8). Recent studies indicate that DC-SIGN� DCs in-crease in the oral mucosal disease chronic periodontitis (CP) (9–11). DC-SIGN is one of a family of calcium-dependent C-typelectins that bind to carbohydrate motifs and to Lewis blood groupAgs (12, 13). Although lacking Toll-IL-1R activation domains,

DC-SIGN has emerged as a key player in the induction of immuneresponses against numerous pathogens via modulation of TLR-induced immune activation (14). This occurs by activation of ERK(15) and by Raf-1 kinase-dependent acetylation of transcriptionfactor NF-�B (16, 17). Mycobacterium tuberculosis (18), Myco-bacterium leprae (19), and Helicobacter pylori (20) target DC-SIGN to gain entry into DCs, disrupt full DC maturation, andinhibit Th1 effector cell polarization. Neisseria meningitidis andLactobacillus spp., in contrast, target DC-SIGN to modulate theimmune response toward Th1 (21) or Treg (22), respectively.

The immunopathogenesis of CP has been linked to negativeregulation of TLRs (23–25) and to the presence of Th2 effector Tcell populations (reviewed in Ref. 26), but the specific role of oralmucosal pathogens in the induction of Th2 effector responses isjust beginning to be identified (9). The oral mucosa in CP containsorganized lymphoid aggregates called oral lymphoid foci or OLF(27). OLF contain immune conjugates consisting of dermal DCsand CD4� T cells, as well as B cells (28). Of particular interest isthe presence of an intense infiltrate of DC-SIGN� DCs in thelamina propria of CP combined with evidence that DCs in thelesions appear to mobilize toward the capillaries (28). This hasfueled speculation that, as with gut lamina propria DCs (29), spe-cific microbiota in the oral mucosa target lamina propria DCs thatcan direct the T cell effector responses (30, 31).

Porphyromonas gingivalis (Pg) is one of several intracellularpathogens implicated in CP (reviewed in Ref. 32). Most pathogens,Pg included (33), express different pathogen-associated molecularpatterns that can trigger distinct classes of PRRs on a single cellsimultaneously (14). Of particular relevance are the two adhesinsof Pg, termed the mfa-1 (minor) and fimA (major) fimbriae. Theadhesion of pathogens to host tissues and the subsequent invasionare important early events in mucosal pathogenesis (34). The mi-nor and major fimbriae of Pg have been shown in the rat model to

*Department of Periodontics and Implantology, School of Dental Medicine and †De-partment of Molecular Genetics and Microbiology, Stony Brook University, StonyBrook, NY 11794

Received for publication March 31, 2009. Accepted for publication August 31, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was supported by U.S. Public Health Service, National Institutes ofHealth/National Institute of Dental and Cranial Research Grants R01 DE014328 (toC.W.C.), F31 DE020014 (to A.E.Z.), and K23 DE018187 (to J.C.).2 A.E.Z. and R. J. contributed equally to this work.3 Address correspondence and reprint requests to Dr. Christopher W. Cutler, Depart-ment of Periodontics, School of Dental Medicine, Stony Brook University; StonyBrook, NY 11794-8703. E-mail address: ccutler@notes.cc.sunysb.edu4 Abbreviations used in this paper: DC, dendritic cell; CBA, cytometric bead array;CM, Chiang Mai; CP, chronic periodontitis; DC-SIGN, DC-specific ICAM-3 grab-bing nonintegrin; maj, major; min, minor; MoDC, monocyte-derived DC; MOI, mul-tiplicity of infection; OLF, oral lymphoid foci; Pg, Porphyromonas gingivalis; PRR,pattern recognition receptor; Raji-DCS, DC-SIGN positive Raji cell.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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play roles in the pathogenesis of periodontal disease (34). The twofimbriae are distinct antigenically by amino acid composition andby size (35, 36). The major fimbriae are composed of a 41-kDaprotein encoded by the fimA gene (37). Much is known of thePRRs targeted by the major fimbriae (38–42) and of the intracel-lular signaling pathways that are activated (43, 44). In contrast,little is known of the cellular receptors targeted by the 67-kDaminor fimbriae encoded by the mfa1 gene. The expression of bothfimbriae is regulated under different environmental conditions (45–47). Understanding the immunobiological properties of these twofimbriae could help in understanding how this oral mucosal patho-gen evades the immune response and induces periodontal disease,described as a Th2 type disease (24).

The purposes of the present study were as follows: 1) to deter-mine the role of DC-SIGN in the binding and uptake of isogenicminor and major fimbria-deficient mutants of Pg using stablytransfected Raji (B) cell lines and monocyte-derived dendritic cells(MoDCs) and; 2) to determine how minor/major fimbriae influenceDC maturation, cytokine secretion, and the T cell effector re-sponses induced by MoDCs. Our results show that the minor fim-briae of Pg are required for binding to the endocytic receptor DC-SIGN, leading to internalization in DC-SIGN-rich compartments.This uncouples cytokine secretion from the maturation of DCs andelicits a Th2-biased effector T cell response. Overall, these resultsmay help explain how this oral pathogen evades and suppresses theimmune response.

Materials and MethodsBacterial strain, growth conditions, bacterial labeling, anduptake experiments

Wild-type (WT) Pg381, which expresses both minor (min) and major (maj)fimbriae (Pgmin�/maj�), isogenic minor fimbria-deficient mutant MFI,which expresses only the major fimbriae (Pgmin/�maj�), isogenic majorfimbria-deficient mutant DPG3, which expresses only the minor fimbriae(Pgmin�/maj�, and the double fimbriae mutant MFB (Pgmin�/maj� weremaintained anaerobically (10% H2, 10% CO2, and 80% N2) in a FormaScientific anaerobic system glove box model 1025/1029 at 37°C (48, 49) inDifco anaerobe broth MIC. Erythromycin (5 �g/ml) and tetracycline (2�g/ml) were added according to the selection requirements of the strains.Bacteria were pelleted, washed once in PBS and, for FACS-based analyses,stained with CFSE (Molecular Probes) as described (50). Briefly, bacteriain PBS were stained with CFSE at a final concentration of 5 �M for 30 minat 37°C and protected from light. The bacterial suspension was washed fivetimes in PBS and Pg was resuspended to an OD at 660 nm of 0.11, whichpreviously determined to be equal to 5 � 107 CFU (51). MoDCs and Rajicells were pulsed with Pg strains at 5:1 or 25:1 multiplicity of infection(MOI) for 1.5–18 h. Low MOI values were used to mimic a natural in-fection as well as to avoid overwhelming the host response. The percent-ages of viable MoDC or Raji (typically �90% after 18 h) were monitoredby trypan blue exclusion and did not differ between the strains (not shown).MoDCs and Raji cells were fixed and stained for FACS analysis of thepercentage of Pg-CFSE� cells as described in the figure legends.

Raji and Raji DC-SIGN� cells and Abs

Raji cell lines (52) were obtained courtesy of D. R. Littman (Skirball Instituteof Biomolecular Medicine, New York University, New York, NY), main-tained in 10% heat-inactivated FBS (Invitrogen), RPMI 1640 with L-glu-tamine, and NaHCO3 (Sigma-Aldrich) in a 5% CO2 incubator at 37°C. Stain-ing to verify surface receptor expression was performed using the followingmAbs: anti-CD14-FITC (BD Biosciences catalog no. 555397); anti-CD209(BD Biosciences catalog no. 551264); anti-E-cadherin (BD Biosciences cata-log no. 612130); anti-CD19 (BD Biosciences catalog no.557697); IgG1 iso-type (BD Biosciences catalog no. 349041); anti-CD80 (BD Biosciencescatalog no. 557226); IgG2a isotype (BD Biosciences catalog no. 349051);anti-CD29-PE (BD Biosciences catalog no. 555443); anti-CD209 (BD Bio-sciences catalog no. 551265); anti-CD18 (BD Biosciences catalog no.555924); anti-CD206 (BD Biosciences catalog no. 555954); anti-HLA-DR(BD Biosciences catalog no. 555812); anti-CD86 (BD Biosciences catalogno. 555657); anti-CD11a (BD Biosciences catalog no. 555379); IgG1 iso-type (BD Biosciences catalog no.349043); anti-CD205- allophycocyanin

(BD Biosciences catalog no. 558156); anti-CD11b (BD Biosciences cata-log no. 550019); IgG1 isotype (BD Biosciences catalog no. 557732); anti-human TLR4-PE (eBioscience catalog no. 12-9917-73); anti-human TLR4-FITC (eBioscience catalog no. 53-9917-73); anti-human TLR2-FITC(eBioscience catalog no. 11-9922-73); anti-CD83-FITC (Immunotech cat-alog no. PN IM2410); anti-CD207-PE (Immunotech catalog no. IM3577);anti-CD11c-PE (Invitrogen catalog no. 349863a); and anti-CD1a-FITC(DakoCytomation catalog no. F7141).

Raji cell adhesion assay

CFSE- or Syto-stained bacteria were added at an MOI of 5:1 to either Rajior Raji DC-SIGN cells (52, 53). Binding was conducted on ice to preventloss of surface DC-SIGN, which cycles rapidly on Raji cells at 37°C (52–54). Cells were pulsed with CFSE-stained bacteria for 1–18 h either at37°C, on ice (48), or in the presence of cytochalasin D at 0.5 �M (theminimal concentration needed to arrest cytoskeletal rearrangements in Rajicells). Cells were washed to remove unbound bacteria, fixed in 1% for-malin, and analyzed by FACS. Association was quantified via FACS aspreviously described (19, 55, 56). In brief, 10,000 Raji cells were gated onforward and side scatter characteristics based on size and to exclude debrisand unbound bacteria and then the percentage of CFSE-positive cells in theFITC channel was quantitated.

Carbohydrate/Ab/gp120 blocking assay

The carbohydrates D-mannose (Sigma-Aldrich catalog no. M 6020); L-fu-cose (Sigma-Aldrich catalog no. F 2252–5G); D-fucose (Sigma-Aldrichcatalog no. F 8150), and mannan from Saccharomyces cerevisiae (Sigma-Aldrich catalog no. M 7504-5g) were all diluted in 2% heat inactivatedFBS (Invitrogen) and PBS and filter sterilized. The carbohydrates (5–100�g) were added to block the DC-SIGN receptor. The reagent, HIV-1 gp120Chiang Mai (CM) envelope protein (1.5–9 �g) was obtained through theNational Institutes of Health AIDS Research and Reference Reagent Pro-gram, Division of AIDS, National Institute of Allergy and Infectious Diseases,National Institutes of Health (catalog no. 2968) and added to block the DC-SIGN receptor. The blocking Ab to human DC-SIGN (clone 120507l; R&DSystems catalog no. MAB161), the IgG2B isotype control Ab (clone 20116;catalog no. MAB004), the anti-human integrin �2 mAb (CD18; Chemiconcatalog no. CBL158), the anti-human integrin �1 mAb (Chemicon catalog no.MAB1987Z), the anti-human CD11c mAb (clone B-ly6; BD Pharmingen cat-alog no. 555390), and the IgG1 isotype control (Chemicon catalog no.CBL600) were added at a final concentration of 10 �g/ml. Raji and RajiDC-SIGN cells were preincubated with blocking Abs or carbohydrates for atleast 30 min on ice. CFSE-stained bacteria were then cocultured for 1 h on iceand binding was measured via FACS as described in the adhesion assay.MoDCs were preincubated with carbohydrates, HIV-1 gp120 CM envelopeprotein, or Abs for at least 30 min at 37°C before being cocultured with CFSE-stained bacteria. Cocultures proceeded for 3 h at 37°C and association wasmeasured via FACS as described in the Raji adhesion assay.

DC cultures and multiparameter flow cytometry analysis

MoDCs were generated as we have described previously (10, 11, 27).Briefly, monocytes were isolated from mononuclear fractions of the pe-ripheral blood of healthy donors and seeded in the presence of GM-CSF(100 ng/ml) and IL-4 (25 ng/ml) at a concentration of 1–2 � 105 cells/mlfor 6–8 days, after which flow cytometry was performed to confirm theimmature DC phenotype (CD14�CD83�CD1a�DC-SIGN�). Cell surfacemarkers of DCs were evaluated by four-color immunofluorescence stainingwith the following mAbs: CD1a- FITC (Biosource), CD80-PE (BD Bio-sciences), CD83-PE (Immunotech), CD86- PE (BD Pharmingen), HLA-DR-PerCP (BD Biosciences), and CD14-allophycocyanin (Caltag). After30 min at 4°C and washing with staining buffer (PBS (pH 7.2), 2 mMEDTA, and 2% FBS), cells were fixed in 1% paraformaldehyde. Analysiswas performed with a FACSCalibur flow cytometer (BD Biosciences).Marker expression was analyzed as the percentage of positive cells in therelevant population defined by forward scatter and side scatter character-istics. Expression levels were evaluated by assessing mean fluorescenceintensity indices calculated by relating the mean fluorescence intensitynoted with Pg-CFSE or the relevant mAb to that of the isotype control mAbfor samples labeled in parallel and acquired using the same setting.

Cytokines from MoDCs

Culture supernatants were collected from MoDCs pulsed with Pg strainsfor 3 and 18 h. Culture supernatants were analyzed by flow cytometry usinga cytometric bead array (CBA) kit (BD Biosciences) following the man-ufacturers’ instructions. A standard curve was achieved for each cytokine;the CBA software calculates levels in picograms per milliliter.

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T cell priming

For T cell priming experiments, the responder cells were autologous CD4�

naive T cells purified from a mononuclear fraction of human buffy coats bypositive selection, using anti-CD4 mAb and goat anti-mouse IgG-coatedmicrobeads (Miltenyi Biotec) as described previously (10). Briefly, isola-tion of CD4� cells was achieved using MiniMACS separation columns(Miltenyi Biotec) as described by the manufacturer. In all experiments, theisolated cells were 80–90% CD4� as determined by staining with FITC-conjugated anti-CD4 mAb followed by flow cytometry analysis (results notshown). MoDCs were washed extensively after an 18-h pulsing with Pgstrains and cultured at graded doses (1,000, 500, and 50 DCs, all per 200�l) in complete RPMI 1640 medium with 10% heat-treated FCS and au-tologous T cells (50,000 cells/200 �l). Proliferation was determined after5 days by loss of CFSE staining.

ResultsDC-SIGN-mediated binding of Pg to Raji cells is dependent onminor fimbriae

Pg has been previously shown to associate in situ with DCs inhuman oral mucosa from chronic periodontitis patients (57) and tobe taken up by MoDCs in vitro (11), but the cellular receptors used

for binding to MoDCs and uptake are unclear. DC-SIGN is ofparticular interest, as DC-SIGN� DCs increase in inflamed oralmucosa in chronic periodontitis (9, 30). To determine the ability ofWT Pg381 to bind to DC-SIGN, stably transfected DC-SIGN-pos-itive (Raji-DCS) and -negative Raji cells (Raji) were obtained andthe phenotype verified by flow cytometry (Fig. 1A). Our resultsindicate that Raji-DCS and Raji are positive and negative, respec-tively, for DC-SIGN, as previously reported (52). The expressionlevel of other relevant cell surface receptors are also shown in Fig. 1A.To determine the binding of WT Pg381 (Pgmin�/maj�) to Raji-DCSand Raji, bacteria were Syto- or CFSE-labeled and binding wasanalyzed qualitatively by image-enhanced fluorescence micros-copy at low magnification (Fig. 1B, panels 1–4) and at highermagnification (Fig. 1B, panels 5 and 6). Our results indicate thatDC-SIGN is required for optimum binding of WT Pg381 to Rajicell lines. This disparity in binding to Raji-DCS vs Raji was quan-titated by flow cytometry, with binding analyzed at 1.5 h, 3 h, and6 h. Optimum binding was achieved at 1.5 h. Shown in Fig. 1C arethe percentages of Raji-DCS and Raji that were associated with

FIGURE 1. Pg binding to Raji cells dependent on DC-SIGN expression and on minor fimbriae. A, Phenotype of stably transfected Raji-DCS (DC-SIGNpositive) and Raji (DC-SIGN negative) B cell lines. The shift to the right of the histograms indicates increased mean fluorescence intensity of Raji-DCS(red tracing) relative to Raji (green tracing) or relative to isotype control Abs (iso) (blue and purple tracings). B, Binding of Syto-labeled (panels 1-4) orCFSE-labeled (panels 5 and 6) Pg to Raji-DCS and Raji was visualized by epifluorescence microscopy at final magnifications of �40 (panels 2 and 4) and�400 (panels 5 and 6). Cells were also examined by phase contrast (differential interference contrast (DIC)) microscopy (panels 1 and 3). C, FACS analysisof the percentage of Pg� Raji-DCS (Raji positive) for CFSE-labeled Pg381 (Pgmin�/maj�) at 1.5 h. Data are representative of a minimum of threeexperiments. The means � SEM, in triplicate, were analyzed by Students t test and the significant differences between Raji-DCS and Raji are shown. D,Percentage of the loss of Pg� Raji relative to Raji-DCS was calculated as follows: (percentage of Pg� Raji DCS � percentage of Pg� Raji)/(percentageof Pg� Raji-DCS) � 100. Strains shown are Pg381 (Pgmin�/maj�; light blue), DPG-3 (Pgmin�/maj�; yellow), MFI (Pgmin�/maj�; green), and MFB(Pgmin�/maj�; purple). The means � SEM in triplicate, were analyzed by Student’s t test and the significant differences between Raji-DCS and Raji areshown.

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WT Pg381 at 1.5 h. The results indicate a significantly decreasedbinding of WT Pg381 to Raji vs Raji-DCS (243%; p � 0.05,Student’s t test; Fig. 1, C and D). This decrease in binding to Rajiwas greater (271%) with strain DPG-3. In contrast, there was nodifference in the binding of MFI or MFB to Raji vs Raji-DCS.Binding of MFI to Raji likely depends on CD29, which is ex-pressed equally by Raji-DCS and Raji (Fig. 1A) and was previ-ously shown to bind major fimbriae (38). MFB failed to associatewith either Raji-DCS or Raji (Fig. 1D). To further verify the rolefor DC-SIGN in the binding of minor vs major fimbriated Pgstrains to Raji-DCS, we blocked DC-SIGN with L-fucose, man-nose, and mannan (Fig. 2). D-Fucose, a stereoisomer of L-fucosethat does not block DC-SIGN (58), was used as a negative controlfor sugar blocking. Preliminary studies established the optimumdose (50 �g/ml) of sugars for the blocking of DC-SIGN on Raji-DCS by FACS analysis (not shown). We show that binding of WTPg381 and DPG-3 to Raji-DCS was blocked by L-fucose, D-man-nose, and mannan, but not control D-fucose (Fig. 2). The MFIstrain was not blocked by any of the sugars, nor did the sugarsresult in significant blocking of any of the strains to Raji cells (notshown). Blocking of MFB with sugars was not performed, as thisstrain did not bind to either Raji cell line.

DC-SIGN-mediated uptake of Pg by MoDCs is mediated byminor, not major fimbriae

Phenotypic analysis of day 6 MoDCs by flow cytometry indicatesthat MoDCs express surface DC-SIGN as well as CD29, CD11b,CD11c, CD18, and DEC-205 (Fig. 3A). Human MoDCs werepulsed with the four CFSE-labeled strains. The results of flowcytometry analysis (Fig. 3B) indicate that association of three ofthe Pg strains with MoDCs occurred within 3 h in the followingorder: MFI � WT Pg381 � DPG-3. MFB did not bind to MoDCsover a background of 3%. Ab blocking studies were thus per-formed with all strains except MFB. Shown in Fig. 4A are resultswith WT Pg381. Ab blocking studies revealed that anti-DC-SIGN,but not anti-CD11c, anti-CD18 or anti-CD29, resulted in a signif-icant reduction in the association of WT Pg381 with MoDCs. En-docytosis of FITC-dextran (59) was unaffected by the anti-DC-SIGN Ab (not shown), indicating that phagocytosis was still intact.

The use of cytochalasin D, which inhibits the actin polymerizationrequired for internalization but not binding, demonstrates that Pg isbeing internalized by MoDCs. The DC-SIGN-blocking sugarsmannose and mannan diminished the uptake of WT Pg381 (Fig.4A) (13). Because mannose is a minor component sugar of the LPSof Pg (60, 61), we tested the ability of Pg LPS to block the uptakeof WT Pg381, but there was no effect (data not shown). To furtherconfirm the role of DC-SIGN on MoDCs in binding to Pg, we usedthe DC-SIGN-targeting HIV-1 glycosylated envelope proteingp120 as a blocking agent (Fig. 4B). We show that gp120 resultedin a dose-dependent loss of uptake of WT Pg381 and DPG-3, butnot MFI, to MoDCs.

Attachment of Pg to DC-SIGN on MoDCs is followed by uptakeinto DC-SIGN-rich intracellular compartments.

To visualize extracellular and intracellular association of Syto-la-beled WT Pg381, MoDCs were probed with FITC-labeled anti-DC-SIGN at 1 h (Fig. 5A) and 6 h (Fig. 5, B and C) and thenanalyzed by image enhanced fluorescence microscopy, aided bydeconvolution analysis. Early attachment to surface DC-SIGN(Fig. 5A, arrows) is followed by intracellular localization of Pgwith DC-SIGN in MoDCs (Fig. 5, B and C, arrows). At later timepoints (18 h), large numbers of essentially intact WT Pg381 weredetected within as yet undefined intracellular compartments (Pg-containing vesicles or PgCV) of MoDCs (Fig. 5D).

Inflammatory cytokine production induced by major fimbriae,regulated by DC-SIGN-targeting minor fimbriae

Microbial DC-SIGN ligands reportedly dampen TLR-dependentproduction of inflammatory and Th1-biasing cytokines by MoDCs(20). We therefore analyzed the production of inflammatory cyto-kines by MoDCs pulsed with the Pg strains for 3 h (Fig. 6A) and18 h (Fig. 6B). We show that MFI was the most potent inducer ofIL-1�, IL-8, IL-6, and TNF-� at 3 h and of IL-1�, IL-10, IL-12p70, IL-8, and TNF-� at 18 h. In contrast, the DC-SIGN-tar-geting strain DPG-3 induced no detectable TNF-� and signifi-cantly lower levels of IL-1� (vs WT Pg381 or MFI) at 3 h andlevels of IL-1�, IL-12p70, IL-8, IL-6, and TNF-� (vs MFI) at 18 h.Double mutant MFB induced the lowest levels of nearly all cyto-kines at 3 and 18 h. To further confirm the influence of DC-SIGNon the regulation of inflammatory cytokine production by Pg, DC-SIGN was blocked with HIV-1 gp120 before pulsing with allstrains except the double mutant. The results for WT Pg381 (Fig.6C) indicate that blocking DC-SIGN enhances induction of theinflammatory cytokines IL-12p70, IL-8, and IL-6, but not IL-10. Incontrast, in the absence of the TLR-activating major fimbriae (i.e.,DPG-3), blocking DC-SIGN with gp120 does not increase cyto-kines IL-12p70, TNF-�, and IL-6. Interestingly, HIV-1 gp120blocking of DC-SIGN also deregulated inflammatory cytokineproduction by MFI, which does not express the DC-SIGN ligand.HIV-1 gp120 alone did not induce inflammatory cytokines but didinduce IL-10, as previously reported (62).

DC maturation induced by major fimbriae and regulated byminor fimbriae

DCs were further analyzed for maturation status at 18 h (Fig. 7).We show that although HLA-DR induction was nearly equivalentfor all strains, strain MFI was the strongest inducer of CD80 andCD83 and MFB was the weakest inducer of all costimulatory andmaturation markers. Compared with MFI, strain DPG-3 was a rel-atively weak inducer of CD80, CD83, and CD86, with WT Pg381falling somewhere between DPG-3 and MFI (i.e., in induction ofCD80 and CD86). Blocking DC-SIGN with HIV-1 gp120 inhib-ited MoDC maturation, as previously published by Shan et al. (62).

FIGURE 2. Specific carbohydrate blocking of DC-SIGN inhibits bind-ing of minor fimbriated Pg strains to Raji cells. Raji-DCS were pretreatedwith carbohydrates at the doses shown for 30 min, after which Pg381(Pgmin�/maj�), DPG-3 (Pgmin�/maj�), MFI (Pgmin�/maj�) were added toRaji-DCS and Raji (not shown) for 1 h and binding was analyzed by FACS.The percentage of the loss of Pg binding in the presence of carbohydratewas calculated as follows: (percentage of Pg� Raji DCS � percentage ofPg� Raji DCS in presence of carbohydrate)/(percentage of Pg� RajiDCS) � 100. Data are representative of a minimum of three experiments.The means � SEM, in triplicate, were analyzed for significant differencesbetween no blocking (none) and sugar blocking by Student’s t test.

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Furthermore, preincubation with HIV-1 gp120 before coculturewith the Pg strains inhibited the MoDC maturation induced by theDC-SIGN-targeting strains WT Pg381 and DPG-3, unlike the in-flammatory cytokine response, which was enhanced by HIV-1gp120 (Fig. 6C). The presence of HIV1 gp120 inhibited the up-regulation of the DPG-3-induced costimulatory molecules CD80,CD86, and CD83 the strongest, followed by WT Pg381. BlockingDC-SIGN did not block the up-regulation of HLA-DR, CD80, orCD83 induced by the non-DC-SIGN targeting strain MFI (Fig. 7,

B and C). These results suggest an uncoupling of DC maturationfrom the inflammatory cytokine response when DCs phagocytosewhole live bacteria that express a DC-SIGN ligand.

The minor fimbriated strain induces a Th2 effector response andthe major fimbriated strain induces a Th1 effector response

Previous studies have shown that DC-SIGN ligands can induce aTh2-based effector response (20, 62). In the present study, MoDCswere pulsed with each of the four Pg strains and then cocultured

FIGURE 3. MoDCs express DC-SIGN, bind fimbriated Pg strains. A, Day 7 immature CD1a� MoDCs were analyzed for expression of DC-SIGN andother putative endocytic receptors by FACS analysis. MoDCs were labeled with isotype control mAb (filled histogram) or receptor-specific mAb (openhistogram), showing expression (shift to the right) of DC-SIGN, CD11b, CD11c, CD18, CD29, and DEC-205. B, FACS scatter graph showing thepercentage of binding (to the right of the line) of CFSE-labeled Pg strains to MoDCs at 3 and 18 h.

FIGURE 4. Blocking DC-SIGN on MoDCs with Abor carbohydrates inhibits uptake of Pg. MoDCs werepretreated for 30 min with mAb to DC-SIGN, CD11c,CD29, CD18, isotype control, 50 �g of mannose, or 50�g of mannan at 50:1 MOI (A) or 3 or 6 �g of HIVgp120 (B) before adding CFSE-stained Pg strain 381 at5:1 MOI, after which association was assessed, in trip-licate, at 3 h and 37°C. For A, the means � SEM of thepercentage of the uptake of Pg-CFSE by MoDCs areshown. For B, the percentage of reduction in the pres-ence of gp120 was calculated as follows: (percentage ofPg� MoDC � percentage of Pg� MoDC in the presenceof gp120)/(percentage of Pg� MoDC) � 100.

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with autologous naive CD4� T cells for 7 days, after which T cellcytokines and T cell proliferation were analyzed. We show thatMoDCs pulsed with the DC-SIGN-binding strain DPG-3 induced

the release of significantly higher IL-4 levels from T cells com-pared with all other strains and very low levels of IL-12p70 rela-tive to MFI (Fig. 8A). When expressed as Th1/Th2 cytokine ratios(IFN-�/IL-4 and IL-12p70/IL-4), DPG-3-pulsed MoDCs inducedthe lowest ratios of all strains except for MFB and yielded a verylow Th1 index (Table I). In contrast, MFI-pulsed MoDCs inducedsignificantly lower levels of the Th2 cytokine IL-4, but comparablelevels of IFN-� and very high levels of IL-12p70 from T cells. Thisyielded high ratios of IFN-�/IL-4 and IL-12p70/IL-4 and a rela-tively high Th1 index (8.81) (20). WT Pg381 induced the lowestlevels of IL-4, low levels of IL-12p70, and comparable levels ofIFN-� by T cells. The low levels of IL-4 by Pg381 resulted in thehighest Th1 index (10.63), whereas MFB induced the lowest levels ofIFN-� and IL-12p70 and had the lowest Th1 index (1.15) (Table I).

To determine whether the immunoproliferative ability of Pg-pulsed MoDCs would correlate with the Th1 index, CFSE-labelednaive CD4� T cells were analyzed at various stimulator:effectorratios for 7 days. The MoDC-T cell 1:50 ratio yielded the maxi-mum proliferation. Our results (Fig. 8B) show that MoDCs pulsedwith DPG-3 and MFB induced the weakest T cell proliferativeresponses, whereas WT Pg381 and MFI induced the strongest Tcell proliferative responses. The weak T cell proliferation and cy-tokine secretion exhibited by the double fimbriae knockout MFBcould be attributed to its lack of binding and uptake (Fig. 3B).However, we can attribute the impaired proliferation of coculturedCD4� T cells to the minor fimbriae of DPG-3 interacting with DC-SIGN dampening inflammatory cytokine production leading to re-duced T cell proliferation in the absence of major fimbriae. Linearregression analysis of the percentage of T cell proliferation induced byMoDCs pulsed with each strain showed a significant association (r2 �0.857, p � 0.024; SPSS software, version 15) with the Th1 index.

DiscussionOverall, these results indicate that both the major and minor fimbriaeof Pg are involved in binding of the whole live bacterium to Raji celllines (Fig. 1) and to DCs (Fig. 3); however, the minor fimbria isrequired for binding to DC-SIGN (Figs. 1, 2, and 4). This results in Pgbeing internalized and routed in large numbers into as yet undefinedintracellular vesicles of DCs (Fig. 5). DCs that have internalized Pgstrains lacking major fimbriae are poorly matured (Fig. 7), secretevery low levels of inflammatory cytokines (Fig. 6), and induce a Th2-biased, weak immunoproliferative T cell response (Fig. 8). Althoughthese findings were established using isogenic, fimbria-deficient mu-tants of Pg, expression of the fimbriae have been shown to be regu-lated by growth conditions, including temperature (45, 63) and heminlevels (46) Apparently, a two-component regulatory system (FimS/FimR) controls the two fimbrial genes at different levels depending onheme levels and temperature (47). Other systemic mucosal pathogenssuch as Yersinia pseudotuberculosis (64) and Salmonella enterica (65,66) also regulate their invasive potential under environmental pres-sures. This is of particular relevance here, because the preferred eco-logical niche of Pg, a hemin-requiring anaerobe, are deep bleeding“crypts” in the human oral mucosa called periodontal pockets (31).These pockets are subjacent to OLF and to lamina propria dermalDCs (27), where Pg infects DCs in situ (57). Pg initially colonizessurface mucosa and tooth surfaces, where hemin levels and temper-ature are reduced, forming part of a complex biofilm (31, 67). Themajor fimbriae appear required for initial attachment to host cells (11,48, 68–70), whereas the minor fimbriae appear to play an importantrole in microcolony formation by facilitating cell-cell interactions andpromoting biofilm formation (67, 71, 72). Both fimbriae play essentialroles in the induction of alveolar bone loss (34) and atherosclerosis(73, 74), but the specific mechanisms of these (seemingly) disparateprocesses are unclear. A recent review describes the important role for

FIGURE 5. Pg colocalizes with membrane DC-SIGN and is takeninside DC-SIGN-rich compartments. Day 7 immature MoDCs werepulsed with Syto-labeled (red fluorescence) Pg 381 at a 50:1 MOI andcells were fixed at 1 h (A) and 6 h (B and C) or with DAPI-labeled Pg381 at 18 h (D). A–C, MoDCs were probed with mAb to DC-SIGN(clone DCN46; BD Biosciences), followed by anti-mouse Alexa Fluor488 (Molecular Probes) Abs (green fluorescence). Controls were la-beled with isotype control primary mAb (not shown). B and C, MoDCswere permeabilized before probing with DC-SIGN mAb to visualizeintracellular DC-SIGN-rich vesicles. Slides were analyzed using an epi-fluorescence microscope (Nikon E600) equipped with SPOT charge-coupled device camera integrated with Pentium IV PC running Im-agePro and deconvolution software. Extracellular Pg 381 is shown at1 h (A2); binding to DC-SIGN is shown in merged channels (A3, a andb) (yellow fluorescence). Intracellular localization of Pg 381 (B2 andC2) within DC-SIGN-rich compartments (B3 and C3) (yellow fluores-cence). D, At 18 h, large numbers of Pg 381 accumulate inside vesicles(Pg-containing vesicles (PgCV)) of MoDCs. The viability dye pro-pidium iodide (red) is excluded by the outer membrane of MoDCs,attesting to viability of MoDC and, potentially, Pg. The DAPI-positivenucleus wrapped around the Pg-containing vesicles is also shown. DIC,Differential interference contrast.

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Th2 type responses in the inability of the host to successfully resolveperiodontal disease (26), suggesting clinical relevance to our findingsof Th2 responses biased by the minor fimbriae of Pg.

Relatively little is known about the steps involved in the formationand secretion of minor fimbriae and of the cellular receptors and sig-naling pathways it targets (34–36). In contrast, the major fimbriae are

FIGURE 6. Minor fimbriated strains inducein MoDCs a distinct cytokine response. Super-natants from MoDCs pulsed for 3 h (A) or 18 h(B) with Pg 381, DPG-3, MFI, MFB or no bac-teria (DC control (ctl)) or an 8 h cytokine re-sponse of MoDCs pretreated with 6 �g/mlHIV-1 gp120 CM and pulsed with Pg381,DPG-3, MFI, and no bacteria were analyzed intriplicate by flow cytometry using the cytomet-ric bead array (CBA kit; BD Biosciences) (C).Based on a standard curve for each cytokine,the software calculates levels in picogram permilliliter. Results shown are mean � SEM.

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under intense investigation in this regard (37–42). The major fimbriaeactivate macrophages through TLR2 and TLR4, as well as the com-plement receptor 3 and CD14 (43, 44). Davey et al. showed that boththe major and minor fimbriae specifically bind to chimeric TLR2 andCD14 proteins in a cell-free ELISA (49). TLR2 appears to be partic-ularly important in IL-10-mediated mucosal immune homeostasis inresponse to commensals (75). DC-SIGN ligation (using purified mi-crobial ligands) has also been shown to induce IL-10 by triggeringRaf-1 phosphorylation (16). Combined with the activation of TLR4,DC-SIGN ligation results in enhanced and prolonged NF-�B activa-tion and stronger IL-10 production (16). Our cytokine results corrob-

orate cross-talk between DC-SIGN and TLRs. For example, the mostprofound effect that the blocking of DC-SIGN had on cytokine se-cretion was observed with WT Pg381 (Fig. 6C), which expressesligands for DC-SIGN (minor fimbriae) and TLR2/4 (major fimbriae).With Pg381 we saw an enhancement (deregulation) of IL-12p70,IL-8, and IL-6. In contrast, blocking DC-SIGN did not enhance IL-12p70, or IL-6 in response to strain MFI, which lacks DC-SIGN li-gand minor fimbriae. Moreover, in the absence of major fimbriae(DPG-3), blocking DC-SIGN decreased IL-12p70 and IL-6. TNF-�,a good indicator of NF-�B activation, was consistently dampened bygp120 (Fig. 6C) regardless of the bacterial strain used. Although DC

FIGURE 7. Minor fimbriatedstrains induce in MoDCs a distinct mat-uration profile. A, Differences in up-reg-ulation of HLA-DR, CD80, CD83, andCD86 on MoDCs by FACS analysis af-ter pulsing with Pg381, DPG-3, MFI,MFB (light tracings), or no bacterialcontrol (dark tracings). B, Differences inup-regulation of HLA-DR, CD80,CD83, and CD86 on MoDCs by puls-ing with Pg381, DPG-3, MFI, or nobacterial control on MoDCs pretreatedwith 6 �g/ml HIV-1 gp120 CM. Redtracings represent uninfected MoDCcontrol, green tracings are MoDC con-trol pretreated with HIV-1 gp120 CM,blue lines represent MoDCs that havebeen pulsed with either Pg381, DPG-3or MFI, and orange lines representMoDCs that have been pretreated withHIV-1 gp120 CM and pulsed with ei-ther Pg381, DPG-3, or MFI. Results arerepresentative of three separate experi-ments. C, The average fold changes inmean fluorescence intensity values forMoDC cell surface marker expression.The ratios of the mean fluorescence in-tensity experimental condition overcontrol were calculated for each cellmarker. The mean fluorescence inten-sity values for the uninfected controlconditions were set at 1.0 (log ratio �0). The means of the base 10 logarithmsof the ratios for all conditions were cal-culated. The fold changes of HLA-DR(blue bars), CD83 (purple bars), CD86(green bars), and CD80 (orange bars)are plotted relative to baseline value ofuninfected control. Data shown are themeans � SD of three separate experi-ments performed previously (63).

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maturation and cytokine secretion are both generally attributed to theligation of TLRs leading to NF-�B activation (76), phagocytosis/en-docytosis in itself triggers a family of intracellular signaling pathways(reviewed in Refs. 77–79). Consistent with this concept, we show thatblocking DC-SIGN reduces the phagocytosis of Pg by MoDCs (Fig.4) and reduces the up-regulation of costimulatory molecules (Fig. 7,B and C) but not certain cytokines (Fig. 6). Overall, these resultssuggest an uncoupling between the DC cytokine response and thematuration by DC-SIGN ligands that warrants further analysis withpurified native fimbriae.

In this context, we recently purified the minor fimbriae by HPLCand analyzed the protein sequence by MALDI-TOF. We discoveredthat there are two conserved Asn-Xaa-Ser/Thr N-glycosylation motifson the minor fimbria sequence (80). Putative glycosylation of the67-kDa minor fimbria was further verified by Pro-Q Emerald glyco-protein staining, a periodate- and fluorescence-based reaction (our un-published data). Efforts are underway to analyze the purified minorfimbriae by monosaccharide compositional analysis and liquid chro-

matography-tandem mass spectrometry. There are other reports of arole for glycosylation of the fimbriae in Pg. Knockouts of gftA (awcaE glycotransferase homologue of Escherichia coli) in Pg fail tomake mature fimbriae (81). The gingipains of Pg are apparently gly-cosylated (82), and this glycosylation is regulated by the vimF, vimA,and vimE glycotransferase genes (83, 84). Knocking out these genescauses a failure to glycosylate these gingipains, leading to their inac-tivation (82–84). Kadowaki et al. have found that Arg gingipain ac-tivity is essential for the processing and translocation of mature fim-briae (85). Recently, it was discovered that the RagA proteins of Pgare glycosylated (86). The commonalties among these outer mem-brane proteins is that they encode for an N-terminal, long signal pep-tide that gets cleaved once they enter the inner membrane (87–89).Many mucosal pathogens exhibit glycosylation motifs on their fla-gella, pili, and fimbriae for binding to host cells (90). Glycosylationalso reportedly plays a role in maintaining the protein structure, pro-tecting against proteolytic degradation, and in immune evasion (89,90). Additionally, it was recently determined that the glycosylation of

FIGURE 8. Minor fimbriated mutant induces a Th2response, and the major fimbriated mutant induces aTh1 response. A, Cytokines from naive autologousCD4� T cells cocultured with MoDCs at a 1:50 stimu-lator:responder ratio were analyzed in triplicate by flowcytometry using a CBA kit (BD Biosciences). Based ona standard curve for each cytokine, the software calcu-lates levels in picograms per milliliter. The resultsshown are mean � SEM. B, MoDCs that had beenpulsed with Pg381, DPG-3, MFI, MFB, or no bacteria(CD4� DC control) for 18 h were washed extensivelyand cultured for 5 days at graded doses (5,000 DCs per200 �l, 1000 DCs per 200 �l, and 300 DCs per 200 �lin complete RPMI 1640) with naive autologous CD4�

T cells (50,000 cells/200 �l) prelabeled with CFSE. Thepercentage of proliferation was quantitated by the lossof CFSE (light tracings) compared with CD4� control(dark tracings) using FACS analysis. Results are repre-sentative of a minimum of three separate experiments.

Table I. Alteration in Th1 index and immunoproliferation dependent on minor/major fimbriaea

P. gingivalisStrain

Fimbriae Expression Autologous Naive CD4� T Cell Response

Minor MajorIFN�/IL-4

ratiobIL-12p70/IL-4

ratiocTh1

indexdProliferation

(%)e

Pg381 � � 1027 36.5 10.63 49DPG-3 � – 195 1.69 1.96 35MFI – � 367 514 8.81 47MFB – – 112 3.5 1.15 22CD4-DC control 16 36 0.52 16CD4 control 35 7.8 0.43 12

a Levels of IL-4, IFN-�, and IL-12p70 from DC-T cell cocultures, as depicted in Fig. 8, were analyzed for the IFN-� /IL-4ratio and the IL-12p70/IL-4 ratio as shown, and the Th1 index was calculated and tabulated.

b IFN-� (pg/ml)/IL-4 (pg/ml).c IL-12p70 (pg/ml)/IL-4 (pg/ml).d (IFN-�/IL-4) � (IL-12p70/IL-4)/100.e Percentage of CFSE negative.

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soluble peanut allergen was sufficient for targeting DC-SIGN inMoDCs and that its recognition by DC-SIGN skewed the T cell re-sponse to a Th2 phenotype (91). Finally, it is believed that the car-bohydrate moieties of HIV-1 gp120 confer immunosuppressive ef-fects on MoDCs (62). The detection of glycosylation on the minorfimbriae might enable Pg to prompt its immunosuppressive phenotypein a similar manner.

In conclusion, our results show distinct immunomodulatory rolesfor two adhesins expressed by the mucosal pathogen Pg. We show themajor fimbriae are immunostimulatory and the minor fimbriae areimmunosuppressive. Although coexpressed in WT strains under lab-oratory conditions, the two fimbriae are regulated by environmentalconditions of direct relevance to their preferred niches. Overall, theseresults may help explain how this oral mucosal pathogen evadesand/or suppresses the mucosal immune response, i.e., by uncouplingDC maturation from the cytokine response, leading to anergy.

AcknowledgmentsThe HIV-1 gp120 CM envelope protein (catalog no. 2968) was obtainedthrough the National Institutes of Health AIDS Research and ReferenceReagent Program, Division of AIDS, National Institute of Allergy andInfectious Diseases, National Institutes of Health. We thank Dr. Caroline AGenco for providing us with the isogenic fimbriae mutants (DPG-3, MFI,and MFB). We also thank Dr. D. R. Littman for providing us with the Rajiand the Raji DC-SIGN cell lines. In addition, we thank Todd Rueb at theResearch Flow Cytometry Core facility from Stony Brook University Med-ical Center for input and technical expertise and Baljit Moonga for proof-reading the manuscript.

DisclosuresThe authors have no financial conflict of interest.

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