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INFECTION AND IMMUNITY, June 2006, p. 3213–3221 Vol. 74, No. 60019-9567/06/$08.00�0 doi:10.1128/IAI.01824-05Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Role for Dendritic Cells in Immunoregulation during ExperimentalVaginal Candidiasis

Dana M. LeBlanc, Melissa M. Barousse, and Paul L. Fidel, Jr.*Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana

Received 8 November 2005/Returned for modification 7 December 2005/Accepted 22 February 2006

Vulvovaginal candidiasis (VVC) caused by the commensal organism Candida albicans remains a significantproblem among women of childbearing age, with protection against and susceptibility to infection still poorlyunderstood. While cell-mediated immunity by CD4� Th1-type cells is protective against most forms of mucosalcandidiasis, no protective role for adaptive immunity has been identified against VVC. This is postulated to bedue to immunoregulation that prohibits a more profound Candida-specific CD4� T-cell response againstinfection. The purpose of this study was to examine the role of dendritic cells (DCs) in the induction phase ofthe immune response as a means to understand the initiation of the immunoregulatory events. Immunostain-ing of DCs in sectioned murine lymph nodes draining the vagina revealed a profound cellular reorganizationwith DCs becoming concentrated in the T-cell zone throughout the course of experimental vaginal Candidainfection consistent with cell-mediated immune responsiveness. However, analysis of draining lymph node DCsubsets revealed a predominance of immunoregulation-associated CD11c� B220� plasmacytoid DCs (pDCs)under both uninfected and infected conditions. Staining of vaginal DCs showed the presence of both DEC-205�

and pDCs, with extension of dendrites into the vaginal lumen of infected mice in close contact with Candida.Flow cytometric analysis of draining lymph node DC costimulatory molecules and activation markers frominfected mice indicated a lack of upregulation of major histocompatibility complex class II, CD80, CD86, andCD40 during infection, consistent with a tolerizing condition. Together, the results suggest that DCs areinvolved in the immunoregulatory events manifested during a vaginal Candida infection and potentiallythrough the action of pDCs.

Vulvovaginal candidiasis (VVC) is a significant problem af-fecting 75% of normal healthy women at least once duringtheir reproductive years (39, 40). Candida albicans, the caus-ative agent of VVC, is a dimorphic fungal organism and botha commensal and opportunistic pathogen of the genital andgastrointestinal tracts (11, 16, 25, 27, 32, 36). Symptoms ofVVC include itching, burning, soreness, abnormal discharge,dyspareunia, and vaginal and vulvar erythema and edema (40).Factors contributing to the development of disease includeantibiotic and high estrogen contraceptive use, hormone re-placement therapy, pregnancy, and uncontrolled diabetes mel-litus (15, 22, 39, 40). Although most women experience infre-quent occurrences of VVC, an additional 5 to 10% of womensuffer from recurrent VVC, defined as three or more episodesof infection per annum without any recognizable predisposingfactors (38, 39).

While CD4� Th1-type cell-mediated immunity (CMI) is thedominant host defense mechanism for most Candida infectionsof mucosal tissues (4, 33–35), no protective role for local orsystemic CMI has been demonstrated for vaginal Candida in-fections. Instead, it is postulated that a protective adaptiveimmune response to vaginal Candida infection is prohibited bya local tolerizing condition or immunoregulatory environmentat the vaginal mucosa and the draining lymph nodes. This issupported by the presence of CD4� CD25� regulatory T cellsin the draining lymph nodes (43), �/� T cells in the vagina (42),

and immunoregulatory cytokines (interleukin-10 [IL-10] andtransforming growth factor � [TGF-�]) in the lymph nodes andvaginal tissue (42). Furthermore, there is little evidence forchanges in the percentage or composition of vaginal T-cellpopulations during experimental vaginal candidiasis (12),suggesting a lack of systemic T-cell infiltration or local T-cellexpansion. Immunoregulation at a reproductive site mayreflect a means for the host to avoid chronic inflammation toa commensal organism such as Candida. Yet some form ofhost response is required to keep Candida in a commensalstate and out of a pathogenic condition. Current evidencesuggests this involves innate immune mechanisms by epithe-lial cells (2, 10, 44).

Dendritic cells (DCs) are considered the major antigen-presenting cell (APC) responsible for activation of the adap-tive immune response. Recent studies also suggest a role forDCs in the initiation of immunoregulatory events and mainte-nance of tolerogenic conditions. Different DC subsets appearresponsible for each condition: CD11c� B220� plasmacytoidDCs (pDCs) are involved in tolerance induction, whereasCD11c� B220� myeloid DCs are involved in the induction ofclassical inflammatory responses. pDCs have been shown to bethe predominant DC population in lymph nodes under toler-izing conditions (28), express low levels of major histocompat-ibility complex (MHC) class II and costimulatory molecules(24, 28), and can induce the differentiation of IL-10-producingTreg cells (5, 17, 24, 37). This is in contrast to the myeloid DCsthat express high levels of MHC class II and costimulatorymolecules in their activated state and are associated with Thcell activation (24, 28). Finally, DCs are known to be present inthe vaginal mucosa (18–20, 41, 45), and studies on the inter-

* Corresponding author. Mailing address: Department of Microbiol-ogy, Immunology, and Parasitology, Louisiana State University HealthSciences Center, New Orleans, LA 70112. Phone/Fax: (504) 568-4066.E-mail: [email protected].

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action of Candida with bone marrow-derived DCs have beenreported (1, 6, 26, 31).

The purpose of this study was to investigate the presence ofDCs in the vagina during infection with C. albicans and toexamine the potential role of DCs in the induction phase of thevaginal immune response to Candida in the vagina that in-cludes the immunoregulatory events, using the well-establishedmurine model of experimental vaginal candidiasis.

MATERIALS AND METHODS

Mice. Female CBA/J (H-2k) mice, 8 to 10 weeks of age (purchased from theNational Cancer Institute; Frederick, MD), were used throughout these studies.All animals were housed and handled according to institutionally recommendedguidelines.

Vaginal Candida infection. Mice were injected subcutaneously with 100 �l of0.2 mg/ml estradiol (Sigma Chemical Co., St. Louis, MO) in sesame oil 72 h priorto inoculation. Intravaginal inoculation was performed by introducing 20 �l ofphosphate-buffered saline (PBS) containing 5 � 104 C. albicans blastoconidiafrom a stationary-phase culture (overnight culture at 25°C in Phytone-peptonemedium plus 0.1% glucose) into the vagina. Control animals were estrogenizedand inoculated with PBS only or PBS containing 5 � 104 heat-killed C. albicansblastoconidia (incubated at 60°C for 2 h, washed, and cultured for verification).Separate groups of mice were sacrificed at days 4 and 7 postinoculation. Vaginallavages were conducted using 100 �l of sterile PBS with repeated aspiration for30 to 40 s, and the fluid was cultured at 1:10 serial dilutions on Sabouraud-dextrose agar plates (Becton Dickinson and Co., Cockeysville, MD) supple-mented with gentamicin (Sigma) as previously described (13). CFU were enu-merated after incubation at 35°C for 48 h and expressed as CFU/100 �l.

Specimen collection and processing. Vaginal draining lumbar lymph nodeswere identified on the posterior abdominal wall lateral to the inferior vena cavaand abdominal aorta, respectively. These lymph nodes and/or the vagina wereexcised and embedded in optimum-cutting-temperature (OCT) medium (Tissue-Tek, Torrance, CA) within cryomolds in an orientation that allowed for cross-sectional cutting. Tissue sections were cut (8 to 12 �m), fixed in acetone (10 min),and then washed in PBS (5 min). For dendritic cell enrichment, lumbar lymphnodes were removed and placed in RPMI 1640 medium (Sigma). Single-cellsuspensions were prepared by digesting the nodes in collagenase D (400 U/ml)(Sigma) for 25 min at 37°C and 5% CO2. The collagenase-cell suspension wasincubated for 1 min at room temperature in 0.1 M EDTA, filtered (40-�m poresize), and centrifuged, and the cell pellet was resuspended in PBS supplementedwith 0.5% fetal bovine serum (FBS). Cells were then enumerated with a hemo-cytometer. CD11c� DCs were enriched from the total lymph node cell popula-tion by magnetic bead selection (Miltenyi Biotec, Auburn, CA). Briefly, nonspe-cific protein sites on the cells were blocked by incubation with anti-Fc receptorantibodies. Cell suspensions were next incubated with anti-CD11c-antibody(clone N418)-conjugated microbeads (100 �l) and passed over a magnetic se-lection column (Miltenyi Biotec). After washing the column with PBS-0.5% FBSto remove unlabeled cells, the column was demagnetized and positively selectedcells were eluted and collected by centrifugation. Pelleted cells were resuspendedin PBS-0.5% FBS and enumerated with a hemocytometer. DC enrichment wasconfirmed by flow cytometry, with the final enrichment ranging from 60 to 80%CD11c� cells.

Immunohistochemistry. (i) Chromophore. Vaginal and lymph node tissueswere stained using the R&D Systems tissue staining kit (Minneapolis, MN).Briefly, tissues were blocked with peroxidase (5 min), goat serum (15 min), avidin(15 min), and biotin (15 min) blocking buffers and rinsed in PBS after each step.Tissues were then incubated with purified rat anti-mouse DEC-205 (0.2 �g/ml)(BMA Biomedicals; Rheinstrasse, Switzerland) or plasmacytoid DC marker (1�g/ml) (Miltenyi Biotec) at room temperature for 1 h. Rat immunoglobulin G2a(IgG2a) isotype-specific antibody staining was used as a negative control. Fol-lowing incubation, the slides were washed three times (15 min/wash) in PBS andthen incubated with biotinylated goat anti-rat IgG antibody (5 �g/ml) (BDBiosciences; San Jose, CA) for 30 min. Washed slides were then incubated withhigh-sensitivity streptavidin-horseradish peroxidase conjugate (30 min), washed,and incubated with the substrate 3,3� diaminobenzidine (20 min). Slides werecounterstained with Mayer’s hematoxylin and preserved using aqueous mountingmedium (R&D Systems).

(ii) Fluorescence. Vaginal tissues were blocked with goat serum (20 min),incubated with purified rat anti-mouse DEC-205 (0.2 �g/ml) (BMA Biomedicals)for 1 h, washed in PBS (5 min/wash), and then incubated with biotinylated goat

anti-rat IgG antibody (5 �g/ml) (BD Biosciences) for 30 min. Rat IgG2a isotype-specific antibody staining was used as a negative control. Slides were washed inPBS (5 min/wash) then incubated with streptavidin-conjugated phycoerythrin(PE; 25 �g/ml). Slides were washed in PBS (5 min/wash) and then incubated withcalcofluor white M2R (Molecular Probes; Eugene, OR) for 10 min to stain C.albicans. Slides were washed and visualized by fluorescent microscopy under thetetramethyl rhodamine isocyanate (TRITC) and 4�,6�-diamidino-2-phenylindole(DAPI) filters.

Flow cytometry. Analysis of cell surface molecules on lymph node cells en-riched for DCs was performed by flow cytometry using standard methodology fordirect and indirect immunofluorescence. Briefly, cells were incubated with com-binations of PE-labeled anti-plasmacytoid DC marker (Miltenyi Biotec), fluo-rescein isothiocyanate (FITC)-labeled anti-B220, PE- or biotin-labeled anti-CD11c (clone HL3), biotin-labeled anti-I-Ak, or purified anti-CD80, anti-CD86,or anti-CD40 antibodies (BD Biosciences) at optimal concentrations (2.0 to 2.5�g/ml) for 20 min at 4°C. Cells were washed in buffer, and those labeled withpurified antibodies were then incubated with a secondary biotinylated anti-ratIgG antibody (2.5 �g/ml) for 20 min at 4°C. Cells were washed and incubated instreptavidin-conjugated Cy-Chrome for 20 min at 4°C. After incubation, the cellswere washed and fixed in Poly-Lem fixative (200 �l) (Polysciences, Inc., War-rington, PA). Samples were analyzed on a FACS Vantage SE flow cytometer(Becton Dickinson; Franklin Lakes, NJ) using Cellquest software at the LSUHealth Sciences Center Core Labs Facility. Cells incubated with either bufferalone or fluorochrome-conjugated isotype control antibodies (rat IgG2a) wereused to determine background staining. For data analyses, cells were evaluatedfrom a predominantly DC population identified by gating on large, forward-scattering cells with isotype-specific antibody staining as a negative control.Compensation for each fluorochrome was determined by parallel single-coloranalysis of cells labeled with each fluorochrome-conjugated antibody.

Statistical analysis. The unpaired Student’s t test was used to analyze data.Significant differences were defined as a confidence level at which P was 0.05,using a two-tailed test. All statistics were evaluated using GraphPad Prism(GraphPad Software, San Diego, CA).

RESULTS

Effects of vaginal candidiasis on draining lymph node ar-chitecture. In initial studies, we sought to evaluate the DCpresence and distribution in vaginal draining lumbar lymphnodes in uninfected mice, mice inoculated with killed Candidablastoconidia, and mice inoculated with live Candida albicansblastoconidia. For this, lymph node tissue sections were stainedfor the lymphoid DC marker DEC-205 and visualized microscop-ically. Lymph nodes from mice given PBS intravaginally showedDCs evenly dispersed throughout the lymphoid tissue (Fig. 1A).The presence of live Candida cells in the vagina induced a pro-found cellular reorganization in draining lymph nodes within 4days, with DCs becoming concentrated in the T-cell zones of thelymph nodes (Fig. 1B). Lymph nodes from mice given killedCandida intravaginally showed no signs of lymph node cellularmigration and rearrangement, similar to PBS-treated mice (Fig.1C). Tissue stained with isotype control antibodies (rat IgG2a)showed no staining (Fig. 1D). Vaginal infection with live Candidablastoconidia was verified based on a vaginal lavage and quanti-tative plate counts. There was no fungal growth from lavages ofmice treated with PBS or killed Candida, consistent with the lackof yeast colonization in mice (data not shown). Henceforth, PBSwas used as the negative control.

Analysis of dendritic cell subsets in vaginal draining lymphnodes. Since immunohistochemical staining clearly indicatedDC reorganization in the lymph nodes of infected mice sug-gestive of DC–T-cell interaction, we next sought to analyze theproperties of the DC populations in the vaginal draining lymphnodes of uninfected (baseline) and infected mice using flowcytometry. Based on the differential expression of B220 onmyeloid and plasmacytoid DCs as evidence of an inflammatory

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versus tolerizing condition (28), we first evaluated the presenceof B220 on forward-scattering (large) cells and then confirmedthem as CD11c� MHC class II� DCs. Results demonstratedthat a higher percentage of the forward-scattering CD11c�

MHC class II� DCs (Fig. 2A, left frame) were B220� (plas-macytoid) DC (Fig. 2A, center frame) and were confirmed bythe smaller percentage of B220� (myeloid) DC (Fig. 2A, rightframe). We next determined that the majority of the B220�

cells were putative pDC, indicated by the high percentage ofB220� cells that stained positive for both pDC marker andCD11c (Fig. 2B). These results did not appreciably change inmice with vaginal Candida infections through 7 days postin-oculation (data not shown). Analysis of the absolute numbersof DCs per lymph node before enrichment indicated that de-spite increased numbers of cells per lymph node during infec-

tion (P 0.008), the percentage of DCs, including pDCs,remained similar (P � 0.05) (Table 1).

Dendritic cells in vaginal tissue. We next examined thepresence of DCs in the vagina before and after inoculationwith Candida. Immunohistochemical staining for DEC-205 re-vealed the presence of DCs dispersed throughout the vaginallamina propria and epithelium in both PBS-treated and in-fected mice (Fig. 3A and B). However, in infected mice (days4 and 7 postinoculation), DCs were also observed at the outerepithelium with dendrites extending into the vaginal lumen(Fig. 3B). Tissue stained with isotype control antibodies (ratIgG2a) showed no staining (data not shown). Concurrent im-munofluorescent staining of DEC-205� DCs and C. albicanschitin revealed colocalization of DCs with Candida at the sur-face of the vaginal epithelium (Fig. 3C). Staining for vaginal

FIG. 1. Effects of vaginal candidiasis on draining lymph node architecture. Frozen lumbar lymph node tissue sections from mice inoculated withPBS (uninfected) (A), live C. albicans blastoconidia (B), or killed C. albicans blastoconidia (C) 7 days postinoculation were stained withanti-DEC-205 (�-DEC-205) antibody (0.2 �g/ml) or isotype control antibodies (rat IgG2a) (D). The figure shows representative images of threerepeats with three mice per group. Images are shown at a magnification of �100.

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FIG. 2. Analysis of DC subsets in vaginal draining lymph nodes.Lymphocytes were isolated from draining lumbar lymph nodes of un-infected mice and enriched for CD11c� cells. (A) Cells were labeledwith B220-FITC, CD11c-PE, and MHC class II-Cy-Chrome and ana-lyzed by flow cytometry. Cells were stratified by gating on the entireforward-scattering population, forward-scattering B220� cells, or for-ward-scattering B220� populations (upper panel) and then determin-ing the percentage of DC (CD11c� MHC class II�) in the gated region(lower panel). (B) Cells were labeled with B220-FITC, CD11c-Cy-Chrome, and plasmacytoid DC (P-DC) marker-PE and analyzed byflow cytometry. The forward-scattering B220� cells (upper panel) thatwere also CD11c� plasmacytoid DC marker positive (lower panel)were used to determine the percentage of plasmacytoid DCs. Cellslabeled with isotype control antibodies were used to set the initialgates. Panels A and B each show a representative result from threerepeats using pooled cells from five mice per group.

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pDCs in uninfected mice revealed positively stained cells sim-ilarly dispersed throughout the vaginal epithelium (Fig. 3D).Under infected conditions (days 4 and 7 postinoculation),pDCs were found accumulated at the interface of the laminapropria and the epithelium (Fig. 3E).

Effects of vaginal Candida infection on dendritic cell activa-tion markers in the draining lymph nodes. Next we examinedthe effect of vaginal Candida infection on MHC class II andcostimulatory molecule expression on DCs in the draininglymph nodes. Controls included mice given PBS intravaginally.Lymph nodes were removed at days 4 and 7 postinoculation,and total lymph node cells were enriched for DCs. Figure 4Ashows a representative flow cytometric result for the expres-sion of the four markers on B220� cells under uninfected andinfected conditions. Cumulative results of the total forward-scattering DC population (total DC) for several experimentsrevealed no statistical differences in mean fluorescence inten-sity (MFI) for MHC class II, CD80, CD86, and CD40 with atrend toward downregulation on DCs from infected mice onboth days 4 and 7, as compared to DCs from control animalsgiven PBS and expressed as percent change (Fig. 4B). Whenstratified by the B220 marker specifically, it was determinedthat the downregulatory trend for each of the markers wasassociated with the B220� cells rather than the B220� cells(Fig. 4C and D), although only the changes for CD86 reachedstatistical significance (P 0.0029).

DC subsets in nonvaginal draining lymph nodes. Since pDCswere found to be the predominant DC subset of the vaginaldraining lymph nodes and have been found by several groups tohave immunoregulatory potential (5, 17, 23, 24, 28), we nextsought to determine if the tolerizing DC profile was unique tovaginal draining lymph nodes or common to all lymph nodesunder naive conditions. To investigate this, the axillary and bra-chial lymph nodes were analyzed for DC subsets and compared toDC subsets in lumbar lymph nodes of naı̈ve mice. Flow cytometricanalysis revealed an equivalent percentage of pDCs among lymphnode cells from all sites (47% 5% for vaginal draining lymphnodes versus 55% 8% for nonvaginal draining lymph nodes).

DISCUSSION

Although Th1-type CMI is protective against Candida infec-tion at most mucosal sites, there is no evidence for protectionagainst vaginal candidiasis. Instead, considerable evidence sug-gests that immunoregulation compromises such protection.The aim of this study was to investigate the role of dendriticcells in the initiation of these immunoregulatory processes.

Immunohistochemical analysis of DCs in vaginal draininglymph nodes revealed a profound cellular reorganization, withDCs becoming concentrated in the T-cell zones of lymph

nodes following an experimental vaginal Candida infection, butnot in control animals given PBS or killed Candida. This cel-lular rearrangement under infected conditions was evidencefor initiation of classical cellular immune activation events, assuggested by our in vitro studies (14), with significant DCinvolvement in the immune response to a vaginal Candidainfection.

In contrast to the implications surrounding the cellular re-organization in the draining lymph nodes of infected mice,examination of specific DC subsets revealed that plasmacytoidDCs (CD11c� B220�) predominated prior to and throughoutthe vaginal Candida infection. While it is common to havepDCs in the lymphoid organs under naive conditions, as con-firmed in the present study, the continued presence duringCandida infection is indicative of a tolerizing condition. Forexample, in a murine airway inflammation model, Oriss et al.(28) demonstrated that while mDCs became the predominantDC subset under inflammatory conditions, pDCs remained thepredominant subset after stimulation with antigen under toler-izing conditions (28). Similar lymph node DC dynamics havebeen demonstrated in studies with other fungal immunogens.Bauman et al. showed that in mice immunized with an immuno-gen that induced a protective response to systemic infection withCryptococcus neoformans, mDCs were the predominant DC pop-ulation in lymph nodes draining the site of immunization (3).Conversely, in mice given nonprotective cryptococcal immuno-gen, the lymphoid DC population (now considered plasmacytoid)was predominant (3). Thus, considerable evidence suggests thatthe myeloid-plasmacytoid DC balance in draining lymph nodes inresponse to specific antigen is important for the development ofprotective or tolerizing CMI conditions.

Flow cytometric analysis of MHC class II and costimulatorymolecules (CD80, CD86, and CD40) on lymph node DC fol-lowing a vaginal Candida infection, which revealed a lack ofupregulation of these molecules, is consistent with the pres-ence of pDCs in the vaginal draining lymph nodes (known toexpress low levels of MHC class II and costimulatory mole-cules). In fact, when focusing on the B220� pDCs, there wasevidence for substantial downregulation of the respectivemarkers, although statistical significance was only obtained forCD86 at one time point (day 7). This failure to upregulateMHC class II and costimulatory molecules and trend toward adownregulation further suggest a deviation from events asso-ciated with a classical inflammatory response and the estab-lishment of a tolerizing condition.

Analysis of DCs in the vaginal tissue revealed the presenceof DEC-205� DCs at the edge of the epithelium of infectedmice with dendrites extending into the lumen. Evidence for theextension of dendrites through the epithelium for the purpose

TABLE 1. Dendritic cells in draining lumbar lymph nodes during experimental vaginal candidiasis

Sample No. of cells/lymphnodea % CD11c� No. of DCs/lymph

nodeb % B220� No. of pDC/lymphnodec

Uninfected 8.5 � 105 1.8 � 105 2.6 0.43 �2.3 � 104 1.6 0.1 �1.36 � 104

7 days postinfection 3.0 � 106 4.0 � 105 3.3 0.83 �9.68 � 104 1.9 0.2 �5.7 � 104

a Values are mean standard error of the mean for three experiments using lymphocytes pooled from five mice per group.b Calculated from percent CD11c�.c Calculated from percent B220�.



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of sampling the microenvironment has been shown previouslyin the gastrointestinal tract (29). Furthermore, pDCs that arenot DEC-205� were seen to aggregate at the interface of thelamina propria and the epithelium. DC positioning in the vag-inal tissue during Candida infection suggested that DEC-205�

DCs were in close proximity to Candida in the vaginal lumenand that pDCs at least migrated towards the epithelium. Al-though it is not known why the pDCs failed to reach the outer

epithelium or what role the interaction of DEC-205� DCs withCandida may ultimately play in the inflammatory versus im-munoregulatory outcome, it is possible that the pDCs will orhave had contact with Candida through processed antigen inthe tissues or from Candida hyphae that may have invaded intothe epithelium. Alternatively, the tissues may not have beensampled at the optimal time to observe complete migration ofpDCs to the outer epithelium.

FIG. 3. Dendritic cells in vaginal tissue. Frozen vaginal tissue sections from estrogenized mice intravaginally inoculated with PBS (frames A andD) or live Candida (frames B, C, and E) were labeled with anti-DEC-205 (frames A and B), anti-DEC-205-PE, and calcofluor white (frame C)or anti-plasmacytoid DC marker (frames D and E). Tissue regions are labeled. White arrows indicate DC staining in vaginal tissue. The small insertfor frame C represents a standard image of tissue revealing the orientation for the confocal image. The figure shows representative images of threerepeats with three mice per group. The magnification is �400 for all images.



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As additional evidence for a direct interaction of DCs andCandida in the vagina, immunofluorescent confocal micros-copy demonstrated the close proximity of DCs and Candidahyphae at the epithelial surface. This putative interaction pro-vides strong evidence of a role for DCs in the initiation ofcellular manifestations observed in the draining lymph nodesin the present study. Interactions of bone marrow-derived DCswith Candida have been demonstrated previously in vitro andare shown to be receptor mediated, depending on fungal mor-phology (i.e., yeast versus hyphae) (6, 26, 31). We postulatethat after interaction with Candida in the vagina, vaginal DCstake up Candida antigen and travel to the draining lymphnodes where they are able to induce activation of CD4�

CD25� Treg cells observed in previous studies (43) and initiateregulatory cytokine production (i.e., TGF-� and IL-10) (42),which together play a potential role in the decrease of T cellsexpressing homing receptors required to migrate and enter thevaginal mucosa via tissue-associated adhesion molecules (43).From this, a general state of tolerance is created, resulting in alack of protective CMI against VVC.

A paradoxical observation as alluded to earlier is that cellsisolated from the draining lymph nodes of mice inoculatedintravaginally with Candida responded to Candida antigen invitro by the production of Th1-type cytokines (IL-2 and gamma

interferon [IFN-�]) as evidence of a classical inflammatoryresponse (9). This is in contrast to the evidence for DC-in-duced immunoregulation and lack of Th1-type CMI responseobserved in vivo (12, 42, 43). The in vitro result may be ex-plained by the removal of the responsive cells from the localimmunoregulatory cytokine milieu whereby the T cells can bestimulated to produce a more classical inflammatory response.

Other groups have also demonstrated that exposure of DCsto antigen in the vagina failed to induce DC activation. Fauschand colleagues have shown that human papillomavirus-likeparticles (HPV VLP) failed to induce upregulation of Langer-hans cell activation markers and cytokine production despiteantigen uptake, although other DCs (non-Langerhans) becameactivated by HPV VLP (7, 8). This is significant because Lang-erhans cells are found in vaginal epithelium and would be thefirst APCs to come in contact with a vaginal pathogen. Studiesby Zhao et al. using a murine model of vaginal herpes simplexvirus type 2 infection provide yet another example of the lackof epithelium-associated Langerhans cell activation in response toa vaginal pathogen, although submucosal DCs exposed to antigenwere able to induce an inflammatory response in the draininglymph nodes (45). These results emphasize the tolerogenic natureof Langerhans cells in response to vaginal pathogens associatedwith the epithelial layer, whereas submucosal DCs may not be

FIG. 4. Effects of vaginal Candida infection on DC in the draining lymph nodes. Lymphocytes were isolated from draining lymph nodes of micegiven PBS or live Candida intravaginally, harvested at day 4 or 7 postinoculation, and enriched for CD11c� cells. Cells were labeled withB220-FITC, CD11c-PE, and MHC class II-Cy-Chrome, CD80-Cy-Chrome, CD86-Cy-Chrome, or CD40-Cy-Chrome and analyzed by flow cytom-etry. (A) Representative result of MFI for each marker of three experiments performed. B220� cells were identified from total DCs (CD11c�).(B) Percent change in MFI standard error of the mean (SEM) for total DCs (CD11c�) for three experiments. (C) Percent change in MFI SEM for B220� DCs for three experiments. (D) Percent change in MFI SEM for B220� DCs for three experiments. Controls were labeled withisotype control antibodies to set gates. The figure shows results from pooled cells using five mice per group.



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inherently tolerogenic. It would be interesting to determinewhether tolerance to Candida can be broken in mice by injectionof Candida into the vaginal submucosa or by coinfecting micewith another vaginal pathogen that stimulates an inflammatoryresponse. The latter is unlikely, however, based on a previousstudy where responses, or lack thereof, following coinfection withCandida and Chlamydia trachomatis remained independent (21).

Mucosal sites, especially the vaginal mucosa, are consideredvery immunotolerant environments. This is important in lightof the many commensal organisms of these tissues and theneed to distinguish between pathogen and commensals. Atolerizing microenvironment in the vagina protects against un-wanted inflammatory responses at a site that is constantlyexposed to foreign antigens and creates avoidance of states ofchronic inflammation. Furthermore, immune tolerance in thereproductive tract is especially important for fertilization andpregnancy, and substantial evidence for immunoregulation in-volving immunoregulatory cytokines and possible cellular me-diators in the upper reproductive tract has been demonstrated(reviewed in reference 30). Future work on the role of DCs inthe initiation of immunoregulation during vaginal candidiasismay include studies of DC-specific cytokine production in thedraining lymph nodes and studies to elucidate the intracellularsignaling mechanisms involved in DC-mediated immunoregu-lation similar to what has been shown for human papillomavi-rus (8). Noteworthy for such studies is the fact that the murinemodel is distinct from the human condition by virtue thatCandida is not a commensal in rodents. Thus, the results fromthe murine model may not be directly applicable to humans,although for issues related to immunoregulation at the vaginalmucosa, data from the animal model have mirrored data fromhumans very well (10). Thus, there is confidence that the mu-rine model is adequate and appropriate for such studies andwill eventually be confirmed in humans.

In summary, we have demonstrated that pDCs are a majorsubset of DCs in the vagina and draining lymph nodes andremain so throughout the course of an experimental vaginalCandida infection. Additionally, vaginal infection with C. albi-cans fails to induce the upregulation of MHC class II antigenand costimulatory molecules on DCs that is consistent with apredominant immunoregulatory microenvironment. We hy-pothesize that initiation of T-cell tolerance and immunoregu-lation that begins in the vagina and continues in the draininglymph nodes during vaginal candidiasis occurs through an es-tablished mechanism of antigen presentation involving DCs,and in particular pDCs, in the absence of costimulation.

ACKNOWLEDGMENT

This study was supported by Public Health Service grant AI32556from the National Institute of Allergy and Infectious Diseases at theNational Institutes of Health.

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