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Research Article

Association of Neisseria gonorrhoeae OpaCEA with Dendritic Cells Suppresses Their Ability to Elicit an HIV-1-Specific T Cell Memory Response

  • Qigui Yu mail,

    andyu@iupui.edu (QY); scott.gray.owen@utoronto.ca (SG)

    Affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America

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  • Edith M. C. Chow,

    Affiliation: Department of Molecular Genetics, and 3Clinical Sciences Division, University of Toronto, Toronto, Ontario, Canada

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  • Shannon E. McCaw,

    Affiliation: Department of Molecular Genetics, and 3Clinical Sciences Division, University of Toronto, Toronto, Ontario, Canada

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  • Ningjie Hu,

    Affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America

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  • Daniel Byrd,

    Affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America

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  • Tohti Amet,

    Affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America

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  • Sishun Hu,

    Affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America

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  • Mario A. Ostrowski,
  • Scott D. Gray-Owen mail

    andyu@iupui.edu (QY); scott.gray.owen@utoronto.ca (SG)

    Affiliation: Department of Molecular Genetics, and 3Clinical Sciences Division, University of Toronto, Toronto, Ontario, Canada

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  • Published: February 12, 2013
  • DOI: 10.1371/journal.pone.0056705

Abstract

Infection with Neisseria gonorrhoeae (N. gonorrhoeae) can trigger an intense local inflammatory response at the site of infection, yet there is little specific immune response or development of immune memory. Gonococcal surface epitopes are known to undergo antigenic variation; however, this is unlikely to explain the weak immune response to infection since individuals can be re-infected by the same serotype. Previous studies have demonstrated that the colony opacity-associated (Opa) proteins on the N. gonorrhoeae surface can bind human carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on CD4+ T cells to suppress T cell activation and proliferation. Interesting in this regard, N. gonorrhoeae infection is associated with impaired HIV-1 (human immunodeficiency virus type 1)-specific cytotoxic T-lymphocyte (CTL) responses and with transient increases in plasma viremia in HIV-1-infected patients, suggesting that N. gonorrhoeae may also subvert immune responses to co-pathogens. Since dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a key role in the induction of an adaptive immune response, we investigated the effects of N. gonorrhoeae Opa proteins on human DC activation and function. While morphological changes reminiscent of DC maturation were evident upon N. gonorrhoeae infection, we observed a marked downregulation of DC maturation marker CD83 when the gonococci expressing CEACAM1-specific OpaCEA, but not other Opa variants. Consistent with a gonococcal-induced defect in maturation, OpaCEA binding to CEACAM1 reduced the DCs’ capacity to stimulate an allogeneic T cell proliferative response. Moreover, OpaCEA-expressing N. gonorrhoeae showed the potential to impair DC-dependent development of specific adaptive immunity, since infection with OpaCEA-positive gonococci suppressed the ability of DCs to stimulate HIV-1-specific memory CTL responses. These results reveal a novel mechanism to explain why infection of N. gonorrhoeae fails to trigger an effective specific immune response or develop immune memory, and may affect the potent synergy between gonorrhea and HIV-1 infection.

Introduction

Gonorrhea, caused by the Gram-negative intracellular diplococcus Neisseria gonorrhoeae (N. gonorrhoeae), is one of the most prevalent sexually transmitted diseases (STDs) of humans, with over 88 million new cases reported globally each year (http://whqlibdoc.who.int/hq/2011/WHO_RHR​_11.14_eng.pdf). N. gonorrhoeae infection initiates with the physical attachment of the bacterial surface appendages called pili to the apical side of the host mucosal cells [1], [2]. This loose attachment is followed by a more intimate association involving integral outer membrane protein adhesins, including the colony opacity-associated (Opa) proteins on the bacterial surface [3], [4]. In addition to mediating bacterial attachment, certain Opa variants also promote the transmigration of gonococci across the epithelial layer into the submucosa [4]. A single strain of N. gonorrhoeae encodes up to 11 related but antigenically distinct Opa alleles [5]. The expression of each Opa allele is randomly phase-variable and can turn on and off independently [6], [7]. Although a minority of Opa variants can bind to heparan sulfate proteoglycans (HSPG) [7][9], the majority of Opa proteins characterized to date target members of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family of receptors [10][15], which are expressed not only on various epithelial and endothelial tissues, but also on immune cells [16][18].

CEACAMs belong to the immunoglobulin (Ig) superfamily and contribute to the adhesive properties of cells. The human CEACAMs comprise seven members (CEACAM1 and CEACAM3 through CEACAM8) that are characterized by a single amino-terminal Ig variable-like domain and a varying number (0 to 6) of IgG2 constant-like domains [19], [20]. CEACAM1 has the broadest tissue distribution of all characterized CEACAMs and its expression can be induced by neisserial infection or stimulation with a variety of stimuli including 12-O-tetradecanoylphorbol-13-acetate (TPA), calcium ionophore and retinoids [18], [21][23]. CEACAM1 has two major isoforms, CEACAM1-L and CEACAM1-S, which differ in their cytoplasmic domains. The cytoplasmic domain of CEACAM1-L consists of 73 amino acids and has 2 immunoreceptor tyrosine-based inhibition motifs (ITIM) that can be phosphorylated to play an inhibitory role through downregulation of intracellular signaling events such as calcium ion influx [24]. CEACAM1-S has a cytoplasmic domain of only 10 amino acids and lacks ITIM motifs [25]. CEACAM1-L and CEACAM1-S are coexpressed at different ratios in different cell types and in different functional states of a cell over time [26].

Despite the availability of effective antibiotic therapies, gonorrhea incidence is rising in the United States and globally after a steady decline for the past two decades [27][29]. Although increased screening, use of more sensitive diagnostic tests, and improved reporting may account for a portion of the recent increase, true increases in disease in some populations and geographic areas are also occurring. The success of N. gonorrhoeae appears to rely on its ability to avoid the normal development of a memory immune response that would otherwise protect an individual from reinfection during a subsequent exposure. In addition, N. gonorrhoeae can subvert immune responses to co-pathogens, such as HIV-1. For example, studies have shown that gonococcal infection is associated with enhanced HIV-1 acquisition and impaired HIV-1-specific CTL responses [30], decreases in blood CD4+ T cell count [31], and increased semen and plasma viral loads [31][33] in HIV-1 infected individuals, and these are often found to be return to normal with effective gonococcal therapy.

Gonococcal surface epitopes frequently undergo antigenic variation, but this is unlikely to explain the weak immune response to infection as individuals can be re-infected by the same serotype of gonococcal strains [34][36]. Recent studies have demonstrated that N. gonorrhoeae can directly subvert the natural immune response [37][39]. N. gonorrhoeae expressing Opa variants that can bind CEACAM1 (herein referred to as OpaCEA) were seen to engage this receptor on the surface of primary human CD4+ T cells and suppress their activation and proliferation in response to a variety of stimuli [37]. The OpaCEA-CEACAM1 binding triggers phosphorylation of CEACAM1 on the tyrosine residuals within the ITIM apparent in the cytoplasmic domain [37], [39]. This allows the recruitment and subsequent activation of the Src homology domain 2 (SH2)-containing tyrosine phosphatases SHP-1 and SHP-2 at the site of bacterial attachment, which prevents the normal tyrosine phosphorylation of the CD3zeta-chain and ZAP-70 kinase in response to T cell receptor (TCR) engagement. This dynamic response allows the bacteria to effectively harness the natural coinhibitory function of CEACAM1 to suppress the adaptive immune response at its earliest step [37][39].

Interestingly, CEACAM1 can also inhibit NK cell cytotoxicity when co-ligated with NK cell-activating receptors and the inhibitory effect is mediated by heterophilic interaction between carcinoembryonic antigen (CEA) and CEACAM1 or CEACAM1-CEACAM1 homophilic interaction [40], [41]. Notably, in certain conditions, CEACAM1 is also able to deliver activation signals during an immune response. Triggering CEACAM1 with the monoclonal antibody (mAb) AgB10 can enhance activation and proliferation of murine T cells and B cells, resulting in amplification of immune responses [42][44]. In addition, the same mAb (AgB10) induces maturation and chemokine/cytokine secretion of murine dendritic cells (DCs) [25]. It is most likely that the CEACAM1-mAb interaction triggers the activation of these immune cells through activation of the c-Jun NH2-terminal kinase (JNK) pathway [44]. Taken together, these data suggest that CEACAM1 delivers both activation and inhibitory signals, depending on the binding ligand, ratio and activation of the expressed CEACAM1-L and CEACAM1-S isoforms, and the metabolic state of the cell, and that has the capacity to regulate cellular functions of multiple cell types during an immune response.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a critical role in initiating and regulating the adaptive immune response. In recent years, using new cell markers, it has become increasingly apparent that DCs are particularly abundant at mucosal sites and are recruited during infections to the site of mucosal inflammation [45]. They perform a sentinel function for the recognition of invading pathogens and a regulatory function to control mucosal immunity. DCs have been observed to express both long and short forms of CEACAM1, both in intro and in vivo [25], suggesting that these should be available to bind the neisserial Opa proteins [10], [13], [14]. There is currently no knowledge about the nature of N. gonorrhoeae Opa interactions with CEACAM1 on DCs or the functional consequences of these interactions. This study investigates effects of Opa variants on human DC activation and function. We demonstrate that gonococcal expression of Opa­CEA allows bacterial binding to DCs, and downregulates DC maturation in response to infection. Consistent with these effects, DCs infected with Opa­CEA-expressing gonococci had a diminished capacity to induce proliferation of allogeneic T cells and were defective in their ability to induce an HIV-1-specific memory CTL response. These effects, when combined with the suppressive effect that Opa-CEACAM1 binding has on primary human T cells, provides an effective strategy by which the bacteria may halt the development of an adaptive immune response.

Methods and Materials

Bacterial strains

N. gonorrhoeae were grown from frozen stocks on Difco GC agar supplemented with 1% (vol/vol) IsoVitaleX enrichment (BD Biosciences, Mississauga, Ontario, Canada). Isogenic gonococcal strains N302 (Opa-negative, pilus-negative or Opa-/P-), N303 (constitutively expressing heparan sulfate proteoglycan-specific Opa50, herein referred to as OpaHSPG), N309 (constitutively expressing CEACAM1 receptor-specific Opa52, herein referred to as OpaCEA), and N496 (Opa-/P+) were described previously [14], [46] and were a generous gift from Professor Thomas Meyer (Berlin, Germany). These Opa genes are expressed in a derivative of strain MS11 containing mutations that abolish the expression of the natural chromosomally-encoded HSPG receptor-specific Opa30. The ligands recognized by these various Opa variants were previously described [3]. These gonococcal strains were subcultured from frozen stocks and a binocular microscope was used to monitor colony opacity phenotype. Opa expression and variant type were routinely confirmed by SDS-PAGE (10%) and resolved proteins were transferred onto Immobilon P membranes (Millipore, Bedford, MA) and probed with an Opa cross-reactive mAb 4B12/C115 [47]. Escherichia coli (E.coli) DH5α strain, which was used as a control for gonococcal effects on DC activation and function throughout this study, was obtained from Invitrogen (Carlsbad, CA) and grown on LB agar and LB broth (Sigma-Aldrich, St. Louis, MO).

Antibodies and reagents

The mouse mAb D14HD11 (IgG1, cross-specific for human CEACAM1, CEACAM3, CEACAM5, and CEACAM6) was a gift of Dr. Fritz Grunert (University of Freiburg, Germany). The murine MOPC-21 IgG1 mAb, used as an isotype control throughout this study, was purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO). Unless otherwise indicated, anti-human mAbs or polyclonal Abs conjugated with fluorochrome were purchased from BD PharMingen (San Diego, CA): anti-CD3APC, anti-CD3PerCP, anti-CD8FITC, anti-CD14FITC, anti-CD1aFITC, anti-CD80FITC, anti-CD83PE, anti-CD86FITC, anti-HLA-DRAPC, anti-IFN-γPE and anti-IL-12PE (p40/p70), and matched-isotype control Abs conjugated with FITC, PE, PerCP, or APC. Recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) were purchased from ProTech (Rocky Hill, NJ). CD40 ligand trimer (CD40LT) was obtained as a gift from Immunex (Seattle, WA). Cy5-conjugated secondary Abs were obtained from Jackson ImmunoResearch Laboratories (Mississauga, Ontario, Canada). Texas red-X-succinimidyl ester (TRXSE), Bodipy FL-conjugated secondary Abs, and Texas red-conjugated phalloidin were purchased from Invitrogen (Carlsbad, CA).

Peripheral blood mononuclear cells, monocytes, monocyte-derived DCs, and HIV-1-specific peptides

Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech AB, Uppsala, Sweden) from heparinized human peripheral blood obtained from healthy blood donors, and frozen at –152°C until use. Monocytes were separated from the PBMCs using multistep Percoll (Sigma-Aldrich, St. Louis, MO) gradient centrifugation, and then purified by depletion of contaminating B cells, T cells, NK cells, and granulocytes using Ab-conjugated magnetic beads in the Monocyte Negative Isolation Kit (Dynal, Oslo, Norway) according to the manufacturer’s guidelines. The resulting cell preparations contained more than 95% monocytes and virtually no residual T and B lymphocytes, as assessed by CD14, CD3 and CD19 staining and flow cytometric analysis. Monocyte-derived DCs (MDDCs) were generated from the purified monocytes by a modification of a method previously described [48], [49]. Briefly, purified monocytes were cultured at 1×106 cells/ml in complete RPMI 1640 medium consisting of RPMI 1640 medium plus 10% fetal bovine serum (FBS), 2 mM glutamine, 25 mM HEPES, and antibiotics in the presence of 50 ng/ml recombinant human GM-CSF and 100 ng/ml recombinant human IL-4 (PeproTech, Rocky Hill, NJ). GM-CSF and IL-4 were added again on days 3 and 5 with the fresh complete RPMI 1640 medium. After 7 days of culture, more than 50% of the cells were CD1ahigh, MHC class II+, CD80low, and CD14-, which represents an immature DC phenotype. The immature MDDCs (iMDDCs) were used for bacterial binding and infection.

Five untreated HIV-1-seropositive individuals (Pt#1 – Pt#5, Table 1) at different stages of disease were recruited for studying gonococcal effects on HIV-1-specific CTL responses. The clinical profiles of these participants are depicted in Table 1. Leukopheresis was performed to obtain large amounts of PBMCs from each of these patients. Prior to the study, individuals were class I HLA-typed and screened for HIV-1-specific CTL by culturing PBMCs with HLA-restricted HIV-1 peptides and detecting IFN-γ-producing CD8+ T cells by ELISPOT assay as previously described [48][50]. HLA-restricted HIV-1 peptides were purchased from the PeproTech (Rocky Hill, NJ) and dissolved in RPMI 1640 medium or DMSO. The HIV-1 peptides for individual participants Pt#1 to Pt#5 are shown in Table 1. Informed consent was obtained from each of all participants including healthy blood donors and HIV-1-seropositive individuals in accordance with the guideline for conduction of clinical research at the University of Toronto and St. Michael’s Hospital, Canada. All investigational protocols were approved by the University of Toronto and St. Michael’s Hospital institutional review boards.

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Table 1. Profiles of HIV-1-infected participants.

doi:10.1371/journal.pone.0056705.t001

Analysis of CEACAM expression

To analyze the surface expression of CEACAM1 on monocytes, iMDDCs and mature MDDCs, the cell suspension was first stained with mAb D14HD11 or a non-specific isotype control mAb as primary Ab, followed by goat anti-mouse IgG1APC, and then stained with anti-human CD14FITC, or HLA-DRPerCP as indicated. HeLa cell lines transfected with either a human CEACAM1 cDNA-encoding plasmid or the empty pRC/CMV expression vector were used as positive and negative controls for CEACAM1 surface expression, respectively [51]. Peripheral B lymphocytes (CD19+) in the PBMCs from each individual were also stained for CEACAM1 expression as these cells are known to display high CEACAM1 surface expression, which could be used for the comparison of CEACAM1 expression on the surface of iMDDCs or mature MDDCs. A human B cell line C1R (a kind gift from Dr. MacDonald K.S. at the University of Toronto) was also used as a positive control for CEACAM1 expression analysis. Stained cells were subjected to flow cytometric analysis to examine surface expression of CEACAM1.

Western blot analysis of CEACAM1 expression was performed as described in our previous report [52]. Briefly, cells were washed once with ice-cold phosphate-buffered saline (PBS) prior to their resuspension in 100 µl of cell lysis buffer (Cell Signaling Technology, Pickering, Ontario, Canada) containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Cells were incubated for 15 min at 4 °C on an orbital shaker and then centrifuged to pellet the cellular debris. The protein-containing supernatant was stored at -80 °C until use. Equal amounts (15 µl per lane) of the protein-containing supernatants were mixed with 5 µl of 4 X NuPAGE LDS Sample Buffer (Invitrogen, Carlsbad, CA), boiled for 5 min, subjected to NuPAGE Novex high performance gel electrophoresis (Invitrogen, Carlsbad, CA), and then blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were blocked with 5% nonfat dry milk in TBS-T buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature (RT) and then incubated at 4 °C overnight with CEACAM-specific mAb D14HD11 as a primary Ab. These blots were detected using horseradish peroxidase (HRP)-conjugated secondary Ab (Southern Biotechnology Associates, Birmingham, AL) and visualized using the ECL detection system (Pierce, Rockford, IL). These blots were reprobed with β-actin Abs (Abcam, Cambridge, MA) as loading controls.

Bacterial binding, internalization and infection

Binding of isogenic gonococcal strains of N302, N303, N309 and N496 to iMDDCs was evaluated using FITC-labeled bacteria. For comparison, FITC-labeled E.coli DH5α was also studied side-by-side for bacterial binding. Bacteria (109/ml) were labeled by incubation of 0.5 mg FITC (Sigma-Aldrich, St. Louis, MO) per ml in phosphate-buffered saline (PBS, pH 7.4) at RT for 1 h. The FITC-pulsed bacteria were washed five times with PBS to remove unbound FITC. To test bacterial binding, iMDDCs were incubated with FITC-labeled bacteria at a multiplicity of infection (MOI) of 10 for 30 min at RT. Bacterial binding was determined by measuring the percentage of cells that bound FITC-labeled bacteria using flow cytometric analysis.

To test whether bacterial binding to iMDDCs is mediated by DC-SIGN on the cell surface, mannan blocking assays were performed site-by-site with the bacterial binding tests. Prior to adding FITC-labeled bacteria, iMDDCs were treated with mannan at 5 - 20 µg/ml, the concentrations that have been shown to completely block DC-SIGN-mediated binding of mycobacterium tuberculosis to DCs [53], [54], followed by incubation with FITC-labeled bacteria at an MOI of 10 for 30 min at RT. Blockage of bacterial binding was determined by measuring the percentage of bacterial binding deduction (% of cells that bound FITC-labeled bacteria in the absence of mannan minus % of cells that bound FITC-labeled bacteria in the presence of mannan).

Internalization of bacteria by iMDDCs was also investigated. Immature MDDCs were allowed to adhere onto coverslips pre-coated with 0.2% gelatin (Sigma-Aldrich, St. Louis, MO). These cells were pulsed with gonococcal strains (MOI = 100) prelabeled with Texas red-X-succinimidyl ester (Invitrogen, Carlsbad, CA) at 37°C for 1 h. Cells were immersed for 30 min at 37°C in 3.5% paraformaldehyde solution. Extracellular bacteria were then labeled with the polyclonal anti-gonococcal serum (UTR01), which was raised against N. gonorrhoeae N302 (Opa-), followed by a staining with a BODIPY-FL-conjugated secondary Ab (Invitrogen, Carlsbad, CA). Immature MDDCs were then permeabilized with 0.4% Triton X-100 and stained with Phalloidin-FITC (Sigma-Aldrich, St. Louis, MO) for visualizing cellular actin filaments. Intracellular (red) versus extracellular (yellow) bacteria with iMDDCs (green) were then distinguished by visualization with a Leica DM-IRBE inverted fluorescence microscope (Leica Microsystems, Toronto, Ontario).

Induction of HIV-1 peptide-specific CTL

The protocol for expanding circulating memory HIV-1-specific CTL ex vivo was described previously [48][50]. Immature MDDCs were infected with individual isogenic gonococcal strain or E.coli DH5α at 0-100 MOI in complete RPMI 1640 medium with an addition of 1 U/ml of endonuclease (Sigma-Aldrich, St. Louis, MO), which was used to prevent gonococcal aggregation mediated by DNA released through bacterial autolysis. Gentamycin was added at a final concentration of 50 µg/ml (Bishop, Burlington, Ontario) to each condition 6 h after the start of infection and maintained throughout the experimental time-course to prevent gonococcal overgrowth during the 2–3 days of cell culture. Infected cells were subjected to centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech AB, Uppsala, Sweden) to remove cellular and bacterial debris prior to co-culture with autologous T cells. In a parallel experiment used as a positive control, iMDDCs were incubated with CD40LT at 1 µg/ml for 2–3 days, which has been shown to significantly expand HIV-1 specific CTLs [48], [49], [55]. Cells from each condition were pulsed with the HIV-1-specific HLA class I-restricted peptide at 40 µg/ml for 1 h at 37°C, then plated in 24-well plates (5×105 pulsed or nonpulsed MDDCs/well) in complete RPMI 1640 medium. Freshly isolated or thawed autologous PBMCs were prepared and then added to the MDDCs at a 1:10 ratio (5×106cells/well in 2 ml medium). The following conditions were included in all experiments: 1) MDDCs not pulsed with peptides; 2) MDDCs pulsed with peptides; and 3) MDDCs infected with N302, N303, N309, N496, or E.coli DH5α, or treated with CD40LT, then pulsed with peptides. On days 3, 5, and 7, the medium was changed. On day 10, duplicate wells were pooled and cells were harvested and tested for HIV-1-specific CTL activity by intracellular IFN-γ staining. Experiments were repeated in Pt#1 and Pt#2 samples.

Flow cytometric analysis

Surface staining of PBMCs, isolated monocytes, and immature or mature MDDCs were performed in PBS/1% FBS/0.02% NaN3 using fluorochrome-conjugated Abs. Events were acquired using FACSCalibur System (BD Biosciences, San Jose, CA) and data were analyzed using FlowJo software (Tree Star Inc., San Carlos, CA). For intracellular staining, cells were permeabilized using reagents in the Cytofix/Cytoperm kit (BD PharMingen, San Diego, CA) in accordance with the manufacturer’s recommendations. Intracellular staining was performed to enumerate the number of IL-12-producing MDDCs or the number of IFN-γ-producing CD8+ T cells, as previously described [48], [49], [55]. For IFN-γ-producing CD8+ T cell staining, 0.25×106 cells were cultured in U-bottom 96-well plates in the presence of peptide-pulsed (1-10 µM) autologous B-lymphoblastoid cell lines (B-LCL) or autologous T cell-depleted PBMCs as stimulator cells; nonpeptide-pulsed stimulator cells were used as background controls. Positive control cells were stimulated with the bacterial superantigen staphylococcal enterotoxin B (SEB, 1 µg/ml) (Sigma-Aldrich, St. Louis, MO). Cells were incubated with peptide-pulsed or nonpeptide-pulsed stimulator cells for 6 h at 37°C in 5% CO2. Monensin was added for the duration of the culture period to facilitate intracellular cytokine accumulation. A total of 50,000–100,000 events were collected from each sample for intracellular IFN-γ assay, and CD8+ T cells were enumerated after gating on CD3-positive cells.

Statistical analysis

Data were compared using the Wilcoxon signed rank test for paired samples. Statistical significant was defined by p< 0.05.

Results

Expression of CEACAM1 by immature and mature MDDCs

To determine whether CEACAM1 expression is affected by DC maturation, we generated MDDCs from ex vivo isolated monocytes by supplementing the primary cultures with recombinant human GM-CSF and IL-4. After 7 days, more than 50% of the cells were CD1ahigh and CD14-, and most cells expressed low or undetectable level of MHC-II molecule HLA-DR and costimulatory molecules CD40, CD80, CD83, and CD86, which together reflects an immature MDDC (iMDDC) phenotype. This population was characterized for CEACAM1 expression, bacterial binding and infection. Flow cytometric analysis using mAb D14HD11, which detects the extracellular domain of human CEACAM1, showed that iMDDCs expressed low, but detectable levels of CEACAM1 on the cell surface (Fig. 1A), which is consistent with the previous observation that mouse DCs express CEACAM1 [25]. CEACAM1 expression on iMMDCs was slightly, but not significantly, down-regulated when compared with the monocytes (Fig. 1A, 1B). The expression of this protein was significantly up-regulated (2- to 3-fold) when the iMDDCs were subcultured in the presence of soluble recombinant human CD40LT at 1 µg/ml for additional 3 days (Fig. 1B), a condition that drives iMDDC maturation. CEACAM1 expression level on monocytes, iMDDCs, or mature MDDCs was lower than that of peripheral CD19+ B cells and C1R cells, which are known to display high CEACAM1 surface expression (Fig. 1A). Different from CEACAM1 cDNA transfected HeLa cells and the C1R cell line, which displayed a clear-cut single CEACAM1-positive peaks (Fig. 1A), primary monocytes, iMDDCs, matured MDDCs and B cells each displayed a gradient of CEACAM1-positive staining on the mass of these cells, indicating that the majority or all cells expressed CEACAM1, but at varying levels.

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Figure 1. Expression of CEACAM1 by immature and mature MDDCs.

A) The histograms show the log fluorescence intensity of CEACAM1 on the surface of immature and mature MDDCs labeled with either mouse anti-human CEACAM1-speciifc mAb D14HD11 (bold line profiles), or an isotype-matched control mAb (solid gray profiles), followed by fluorochrome-conjugated goat-anti-mouse secondary Ab. HeLa cells transfected with CEACAM1 cDNA (HeLa/cDNA) or empty plasmid vector (HeLa/Neo) were used as positive and negative staining controls of CEACAM1 expression, respectively. Peripheral CD14+ monocytes, CD19+ B cells, and C1R cells were also included for comparison of surface expression of CEACAM1 and expression phenotypes. The data show one representative staining from five healthy blood donors. B) Summarized data of CEACAM1 expression determined by flow cytometric analysis from five healthy blood donors. Fluorescence intensity was obtained from CEACAM-1 specific D14HD11 mAb staining subtracted from isotype-matched control mAb staining. The number shows the average fluorescence intensity. C) Western blot analysis of CEACAM1 protein expression. Equal amounts of protein extracts from peripheral CD19+ B cells, C1R cells, purified peripheral CD14+ monocytes, iMDDCs (harvested from day 7 cultures), and CD40LT-matured MDDCs were subjected to Western blot analysis using D14HD11 as a primary mAb and a secondary Ab labeled with HRP. The blot was reprobed with β-actin Ab as loading controls (lower panel). Data are representative for three independent experiments.

doi:10.1371/journal.pone.0056705.g001

The expression of CEACAM1 by monocytes, immature or mature MDDCs was confirmed with a Western blot. Monocytes, iMDDCs from day 7 cultures and MDDCs matured by treatment of CD40LT expressed both CEACAM1 isoforms (Fig. 1C). The presence of the L isoform indicated that CEACAM1 expressed on the surface of MDDCs could potentially deliver inhibitory signals via its ITIM motifs [37]. CEACAM1 is thus expressed on the surface of iMDDCs developed in vitro from monocytes, and is up-regulated to a limited extent on mature MDDC during their in vitro differentiation.

Contribution of Opa proteins and pili to N. gonorrhoeae interactions with iMDDCs

In order to understand whether the gonococcal Opa protein adhesins and/or pili influenced association of N. gonorrhoeae with iMDDCs, isogenic strains of N. gonorrhoeae constitutively expressing defined adhesins were employed. Immature MDDCs were incubated with FITC-labeled isogenic gonococcal strains or E. coli DH5α at an MOI of 10 bacteria per cell at RT for 30 min. After fixation with 1% formaldehyde, bacterial binding to cells was determined by flow cytometric analysis to measure the percentage of cells with bound FITC-labeled bacteria. The iMDDCs efficiently bound N. gonorrhoeae regardless of Opa expression (Fig. 2A), which was consistent with a previous report [56]. Of the four isogenic gonococcal strains examined, the Opa-/P- N302 showed the lowest binding to iMDDCs (20.6±3.6%, n = 3), while N303 (expressing OpaHSPG), N309 (expressing CEACAM1 receptor-specific OpaCEA), and N496 (Opa-/P+) all bound to iMDDCs at high levels, with 53.7±6.9% (n = 3), 51.2±7.3% (n = 3), 47.6±7.8% (n = 3), respectively. Compared with N. gonorrhoeae, E.coli showed substantially less binding to iMDDCs (11.7±3.2 %, n = 3). Combined, the data indicate that gonococcal binding to iMDDCs did not require an interaction between OpaCEA and CEACAM1, as gonococci lacking OpaCEA effectively adhered to the cells. However, CEACAM1 binding could facilitate bacterial association with the iMDDCs since OpaCEA-expressing N309 adhered to a greater extent than did the isogenic Opa-deficient N302. When considered together with previous reports [56], [57], these observations suggest that neither binding nor engulfment of N. gonorrhoeae by DCs was not affected by Opa protein or pilus expression.

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Figure 2. Gonococcal binding and internalization to iMDDCs.

A) Immature MDDCs were incubated with FITC-labeled gonococcal strains N302, N303, N309, and N496, or E.coli DH5α bacteria at an MOI of 10 for 30 min at RT. Bacterial binding was determined by measuring the percentage of cells that bound FITC-labeled bacteria using flow cytometric analysis. The percentage of cells with bound FITC-labeled bacteria is indicated in each condition. B) Bacterial binding to iMDDCs is not mediated by DC-SIGN on the cell surface. Mannan blocking assays were carried out site-by-site with the bacterial binding tests. The percentage of cells with bound FITC-labeled bacteria is indicated in each condition in the presence of mannan at 20 µg/ml. C) Internalization of gonococcal strains and E.coli DH5α into iMDDCs. Immature MDDCs were allowed to adhere onto coverslips pre-coated with 0.2% gelatin. These cells were pulsed with gonococcal strains or E.coli DH5α (MOI = 100) prelabeled with Texas red-X-succinimidyl ester at 37oC for 1 h. Extracellular bacteria were then labeled with the polyclonal anti-gonococcal serum, followed by a staining with a BODIPY-FL-conjugated secondary Ab. Immature MDDCs were then permeabilized with 0.4% Triton X-100 and stained with Phalloidin-FITC. Intracellular (red) versus extracellular (yellow) bacteria with MDDCs (green) were then distinguished by visualization with a Leica DM-IRBE inverted fluorescence microscope. The magnification for all conditions is 40.

doi:10.1371/journal.pone.0056705.g002

Considering that binding occurred without Opa or pilus adhesin expression, we investigated whether N. gonorrhoeae or E. coli bound to iMDDCs through the surface molecule DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin), which structurally contains a mannan-binding lectin domain [58], [59]. DC-SIGN recognizes a large array of pathogens, including HIV-1 [60], Ebola [61], hepatitis C virus (HCV) [62], [63], Dengue virus [64], Leishmania amastigotes [65], Mycobacterium tuberculosis [53], [54], Candida albicans [66], and Aspergillus fumigatus conidia [67], in a mannan-dependent manner. For these pathogens, DC-SIGN relies on the presence of mannose residues on surface oligosaccharide chains. More pertinent for this study, certain lipo-oligosaccharide mutants of N. gonorrhoeae that expose otherwise internal mannose residues are recognized by DC-SIGN [57]. In each case, soluble mannan at 5 - 20 µg/ml will, therefore, block the binding of these pathogens to DCs [53], [54]. We found that bacterial binding to iMDDCs was not affected by 20 µg/ml mannan treatment, as the percentage of cells that bound FITC-labeled bacteria did not significantly decrease. This indicates that gonococcal binding by iMDDCs was not dependent on DC-SIGN for any strain tested (Fig. 2B).

The high efficiency bacterial binding to iMDDCs prompted us to investigate whether the iMDDC-bound bacteria could be internalized by iMDDCs. Immature MDDCs were pulsed with isogenic gonococcal strains or E.coli DH5α that had been prelabeled with Texas red-X, succinimidyl ester (Invitrogen, Carlsbad, CA). Prior to permeabilizing the DC membrane, extracellular gonococcal bacteria were labeled with the polyclonal anti-gonococcal serum (UTR01), which was then detected using a BODIPY-FL-conjugated secondary Ab (Invitrogen, Carlsbad, CA). Intracellular (red) versus extracellular (yellow) gonococci associated with iMDDCs (green) were then visualized by fluorescence microscopy. Both the gonococcal strains and E.coli DH5α could be internalized by iMDDCs (Fig. 2C), and this activity was not blocked by mannan (data not shown). In all four isogenic gonococcal strains tested, both extracellular (yellow) and intracellular (red alone) bacteria could be observed, indicating that the bound bacteria could be internalized. In contrast, only bacteria (red) labeled with Texas red-X, succinimidyl ester were visualized for E. coli DH5α, confirming the specificity of extracellular staining (yellow) with anti-N. gonorrhoeae N302 polyclonal Abs (Fig. 2C).

Association of N. gonorrhoeae OpaCEA with MDDCs down-regulates CD83 expression

Immature DCs are highly efficient in antigen capture and processing, whereas mature DCs are specialized in antigen presentation and activation of naive T cells to evoke cellular immune responses. Immature DCs mature in response to various signals, including bacterial components (lipopolysaccharide or LPS), inflammatory cytokines (TNF-α, PGE2) or co-stimulatory molecules (CD40L). We investigated whether N. gonorrhoeae affected the normal maturation of MDDCs in response to infection. Relative to medium alone, all bacterial infections induced marked changes in the cellular morphology of the iMDDCs, consistent with those occurring in response to the potent inducer (CD40-ligation) of DC maturation (data not shown). The bacterial infections generally also led to changes in expression of surface molecules known to correlate with maturation. As shown in Fig. 3A, the MDDCs cultured with medium alone were HLA-DRlow, expressed high levels of CD1a, and low or undetectable levels of CD80, CD83, and CD86, typical of an immature DC phenotype. Among the bacterial strains, E. coli DH5α was the most potent inducer of DC maturation, as demonstrated by decreased CD1a expression and increased CD80, CD83, and CD86 expression (Fig. 3A, 3B). N. gonorrhoeae infection tended to induce similar changes in MDDC maturation markers regardless of adhesin expression, with the exception that CD83 was unexpectedly down-regulated in the samples infected with OpaCEA-positive gonococcal strain N309 (6.7 ± 2.2, n = 5). In contrast, the Opa-negative gonococcal strain N302 (16.8 ± 3.6, n = 5), OpaHSPG-expressing N303 (14.5 ± 2.8, n = 5), and piliated N496 (15.9 ± 2.7, n = 5) all induced CD83 levels similar to that of the E. coli control, indicating that interaction of OpaCEA with CEACAM1 affected MDDC maturation by suppressing CD83 (Fig. 3A, B). It is pertinent to note that, other than CD83, there were no other apparent differences between N309, the other gonococcal strains or E. coli with regards to its effect on CD1a, HLA-DR, CD80, and CD86 (Fig. 3A, 3B, 3C), suggesting that this was a CD83 specific effect.

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Figure 3. Association of N. gonorrhoeae OpaCEA with MDDCs down-regulated CD83 expression, but did not affect IL-12 induction.

A) A representative experiment from a healthy participant is shown. Immature MDDCs were infected with individual isogenic gonococcal strain or E.coli DH5α at an MOI of 10 in complete RPMI 1640 medium. Medium alone and CD40LT were included for negative and positive controls, respectively. MDDCs were harvested and expression of surface molecules was assayed by flow cytometric analysis. Values represent the mean flurorescence intensity subtracted from the value of matched isotype control mouse mAbs (shaded gray histogram). B) Summary data of CD83 expression on MDDCs surface obtained from all five participants of healthy blood donors are shown. Statistical comparisons of data pooled from five participants were performed between OpaCEA-expressing N309 and other gonococci strains, E.coli DH5α, or CD40LT treatment: N309 vs N302, p < 0.05; N309 vs N303, p < 0.05; N309 vs N496, p < 0.01; N309 vs E.coli, p < 0.005; N309 vs CD40LT, p < 0.0001; CD40LT = CD40 ligand trimer. C) Summary data on expression of CD1a, CD80, CD86, and HLA-DR on MDDCs surface obtained from all five participants of healthy blood donors are shown. D) A representative experiment is illustrated to show the effects of N. gonorrhoeae infection on IL-12 induction by MDDCs. Immature MDDCs were infected with OpaCEA-positive N. gonorrhoeae N309, or OpaCEA-negative gonococcal strains N302, N303, N496 or E.coli DH5α, or treated with CD40LT at 1 µg/ml for 12 h, and IL-12 induction was measured by intracellular staining and flow cytometric analysis. The numbers (inset upper right) represent the percentage of IL-12 p40/p70-positive MDDCs per total MDDCs. Data are taken from one of HIV-1-uninfected participants and are representative of experiments with MDDCs derived from HIV-1-infected and HIV-1-uninfected individuals. E) Pooled data from all participants are shown.

doi:10.1371/journal.pone.0056705.g003

IL-12 is a pleiotropic cytokine that is secreted by activated professional APCs, including DCs. IL-12 can induce Th1-type cellular responses, T cell proliferation, and IFN-γ secretion from activated T cells and NK cells. We and others previously have demonstrated that MDDCs could be induced to produce IL-12 after CD40 ligation [48], [49], [55], [68]. We thus investigated whether OpaCEA could affect IL-12 production from N. gonorrhoeae-infected MDDCs. Immature MDDCs were infected with OpaCEA-positive N. gonorrhoeae N309, or OpaCEA-negative gonococcal strain N302, N303, N496 or E.coli DH5α for 12 h, and IL-12 induction was measured by intracellular staining using flow cytometric analysis. A representative experiment is illustrated in Fig. 3D and summary data from all experiments is shown in Fig. 3E. In comparison with medium alone, all isogenic gonococcal strains, regardless of OpaCEA expression, induced IL-12 production by MDDCs, albeit at somewhat lower levels than did E. coli DH5α or CD40LT. No significant difference between the levels of IL-12 expression was observed among the isogenic gonococcal strains.

Association of N. gonorrhoeae OpaCEA with MDDCs decreases the sensitization of allogeneic mixed lymphocyte proliferation

Activated DCs are potent APCs that sensitize T lymphocytes to both allotypic and other antigens. Considering the role of CD83 in T cell stimulation [69], we sought to test whether OpaCEA expression affected the functional capacity of MDDCs that were matured in response to infection with the various N. gonorrhoeae strains by measuring induced T lymphocyte proliferation in an allogeneic mixed lymphocyte reaction (MLR). Medium alone and CD40LT were included as negative and positive controls, respectively. As reported previously [48], [49], MDDCs that were matured in response to CD40LT strongly enhanced allogeneic T cell proliferation, measured by thymidine incorporation, when compared with medium alone (Fig. 4). MDDCs that were matured in response to most bacterial infections induced T cell proliferation to a similar degree. E.coli DH5α-infected MDDCs were the most potent inducers of T cell proliferation, however OpaCEA-negative strains, including N302, N303 and N496, also induced strong T cell proliferation. Unexpectedly when considering that they appeared to be mature by most measures, the MDDCs that had been exposed to the OpaCEA-positive strain N309 showed markedly lower T cell proliferation, with the difference being both highly reproducible among donor samples and statistically significant when all were combined (Fig. 4).

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Figure 4. Association of N. gonorrhoeae OpaCEA with MDDCs decreased the sensitization of allogeneic mixed lymphocyte proliferations.

Immature MDDCs infected, uninfected, or CD40LT-treated for 3 days were plated into 96-well round-bottom tissue-culture plates with allogeneic PBMCs at a ratio of 1:20 (MDDCs to T cells). Cells were cocultured for 3 days and pulsed with 2 µCi (0.076 Mbq) [3H]thymidine for an additional 6 h. [3H]Thymidine incorporation in harvested cells was assayed by liquid scintillation spectrometry. These data are mean ± SEM of triplicates and representative of four independent experiments with different donors. Statistical comparisons of data pooled from the five participants were performed between OpaCEA-expressing N309 and other gonococci strains, E.coli DH5α, or CD40LT treatment: N309 vs N302, p < 0.05; N309 vs N303, p < 0.05; N309 vs N496, p < 0.05; N309 vs E.coli, p < 0.05; N309 vs CD40LT, p < 0.001; CD40LT = CD40 ligand trimer.

doi:10.1371/journal.pone.0056705.g004

Effects of N. gonorrhoeae infection on HIV-1-specific CTL memory response

After determining that infection with the OpaCEA-expressing N. gonorrhoeae N309 impaired MDDC maturation and decreased MDDC-induced T cell proliferation, we sought to determine if Opa-CEACAM1 interactions would also alter an epitope-specific T cell memory response. To this end, we adapted our established in vitro CTL response assay in which peptide-pulsed MDDCs and T cells from the same individual are cocultured in the absence of exogenous cytokines to expand epitope-specific memory CTL responses [48], [49]. Five HIV-1 seropositive individuals with different rates of disease progression were studied: 2 long-term nonprogressors (LTNPs) (Pt#1 and Pt#4), 1 chronic progressor (Pt#3), and 2 recent seroconvertors (Pt#2 and #5) (Table 1). Immature MDDCs derived from these individuals were infected with the isogenic gonococcal strains or E.coli DH5α at an MOI of 10 for 3 days, and then pulsed with HLA-restricted peptides before co-culturing with autologous PBMCs. After 7–10 days of co-culture, CTL effector activity was assayed by measuring intracellular IFN-γ production after exposure to peptide-pulsed targets (autologous B lymphocyte cell lines or autologous T cell-depleted PBMCs). A representative experiment measuring HIV-1- specific CD8+ T cells producing IFN-γ by intracellular staining and flow cytometric analysis from Pt#1 is illustrated in Fig. 5A. A summary of the HIV-1-specific CD8+ T cells from Pt#2 to Pt#5 producing IFN-γ determined by flow cytometric analysis are illustrated in Fig. 5B. Summary pooled data from all five of HIV-1 seropositive participants are shown in Fig. 5C. Medium alone and CD40LT were included as negative and positive controls, respectively. As reported previously [49], [55], CD40LT-stimulated MDDCs strongly enhanced HIV-1-specific memory CTL responses when compared with medium-treated iMDDCs. In comparison with medium alone, MDDCs infected with the bacterial strains all enhanced HIV-1-specific memory CTL responses to a similar degree, with the exception that the OpaCEA-positive strain N309 induced a relatively weak response. Highlighting the dramatic effect of CEACAM1-specific Opa protein expression, the IFN-γ response elicited by the N309-infected MDDCs often reflected that with the peptide-pulsed but otherwise untreated DCs (986 ± 235, n = 5 vs 553 ± 181, n = 5)(Fig. 5A, 5B, 5C). Thus, infection of OpaCEA-positive gonococcal strain of N309 effectively suppresses the ability of DCs to stimulate HIV-1-specific memory CTL responses. This effect of OpaCEA-positive gonococcal strain of N309 on DC effector function corresponds with its inhibitory effect on CD83 expression and the allogeneic response, suggesting a potent ability of N. gonorrhoeae to prevent the DCs from eliciting a T cell response.

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Figure 5. Effects of N. gonorrhoeae infection on HIV-1-specific CTL memory response.

PBMCs from HIV-1-seropositive individuals were cocultured with HLA-restricted peptide-pulsed or nonpulsed autologous MDDCs that were previously infected for 72 h with N. gonorrhoeae N302, N303, N309, or N496, or E.coli DH5α, or CD40LT treatment at 1 µg/ml. On day 10 of coculture, HIV-1-specific CTL activity was assayed by intracellular staining and flow cytometric analysis of IFN-γ-producing CD8+ T cells. A) representative intracellular IFN-γ data obtained from Pt#1 of HIV-1 seropositive individual are shown. Cells were gated for CD3 and CD8 to enumerate IFN-γ-producing CD8+ T cells only. B) Summary data of intracellular IFN-γ production in CD8+ T cells from Pt#2 – Pt#5 are graphically depicted. C) Summary data of IFN-γ-producing CD8+ T cells from all patients tested are graphically depicted. The experiments from Pt#1 and Pt#2 were repeated with similar results. Statistical comparisons of data pooled from five participants were performed between OpaCEA-expressing N309 and other gonococci strains, E.coli DH5α, or CD40LT treatment: N309 vs N302, p < 0.05; N309 vs N303, p < 0.05; N309 vs N496, p < 0.05; N309 vs E.coli, p < 0.05; N309 vs CD40LT, p < 0.001; DC = DC not pulsed with peptides; DCp = DC pulsed with peptides; CD40LT = CD40 ligand trimer.

doi:10.1371/journal.pone.0056705.g005

Discussion

The success of N. gonorrhoeae as a colonizer of humans stems largely from its ability to persist in core groups of sexually active individuals within the population [70]. The gonococci have an incredible capacity to vary surface antigens, yet this characteristic is not sufficient to explain the absence of protection afforded against reinfection with the same serovar or, in some instances, an apparently identical strain [71]. The gonococcal Opa proteins that bind human CEACAM1 have previously been shown to cause a marked suppression in CD4+ T cell responses to a variety of activating stimuli [37][39], an effect that could presumably affect development and/or persistence of an adaptive response. More recent evidence suggests that bacterial binding to CEACAM1 may also suppress TLR2 (toll-like receptor 2)-mediated innate responses from infected epithelial cells [72], implying that Opa-CEACAM1 may facilitate both early colonization and longer term persistence within an infected individual.

Our current studies demonstrate that human iMDDCs are intrinsically better at binding N. gonorrhoeae than they are at adhering to E. coli, which was used as a prototypical Gram negative bacteria with its spectrum of innate immune agonists, throughout our studies. While gonococci that lack both the Opa and pilus adhesins can associate with the DCs, expression of one or more adhesins clearly facilitates this interaction. Despite this fact, iMDDCs exposed to either the E. coli or N. gonorrhoeae strains tended to show similar gross morphological changes and alteration in surface antigen expression typical of phenotypic maturation initiated in response to purified microbial-associated molecular pattern (MAMP)-containing molecules such as lipopolysaccharide (LPS) or the potent DC agonist CD40LT. In each case, the maturing DCs showed a similar pattern of upregulating their expression of the surface antigens, CD80, CD86, and HLA-DR, and down-regulated expression of CD1a. Strikingly, however, DCs that were infected with the OpaCEA-expressing strain of N. gonorrhoeae did not display any CD83, a commonly used marker of DC maturation and one that was apparent upon exposure to the other stimuli, including the isogenic gonococcal variants that lacked OpaCEA. The affect of OpaCEA expression had functional consequences, since the DCs that matured in response to OpaCEA-expressing gonococci showed significant defects in their ability to elicit an allogeneic response against non-self leukocytes and in their ability to drive a peptide epitope-driven HIV-1-specific CD8+ T cell response against recall antigens.

A causal link for the absence of CD83 expression and defect in T cell stimulatory capacity of the OpaCEA-primed DCs is consistent with the recent demonstration that the specific siRNA-mediated knockdown of CD83 had a marked effect on their ability to stimulate T cell responses [69]. Indeed, even incomplete (~60%) reduction in CD83 protein was sufficient to cause a significant reduction in T cell proliferation in an allogeneic mixed lymphocyte reaction, reflecting the effect seen with the CD83-negative but otherwise mature MDDCs arising following infection with the gonococci expressing OpaCEA. Curiously, several viruses, including herpes simplex virus type 1 (HSV-1) [73][75], human cytomegalovirus (HCMV) [76], HIV-1 [77] and vaccinia virus [78], have been shown to interfere with CD83 expression on the surface of infected DCs and thereby prevent DC-mediated activation and proliferation of T cells [79]. These viruses down-regulate CD83 expression through different mechanisms. For example, HCMV causes CD83 shedding from the surface of mature DCs [76] and the released soluble CD83 competes with membrane-associated CD83 to block DC-T-cell clustering [80], while HSV-1 strongly induces CD83 protein degradation in mature DCs via a process thought to be proteasome-mediated [74], [75]. HSV-1 infection also suppresses de novo expression of CD83 through degradation of cellular mRNA during DC maturation and blocks CD83 mRNA export from the nucleus into the cytoplasm [73], implying the importance of knocking out this protein for viral infection. Similar to the OpaCEA-expressing strain of N. gonorrhoeae, HSV-1 specifically diminishes CD83 without affecting other co-stimulatory molecules, leaving CD80 and CD86 on the surface of mature DCs [74], [75]. The molecular mechanisms by which these co-stimulatory molecules can be differentially regulated by DCs have not been studied. It has, however, been shown that CD83 expression is regulated at the posttranscriptional level by interaction of the shuttle protein HuR with the posttranscriptional regulatory RNA element (PRE) that is located in the coding region of the CD83 transcript [81]. HuR binds in a specific manner to the CD83 PRE region, and this interaction results in significant stabilization of the otherwise highly labile CD83 mRNAs [81]. As a consequence, HuR-CD83 mRNA binding ensures the timely and efficient nuclear export, and thereby protein expression, of CD83 transcripts [81]. When considered together with the N. gonorrhoeae-dependent effects shown herein, it is enticing to consider that CD83 downregulation must be a particularly effective mechanism for both viral and bacterial pathogens to disengage DCs and thereby prevent an effective immune response.

While N. gonorrhoeae can randomly phase vary expression of its eleven Opa genes on and off, Opa-negative variants are rarely found in clinical samples obtained from men [82][84] or women [85]; the only exception to this rule is during menstruation, when transparent (Opa-negative) colonies predominate [85]. The Opa-CEACAM1 dependent effects on DC and T cell effector function would presumably contribute to the absence of effective adaptive memory responses in response to gonorrhea, impacting CD4+ T cells’ role in the adaptive response and CD8+ T cell-mediated killing of N. gonorrhoeae infected cells. Either effect would allow gonococcal persistence in high-risk core groups of sexually active men and women. It may also contribute to the heightened transmission of HIV-1 apparent in co-infected individuals. Indeed, the relationship between these pathogens appears to occur at multiple levels since in vitro studies indicate that N. gonorrhoeae can dramatically enhance HIV-1 replication in humans CD4+ T cells [86] and DCs [56]. Moreover, gonococcal infection of genital epithelial cells promotes their release of pro-inflammatory cytokines that drive further HIV-1 expression [87]. When combined with an ability to suppress DC functions that are known to contribute to HIV-1 immunity, shown herein, and the direct suppression of T cell activation shown previously [37][39], this sets the stage for a dangerous liaison between two major sexually transmitted pathogens.

Acknowledgments

This work was supported in part by the Grand Challenges Explorations (GCE) Phase II grant through the Bill & Melinda Gates Foundation (OPP1035237 to QY), Showalter Research Trust Fund (QY), NIH T32 AI060519 (DB), the Research Facilities Improvement Program Grant Number C06 RR015481-01 from the National Center for Research Resources, NIH to Indiana University School of Medicine, the Canadian Institutes for Health Research grants MOP-15499 (SDG) and HET-85518 (SDG and MAO), and support from the Ontario HIV Treatment Network (SDG and MAO).

Author Contributions

Conceived and designed the experiments: QY MAO SDG. Performed the experiments: QY EC SM NH. Analyzed the data: QY EC SM NH DB TA SH MAO SDG. Contributed reagents/materials/analysis tools: QY MAO SDG. Wrote the paper: QY MAO SDG.

References

  1. 1. Merz AJ, So M, Sheetz MP (2000) Pilus retraction powers bacterial twitching motility. Nature 407: 98–102. doi: 10.1038/35024105
  2. 2. Nassif X, So M (1995) Interaction of pathogenic neisseriae with nonphagocytic cells. Clin Microbiol Rev 8: 376–388.
  3. 3. Dehio C, Gray-Owen SD, Meyer TF (1998) The role of neisserial Opa proteins in interactions with host cells. Trends Microbiol 6: 489–495. doi: 10.1016/s0966-842x(98)01365-1
  4. 4. Sadarangani M, Pollard AJ, Gray-Owen SD (2011) Opa proteins and CEACAMs: pathways of immune engagement for pathogenic Neisseria. FEMS Microbiol Rev 35: 498–514. doi: 10.1111/j.1574-6976.2010.00260.x
  5. 5. Bhat KS, Gibbs CP, Barrera O, Morrison SG, Jahnig F, et al. (1992) The opacity proteins of Neisseria gonorrhoeae strain MS11 are encoded by a family of 11 complete genes. Mol Microbiol 6: 1073–1076. doi: 10.1111/j.1365-2958.1992.tb02172.x
  6. 6. Murphy GL, Connell TD, Barritt DS, Koomey M, Cannon JG (1989) Phase variation of gonococcal protein II: regulation of gene expression by slipped-strand mispairing of a repetitive DNA sequence. Cell 56: 539–547. doi: 10.1016/0092-8674(89)90577-1
  7. 7. van Putten JP, Paul SM (1995) Binding of syndecan-like cell surface proteoglycan receptors is required for Neisseria gonorrhoeae entry into human mucosal cells. EMBO J 14: 2144–2154.
  8. 8. Chen T, Belland RJ, Wilson J, Swanson J (1995) Adherence of pilus- Opa+ gonococci to epithelial cells in vitro involves heparan sulfate. J Exp Med 182: 511–517. doi: 10.1084/jem.182.2.511
  9. 9. Freissler E, Meyer auf der Heyde A, David G, Meyer TF, Dehio C (2000) Syndecan-1 and syndecan-4 can mediate the invasion of OpaHSPG-expressing Neisseria gonorrhoeae into epithelial cells. Cell Microbiol 2: 69–82. doi: 10.1046/j.1462-5822.2000.00036.x
  10. 10. Virji M, Makepeace K, Ferguson DJ, Watt SM (1996) Carcinoembryonic antigens (CD66) on epithelial cells and neutrophils are receptors for Opa proteins of pathogenic neisseriae. Mol Microbiol 22: 941–950. doi: 10.1046/j.1365-2958.1996.01551.x
  11. 11. Virji M, Watt SM, Barker S, Makepeace K, Doyonnas R (1996) The N-domain of the human CD66a adhesion molecule is a target for Opa proteins of Neisseria meningitidis and Neisseria gonorrhoeae. Mol Microbiol 22: 929–939. doi: 10.1046/j.1365-2958.1996.01548.x
  12. 12. Chen T, Gotschlich EC (1996) CGM1a antigen of neutrophils, a receptor of gonococcal opacity proteins. Proc Natl Acad Sci U S A 93: 14851–14856. doi: 10.1073/pnas.93.25.14851
  13. 13. Chen T, Grunert F, Medina-Marino A, Gotschlich EC (1997) Several carcinoembryonic antigens (CD66) serve as receptors for gonococcal opacity proteins. J Exp Med 185: 1557–1564. doi: 10.1084/jem.185.9.1557
  14. 14. Gray-Owen SD, Dehio C, Haude A, Grunert F, Meyer TF (1997) CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria gonorrhoeae and human polymorphonuclear phagocytes. EMBO J 16: 3435–3445. doi: 10.1093/emboj/16.12.3435
  15. 15. Bos MP, Grunert F, Belland RJ (1997) Differential recognition of members of the carcinoembryonic antigen family by Opa variants of Neisseria gonorrhoeae. Infect Immun 65: 2353–2361.
  16. 16. Billker O, Popp A, Gray-Owen SD, Meyer TF (2000) The structural basis of CEACAM-receptor targeting by neisserial Opa proteins. Trends Microbiol 8: : 258–260; discussion 260–251.
  17. 17. Gray-Owen SD, Blumberg RS (2006) CEACAM1: contact-dependent control of immunity. Nat Rev Immunol 6: 433–446. doi: 10.1038/nri1864
  18. 18. Kuespert K, Pils S, Hauck CR (2006) CEACAMs: their role in physiology and pathophysiology. Curr Opin Cell Biol 18: 565–571. doi: 10.1016/j.ceb.2006.08.008
  19. 19. Beauchemin N, Draber P, Dveksler G, Gold P, Gray-Owen S, et al. (1999) Redefined nomenclature for members of the carcinoembryonic antigen family. Exp Cell Res 252: 243–249. doi: 10.1006/excr.1999.4610
  20. 20. Hammarstrom S (1999) The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol 9: 67–81. doi: 10.1006/scbi.1998.0119
  21. 21. Muenzner P, Naumann M, Meyer TF, Gray-Owen SD (2001) Pathogenic Neisseria trigger expression of their carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1; previously CD66a) receptor on primary endothelial cells by activating the immediate early response transcription factor, nuclear factor-kappaB. J Biol Chem 276: 24331–24340. doi: 10.1074/jbc.m006883200
  22. 22. Botling J, Oberg F, Nilsson K (1995) CD49f (alpha 6 integrin) and CD66a (BGP) are specifically induced by retinoids during human monocytic differentiation. Leukemia 9: 2034–2041.
  23. 23. Bamberger AM, Briese J, Gotze J, Erdmann I, Schulte HM, et al. (2006) Stimulation of CEACAM1 expression by 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore A23187 in endometrial carcinoma cells. Carcinogenesis 27: 483–490. doi: 10.1093/carcin/bgi275
  24. 24. Chen T, Zimmermann W, Parker J, Chen I, Maeda A, et al. (2001) Biliary glycoprotein (BGPa, CD66a, CEACAM1) mediates inhibitory signals. J Leukoc Biol 70: 335–340.
  25. 25. Kammerer R, Stober D, Singer BB, Obrink B, Reimann J (2001) Carcinoembryonic antigen-related cell adhesion molecule 1 on murine dendritic cells is a potent regulator of T cell stimulation. J Immunol 166: 6537–6544.
  26. 26. Obrink B (1997) CEA adhesion molecules: multifunctional proteins with signal-regulatory properties. Curr Opin Cell Biol 9: 616–626. doi: 10.1016/s0955-0674(97)80114-7
  27. 27. Gonorrhea rates rising among hardest-hit. AIDS Alert 17: 63–66.
  28. 28. Levine D, McCright J, Dobkin L, Woodruff AJ, Klausner JD (2008) SEXINFO: a sexual health text messaging service for San Francisco youth. Am J Public Health 98: 393–395. doi: 10.2105/ajph.2007.110767
  29. 29. Ciesielski CA (2003) Sexually Transmitted Diseases in Men Who Have Sex with Men: An Epidemiologic Review. Curr Infect Dis Rep 5: 145–152. doi: 10.1007/s11908-003-0051-5
  30. 30. Kaul R, Rowland-Jones SL, Kimani J, Dong T, Yang HB, et al. (2001) Late seroconversion in HIV-resistant Nairobi prostitutes despite pre-existing HIV-specific CD8+ responses. J Clin Invest 107: 341–349. doi: 10.1172/jci10714
  31. 31. Anzala AO, Simonsen JN, Kimani J, Ball TB, Nagelkerke NJ, et al. (2000) Acute sexually transmitted infections increase human immunodeficiency virus type 1 plasma viremia, increase plasma type 2 cytokines, and decrease CD4 cell counts. J Infect Dis 182: 459–466. doi: 10.1086/315733
  32. 32. Winter AJ, Taylor S, Workman J, White D, Ross JD, et al. (1999) Asymptomatic urethritis and detection of HIV-1 RNA in seminal plasma. Sex Transm Infect 75: 261–263. doi: 10.1136/sti.75.4.261
  33. 33. Cohen MS, Hoffman IF, Royce RA, Kazembe P, Dyer JR, et al. (1997) Reduction of concentration of HIV-1 in semen after treatment of urethritis: implications for prevention of sexual transmission of HIV-1. AIDSCAP Malawi Research Group. Lancet 349: 1868–1873. doi: 10.1016/s0140-6736(97)02190-9
  34. 34. Plummer FA, Chubb H, Simonsen JN, Bosire M, Slaney L, et al. (1994) Antibodies to opacity proteins (Opa) correlate with a reduced risk of gonococcal salpingitis. J Clin Invest 93: 1748–1755. doi: 10.1172/jci117159
  35. 35. Fox KK, Thomas JC, Weiner DH, Davis RH, Sparling PF, et al. (1999) Longitudinal evaluation of serovar-specific immunity to Neisseria gonorrhoeae. Am J Epidemiol 149: 353–358. doi: 10.1093/oxfordjournals.aje.a009820
  36. 36. Hobbs MM, Alcorn TM, Davis RH, Fischer W, Thomas JC, et al. (1999) Molecular typing of Neisseria gonorrhoeae causing repeated infections: evolution of porin during passage within a community. J Infect Dis 179: 371–381. doi: 10.1086/314608
  37. 37. Boulton IC, Gray-Owen SD (2002) Neisserial binding to CEACAM1 arrests the activation and proliferation of CD4+ T lymphocytes. Nat Immunol 3: 229–236. doi: 10.1038/ni769
  38. 38. Lee HS, Boulton IC, Reddin K, Wong H, Halliwell D, et al. (2007) Neisserial outer membrane vesicles bind the coinhibitory receptor carcinoembryonic antigen-related cellular adhesion molecule 1 and suppress CD4+ T lymphocyte function. Infect Immun 75: 4449–4455. doi: 10.1128/iai.00222-07
  39. 39. Lee HS, Ostrowski MA, Gray-Owen SD (2008) CEACAM1 dynamics during neisseria gonorrhoeae suppression of CD4+ T lymphocyte activation. J Immunol 180: 6827–6835. doi: 10.4049/jimmunol.180.10.6827
  40. 40. Markel G, Wolf D, Hanna J, Gazit R, Goldman-Wohl D, et al. (2002) Pivotal role of CEACAM1 protein in the inhibition of activated decidual lymphocyte functions. J Clin Invest 110: 943–953. doi: 10.1172/jci0215643
  41. 41. Stern N, Markel G, Arnon TI, Gruda R, Wong H, et al. (2005) Carcinoembryonic antigen (CEA) inhibits NK killing via interaction with CEA-related cell adhesion molecule 1. J Immunol 174: 6692–6701.
  42. 42. Donda A, Mori L, Shamshiev A, Carena I, Mottet C, et al. (2000) Locally inducible CD66a (CEACAM1) as an amplifier of the human intestinal T cell response. Eur J Immunol 30: 2593–2603. doi: 10.1002/1521-4141(200009)30:9<2593::aid-immu2593>3.0.co;2-0
  43. 43. Kammerer R, Hahn S, Singer BB, Luo JS, von Kleist S (1998) Biliary glycoprotein (CD66a), a cell adhesion molecule of the immunoglobulin superfamily, on human lymphocytes: structure, expression and involvement in T cell activation. Eur J Immunol 28: 3664–3674. doi: 10.1002/(sici)1521-4141(199811)28:11<3664::aid-immu3664>3.0.co;2-d
  44. 44. Greicius G, Severinson E, Beauchemin N, Obrink B, Singer BB (2003) CEACAM1 is a potent regulator of B cell receptor complex-induced activation. J Leukoc Biol 74: 126–134. doi: 10.1189/jlb.1202594
  45. 45. Granucci F, Ricciardi-Castagnoli P (2003) Interactions of bacterial pathogens with dendritic cells during invasion of mucosal surfaces. Curr Opin Microbiol 6: 72–76. doi: 10.1016/s1369-5274(03)00007-9
  46. 46. Kupsch EM, Knepper B, Kuroki T, Heuer I, Meyer TF (1993) Variable opacity (Opa) outer membrane proteins account for the cell tropisms displayed by Neisseria gonorrhoeae for human leukocytes and epithelial cells. EMBO J 12: 641–650.
  47. 47. Achtman M, Neibert M, Crowe BA, Strittmatter W, Kusecek B, et al. (1988) Purification and characterization of eight class 5 outer membrane protein variants from a clone of Neisseria meningitidis serogroup A. J Exp Med 168: 507–525. doi: 10.1084/jem.168.2.507
  48. 48. Ostrowski MA, Justement SJ, Ehler L, Mizell SB, Lui S, et al. (2000) The role of CD4+ T cell help and CD40 ligand in the in vitro expansion of HIV-1-specific memory cytotoxic CD8+ T cell responses. J Immunol 165: 6133–6141.
  49. 49. Yu Q, Gu JX, Kovacs C, Freedman J, Thomas EK, et al. (2003) Cooperation of TNF family members CD40 ligand, receptor activator of NF-kappa B ligand, and TNF-alpha in the activation of dendritic cells and the expansion of viral specific CD8+ T cell memory responses in HIV-1-infected and HIV-1-uninfected individuals. J Immunol 170: 1797–1805.
  50. 50. Yu Q, Yue FY, Gu XX, Schwartz H, Kovacs CM, et al. (2006) OX40 ligation of CD4+ T cells enhances virus-specific CD8+ T cell memory responses independently of IL-2 and CD4+ T regulatory cell inhibition. J Immunol 176: 2486–2495.
  51. 51. McCaw SE, Liao EH, Gray-Owen SD (2004) Engulfment of Neisseria gonorrhoeae: revealing distinct processes of bacterial entry by individual carcinoembryonic antigen-related cellular adhesion molecule family receptors. Infect Immun 72: 2742–2752. doi: 10.1128/iai.72.5.2742-2752.2004
  52. 52. Yu Q, Chow EM, Wong H, Gu J, Mandelboim O, et al. (2006) CEACAM1 (CD66a) promotes human monocyte survival via a phosphatidylinositol 3-kinase- and AKT-dependent pathway. J Biol Chem 281: 39179–39193. doi: 10.1074/jbc.m608864200
  53. 53. Tailleux L, Schwartz O, Herrmann JL, Pivert E, Jackson M, et al. (2003) DC-SIGN is the major Mycobacterium tuberculosis receptor on human dendritic cells. J Exp Med 197: 121–127. doi: 10.1084/jem.20021468
  54. 54. Geijtenbeek TB, Van Vliet SJ, Koppel EA, Sanchez-Hernandez M, Vandenbroucke-Grauls CM, et al. (2003) Mycobacteria target DC-SIGN to suppress dendritic cell function. J Exp Med 197: 7–17. doi: 10.1084/jem.20021229
  55. 55. Yu Q, Kovacs C, Yue FY, Ostrowski MA (2004) The role of the p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and phosphoinositide-3-OH kinase signal transduction pathways in CD40 ligand-induced dendritic cell activation and expansion of virus-specific CD8+ T cell memory responses. J Immunol 172: 6047–6056.
  56. 56. Zhang J, Li G, Bafica A, Pantelic M, Zhang P, et al. (2005) Neisseria gonorrhoeae enhances infection of dendritic cells by HIV type 1. J Immunol 174: 7995–8002.
  57. 57. Zhang P, Schwartz O, Pantelic M, Li G, Knazze Q, et al. (2006) DC-SIGN (CD209) recognition of Neisseria gonorrhoeae is circumvented by lipooligosaccharide variation. J Leukoc Biol 79: 731–738. doi: 10.1189/jlb.0405184
  58. 58. Geijtenbeek TB, van Kooyk Y (2003) Pathogens target DC-SIGN to influence their fate DC-SIGN functions as a pathogen receptor with broad specificity. APMIS 111: 698–714. doi: 10.1034/j.1600-0463.2003.11107803.x
  59. 59. Kaufmann SH, Schaible UE (2003) A dangerous liaison between two major killers: Mycobacterium tuberculosis and HIV target dendritic cells through DC-SIGN. J Exp Med 197: 1–5. doi: 10.1084/jem.20021964
  60. 60. Geijtenbeek TB, van Vliet SJ, van Duijnhoven GC, Figdor CG, van Kooyk Y (2001) DC-SIGN, a dentritic cell-specific HIV-1 receptor present in placenta that infects T cells in trans-a review. Placenta 22 Suppl A: S19–23.
  61. 61. Alvarez CP, Lasala F, Carrillo J, Muniz O, Corbi AL, et al. (2002) C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans. J Virol 76: 6841–6844. doi: 10.1128/jvi.76.13.6841-6844.2002
  62. 62. Pohlmann S, Zhang J, Baribaud F, Chen Z, Leslie GJ, et al. (2003) Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR. J Virol 77: 4070–4080. doi: 10.1128/jvi.77.7.4070-4080.2003
  63. 63. Lozach PY, Lortat-Jacob H, de Lacroix de Lavalette A, Staropoli I, Foung S, et al. (2003) DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2. J Biol Chem 278: 20358–20366. doi: 10.1074/jbc.m301284200
  64. 64. Tassaneetrithep B, Burgess TH, Granelli-Piperno A, Trumpfheller C, Finke J, et al. (2003) DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J Exp Med 197: 823–829. doi: 10.1084/jem.20021840
  65. 65. Colmenares M, Puig-Kroger A, Pello OM, Corbi AL, Rivas L (2002) Dendritic cell (DC)-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN, CD209), a C-type surface lectin in human DCs, is a receptor for Leishmania amastigotes. J Biol Chem 277: 36766–36769. doi: 10.1074/jbc.m205270200
  66. 66. Cambi A, Gijzen K, de Vries JM, Torensma R, Joosten B, et al. (2003) The C-type lectin DC-SIGN (CD209) is an antigen-uptake receptor for Candida albicans on dendritic cells. Eur J Immunol 33: 532–538. doi: 10.1002/immu.200310029
  67. 67. Feinberg H, Mitchell DA, Drickamer K, Weis WI (2001) Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR. Science 294: 2163–2166. doi: 10.1126/science.1066371
  68. 68. Cella M, Scheidegger D, Palmer-Lehmann K, Lane P, Lanzavecchia A, et al. (1996) Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J Exp Med 184: 747–752. doi: 10.1084/jem.184.2.747
  69. 69. Prechtel AT, Turza NM, Theodoridis AA, Steinkasserer A (2007) CD83 knockdown in monocyte-derived dendritic cells by small interfering RNA leads to a diminished T cell stimulation. J Immunol 178: 5454–5464. doi: 10.4049/jimmunol.178.9.5454
  70. 70. Plummer FA, Brunham RC (1987) Gonococcal recidivism, diversity, and ecology. Rev Infect Dis 9: 846–850. doi: 10.1093/clinids/9.4.846
  71. 71. Plummer FA, Simonsen JN, Chubb H, Slaney L, Kimata J, et al. (1989) Epidemiologic evidence for the development of serovar-specific immunity after gonococcal infection. J Clin Invest 83: 1472–1476. doi: 10.1172/jci114040
  72. 72. Slevogt H, Zabel S, Opitz B, Hocke A, Eitel J, et al. (2008) CEACAM1 inhibits Toll-like receptor 2-triggered antibacterial responses of human pulmonary epithelial cells. Nat Immunol 9: 1270–1278. doi: 10.1038/ni.1661
  73. 73. Salio M, Cella M, Suter M, Lanzavecchia A (1999) Inhibition of dendritic cell maturation by herpes simplex virus. Eur J Immunol 29: 3245–3253. doi: 10.1002/(sici)1521-4141(199910)29:10<3245::aid-immu3245>3.0.co;2-x
  74. 74. Kruse M, Rosorius O, Kratzer F, Stelz G, Kuhnt C, et al. (2000) Mature dendritic cells infected with herpes simplex virus type 1 exhibit inhibited T-cell stimulatory capacity. J Virol 74: 7127–7136. doi: 10.1128/jvi.74.15.7127-7136.2000
  75. 75. Kummer M, Turza NM, Muhl-Zurbes P, Lechmann M, Boutell C, et al. (2007) Herpes simplex virus type 1 induces CD83 degradation in mature dendritic cells with immediate-early kinetics via the cellular proteasome. J Virol 81: 6326–6338. doi: 10.1128/jvi.02327-06
  76. 76. Senechal B, Boruchov AM, Reagan JL, Hart DN, Young JW (2004) Infection of mature monocyte-derived dendritic cells with human cytomegalovirus inhibits stimulation of T-cell proliferation via the release of soluble CD83. Blood 103: 4207–4215. doi: 10.1182/blood-2003-12-4350
  77. 77. Knight SC, Patterson S (1997) Bone marrow-derived dendritic cells, infection with human immunodeficiency virus, and immunopathology. Annu Rev Immunol 15: 593–615. doi: 10.1146/annurev.immunol.15.1.593
  78. 78. Engelmayer J, Larsson M, Subklewe M, Chahroudi A, Cox WI, et al. (1999) Vaccinia virus inhibits the maturation of human dendritic cells: a novel mechanism of immune evasion. J Immunol 163: 6762–6768.
  79. 79. Lechmann M, Kotzor N, Zinser E, Prechtel AT, Sticht H, et al. (2005) CD83 is a dimer: Comparative analysis of monomeric and dimeric isoforms. Biochem Biophys Res Commun 329: 132–139. doi: 10.1016/j.bbrc.2005.01.114
  80. 80. Lechmann M, Krooshoop DJ, Dudziak D, Kremmer E, Kuhnt C, et al. (2001) The extracellular domain of CD83 inhibits dendritic cell-mediated T cell stimulation and binds to a ligand on dendritic cells. J Exp Med 194: 1813–1821. doi: 10.1084/jem.194.12.1813
  81. 81. Pieper D, Schirmer S, Prechtel AT, Kehlenbach RH, Hauber J, et al. (2011) Functional characterization of the HuR:CD83 mRNA interaction. PLoS One 6: e23290. doi: 10.1371/journal.pone.0023290
  82. 82. Swanson J, Barrera O, Sola J, Boslego J (1988) Expression of outer membrane protein II by gonococci in experimental gonorrhea. J Exp Med 168: 2121–2129. doi: 10.1084/jem.168.6.2121
  83. 83. Jerse AE, Cohen MS, Drown PM, Whicker LG, Isbey SF, et al. (1994) Multiple gonococcal opacity proteins are expressed during experimental urethral infection in the male. J Exp Med 179: 911–920. doi: 10.1084/jem.179.3.911
  84. 84. Schmidt KA, Deal CD, Kwan M, Thattassery E, Schneider H (2000) Neisseria gonorrhoeae MS11mkC opacity protein expression in vitro and during human volunteer infectivity studies. Sex Transm Dis 27: 278–283. doi: 10.1097/00007435-200005000-00008
  85. 85. James JF, Swanson J (1978) Studies on gonococcus infection. XIII. Occurrence of color/opacity colonial variants in clinical cultures. Infect Immun 19: 332–340.
  86. 86. Chen A, Boulton IC, Pongoski J, Cochrane A, Gray-Owen SD (2003) Induction of HIV-1 long terminal repeat-mediated transcription by Neisseria gonorrhoeae. AIDS 17: 625–628. doi: 10.1097/00002030-200303070-00019
  87. 87. Ferreira VH, Nazli A, Khan G, Mian MF, Ashkar AA, et al. (2011) Endometrial epithelial cell responses to coinfecting viral and bacterial pathogens in the genital tract can activate the HIV-1 LTR in an NF{kappa}B-and AP-1-dependent manner. J Infect Dis 204: 299–308. doi: 10.1093/infdis/jir260