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Keywords:

  • Chlamydia trachomatis;
  • chlamydial infectious load;
  • cytokines;
  • flow cytometry;
  • plasmacytoid and myeloid dendritic cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

The mobilization of myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) to the cervix during chlamydial infection is not fully understood, and the role of these cells in immunopathogenesis is largely unknown. As an effective vaccine to control chlamydial infection is currently unavailable, understanding the regulation of the local immune response becomes a necessity. Therefore, mDC and pDC populations were analysed in peripheral blood and cervical samples of controls and Chlamydia-positive women, with or without mucopurulent cervicitis (MPC). Cervical cytokines and C-reactive protein levels in serum were quantified by ELISA and the chlamydial infectious load by culture. Chlamydia trachomatis infection mobilized both mDCs and pDCs to the cervical mucosa. pDCs were recruited more often in women with MPC (p <0.05) and they correlated significantly with the chlamydial load, C-reactive protein levels and cervical interleukin-8 (IL-8) levels. Upregulation of surface expression of co-stimulatory molecules (CD80, CD83 and CD86) on cervical mDCs and pDCs was observed during chlamydial infection but was significant only for mDCs. Significantly higher levels of IL-1β, IL-6 and IL-8 were observed in Chlamydia-positive women with MPC; however, after therapy, IL-8 levels decreased significantly. Median numbers of mDCs after therapy were significantly higher in the cervix and blood of infected women as compared to the numbers of pDCs, which were found to be lower in the cervix after therapy. These results thus suggest that during chlamydial infection, both mDCs and pDCs are recruited to the cervix, but their number and possible immunological functions may differ with the pathological condition. pDCs were associated more often with MPC and inflammatory factors, suggesting that they may possibly be involved in the immunopathogenesis of infections due to Chlamydia.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

As an obligate intracellular bacterial pathogen, Chlamydia trachomatis causes many important human diseases worldwide [1,2]. In India, a high prevalence rate of genital chlamydial infection, up to 40%, has been reported among symptomatic women [3–5]. As the most prevalent sexually transmitted bacterium, C. trachomatis causes urethritis, cervicitis, salpingitis and reproductive disorders, with sequelae often resulting from scarring of the affected genital tissues [6]. The exact pathological mechanism by which C. trachomatis induces scarring is still not well understood.

The mucosal surface of the urogenital tract provides a large site of entry for various pathogens, the immune response to which strongly depends on the presence of different infiltrating immune cells. During chlamydial infection [7], T-cell-mediated adaptive immune responses play a major role in the clearance and resolution of infection.

Dendritic cells (DCs) are potent antigen-presenting cells, playing a crucial role in the initiation and maintenance of T-cell immunity [8]. Besides contributing to adaptive resistance against microbial pathogens [9], DCs have also been reported to be involved in chronic inflammation [10]. In peripheral blood, two major subsets of DCs are present, the CD123+ plasmacytoid DCs (pDCs) and the CD11c+ myeloid DCs (mDCs). Both of these subsets express high levels of HLA-DR and lack the lineage markers CD3 [11], CD14, CD19, CD20, CD16 and CD56 [12]. In humans, pDCs and mDCs have been shown to be associated with various pathological and disease conditions, e.g. viral infections [13–16], bacterial infections [17], parasitic infections [18], fungal infections [19], cancerous conditions [20–23], systemic lupus erythematosus [24], rheumatoid arthritis [25], coronary artery disease [26] and inflammatory skin disease [27].

The cervical mucosa is also reported to contain numerous DCs, interdigitating with the epithelial cells [28] and these DCs are often CD1a-positive [29]. A previous study by Bontkes et al. [30] found both pDCs and mDCs in the female cervix during cervical carcinoma, but no information is available concerning the mobilization of these two subsets to the cervix during bacterial infections. We have previously shown the enhancement in CD4+ T-cells, CD8+ T-cells and dendritic cellular phenotypes in cervical samples from Chlamydia-positive women [31], but the recruitment of these two DC subsets to the cervical mucosa during C. trachomatis infection was not studied.

Therefore, in the present study, the mDC and pDC populations in peripheral blood and cervical mucosa of healthy controls and Chlamydia-positive women, with or without mucopurulent cervicitis (MPC), were analysed to determine their role in inflammation. The populations of these subsets were correlated with the chlamydial load, C-reactive protein (CRP) levels and cytokine levels. The populations of these subsets were further studied in Chlamydia-positive women after full antibiotic therapy, in order to determine their role in the pathogenesis of chlamydial infection.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Study population

Between December 2005 and February 2007, 128 asymptomatic women attending the family planning clinic and 69 women with signs and symptoms of cervicitis (mucopurulent discharge) attending the Gynecology Outpatient Department of Safdarjung Hospital, New Delhi, India were enrolled for the study. Informed written consent was obtained from all the participants. Women with a positive urine pregnancy test result and those with a recent history of antibiotic therapy or any history of previously treated sexually transmitted infection were excluded from the study. All women underwent general physical and speculum examination. As variations in sex hormones are known to influence cytokine concentrations and immune cell populations, including the mDCs and pDCs [32], cervical samples were collected during the mid-menstrual cycle (median 13 days, range 9th to 15th day of the cycle). The study received approval from the hospital’s ethics review committee.

Collection of samples

The vulva was examined for lesions, and the cervix for warts, ulcers, ectopy, erythema or discharge, to determine whether confounding venereal infections were present. After cleaning of the cervix, cervical swabs and vaginal swabs (HiMedia, Mumbai, India) were collected for diagnosis of C. trachomatis and other sexually transmitted pathogens. For collection of cervical cells, a cytobrush was rotated within the cervical canal and then transferred to a sterile centrifuge tube with phosphate-buffered saline (PBS) (pH 7.2), supplemented with 100 U penicillin/mL, 100 μg streptomycin/mL and 100 μg glutamine/mL. All cytobrush samples yielded negative results for blood contamination. Cervical washes in 5 mL of sterile saline and heparinized peripheral venous blood (5 mL) were also collected.

Microbiology

The presence of chlamydiae in samples was confirmed using direct fluorescenceanalysis with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAbs) against the C. trachomatis major outer membrane protein (Microtrak, Palo Alto, USA). A sample was considered to be positive when five to ten elementary bodies were detected. Negative samples were further confirmed by PCR analysis using a primer specific for the 517-bp plasmid of C. trachomatis [33]. Gram-stained cervical smears were examined for the presence of yeast cells (candidiasis), and quantification of polymorphonuclear leukocytes (PMNLs) per high-power field was performed using smears to ensure the presence of cervicitis. Vaginal smears were analysed for clue cells for diagnosis of bacterial vaginosis. Wet mount microscopy was performed for the diagnosis of Trichomonas vaginalis. Neisseria gonorrhoeae, Mycobacterium hominis and Ureaplasma urealyticum were detected by culture.

Isolation of cells from cervical samples

Cervical cells were isolated from the cytobrush by vigorously rotating it against the sides of the transport tube after incubating the sample with 5 mM dl-dithiothreitol (Sigma, St Louis, MO, USA) at 37°C for 15 min (to reduce the mucus component of the sample). The cell suspension obtained was then filtered through a sterile 70=μm nylon cell strainer (BD Biosciences, San Diego, CA, USA) and centrifuged at 300 g for 10 min; the resultant pellet yielded endocervical cells. The viability of cells was determined using a Trypan blue exclusion assay.

Antibodies

DCs were identified by multi-parametric flow cytometry with the following mAbs: FITC-conjugated lineage cocktail LIN-1 (containing anti-CD3, anti-CD14, anti-CD16, anti-CD19, anti-CD20 and anti-CD56), CD14–FITC, CD4–FITC, CD8–FITC, CD123–phycoerythrin (PE) and HLA-DR–peridin chlorophyll protein (BD Biosciences). In addition, allophycocyanin-labelled anti-CD11c was purchased from eBiosciences (San Diego, CA, USA). PE-labelled anti-CD80, CD83 and CD86 were obtained from BD Biosciences. To measure expression of CD80, CD83 and CD86 on CD123+ cells, anti-CD123–PE–Cy5 and HLA-DR–allophycocyanin were purchased from BD Biosciences.

For blood DCs, 100 μL of whole blood was incubated with an antibody cocktail for 20 min at room temperature. Erythrocytes were lysed with FACS Lysing Buffer (BD Biosciences), and cells were washed with PBS containing 0.1% (w/v) bovine serum albumin and 0.1% NaN3 to remove unbound mAb and resuspended in 1% (w/v) paraformaldehyde in PBS. In total, 100 000 cervical cells/tube were incubated with antibody cocktail for 25 min on ice and were washed and fixed following the protocol above. Cell preparations were labelled in parallel, and included all isotype control antibodies (BD Biosciences) appropriate for establishing the demarcation between negative and positive populations.

As previously described [29], cervical specimens exhibit a high level of granularity and autofluorescence, which could be attributed to many factors. Background fluorescence and the presence of lymphocytes was minimized by introduction of an acquisition gate on the forward-scatter vs. side-scatter profile, which included most of the mononuclear cell fraction and provided reliable differentiation of these cells from epithelial cells, lymphocytes and cell debris. Samples were acquired using a FACS Calibur Cytometer and analysed with Cell Quest software (Becton Dickinson, San Jose, CA, USA). The gating strategy used to identify and quantify LIN/DR+ cells is illustrated in Fig. 1.

image

Figure 1.  Quantification of dendritic cell (DC) subsets in cervical mucosa by flow cytometric analysis. (a) Introduction of an acquisition gate (R1) on the forward-scatter (FSC) vs. side-scatter (SSC) profile to select the mononuclear cell population. (b) Cervical DCs were identified within gate R2 as fluorescein isothiocyanate (FITC) cocktail (contains anti-CD3, CD14, CD16, CD19, CD20, and CD56)-negative and HLA-DR-positive. (c, d) Detection of HLA-DR+ CD11c+ myeloid DCs and HLA-DR+ CD123+ plasmacytoid DCs, respectively. PerCP, peridin chlorophyll protein; PE, phycoerythrin; APC, allophycocyanin.

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mDCs and pDCs in cervical secretions were measured as the number of events present per 100 000 cells taken for the experiment. This number was then adjusted according to the total number of cells obtained in that cervical sample. These were finally presented as the number of events per cervical sample. In the case of blood, the DC subsets were counted as events per 100 μL of blood taken, and were then calculated and represented as events per millilitre of blood.

Quantification of cytokines in cervical washes

Quantification of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, tumour necrosis factor (TNF)-α, interferon (IFN)-α and IFN-γ in cervical washes was performed with commercially available ELISA kits (eBiosciences, San Diego, USA and Diaclone, Cedex, France), in accordance with the manufacturer’s instructions.

Quantification of chlamydial infectious load in cervical samples

Chlamydial infectious load in cervical samples was determined as infection-forming units (IFUs)/mL, as described elsewhere [34].

Determination of CRP levels

CRP was determined using high-sensitivity hs-CRP ELISA (Calbiotech, Spring Valley, CA, USA), according to the manufacturer’s instructions, and CRP levels above 2 mg/L were considered to reveal a higher risk of chronic inflammation, according to the manufacturer’s instructions.

Statistical analysis

The Kruskal–Wallis non-parametric test was used to compare continuous variables among multiple groups. The non-parametric Mann–Whitney U-test was used to compare DC populations and cytokine concentrations. The Wilcoxon signed rank test was used to compare DC numbers before and after therapy. Correlation was determined with Spearman’s correlation coefficient.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Study population

Cervical C. trachomatis infection was diagnosed using direct fluorescence analysis in conjunction with PCR in 35 women attending the family planning clinic (asymptomatic) and in 31 women attending the Gynecology Outpatient Department (symptomatic). Fifteen Chlamydia-positive women, co-infected with Candida spp., T. vaginalis, M. hominis, U. urealyticum or N. gonorrhoeae, or having bacterial vaginosis, were excluded from the study. Three Chlamydia-positive patients were excluded, as the mononuclear cell count in the cervical cells was less than 106 cells/mL. On the basis of diagnosis, the women were divided into three groups. Group I (n = 28) comprised uninfected healthy controls selected from among women attending the family planning clinic; group II (n = 23) comprised asymptomatic Chlamydia-positive women without MPC (number of PMNLs <5); and group III (n = 25) comprised Chlamydia-positive women with MPC (number of PMNLs  >30) [35]. All women were age-matched, with no significant difference in their median ages.

Immune cell population in cervical mucosa

Flow cytometric analysis demonstrated the presence of mDCs, pDCs, CD14+ monocytes, CD3+CD4+ T-cells and CD3+CD8+ T-cells in both the Chlamydia-positive groups. There was a significant increase in the mean number of CD4+ T-lymphocytes per 10 000 events in the cervical mucosa of women with MPC, as compared to women without MPC and to controls (573 vs. 375 and 234 respectively; p <0.05). In contrast, in the cervical mucosa, the CD8+ T-cell population was found to be higher during chlamydial infection, but not significantly. The number of CD14+ monocytes per 10 000 events was significantly increased in women with MPC, as compared to women without MPC and to controls (474 vs. 259 and 138; p <0.05). These results are summarized in Fig. 2.

image

Figure 2.  Lymphocyte subsets (CD4+ and CD8+ T-cells) and monocytic cells in cervical mucosa of contols, Chlamydia trachomatis-positive women with mucopurulent cervicitis (MPC) and without MPC detected by flow cytometry and expressed as mean number of cells per 10 000 events on the y-axis.

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mDC population in cervical mucosa and peripheral blood

Three of 28 healthy controls, one of the 23 Chlamydia-positive women without MPC and two of the 25 Chlamydia-positive women with MPC did not have mDCs in their cervical samples. The median and range of absolute numbers of mDCs/cervical sample are given in Table 1. Healthy controls had a significantly lower number of mDCs/cervical sample than patients in the Chlamydia-positive groups (p <0.01). The frequency (%) of mDCs in cervical samples of patients (without MPC, median 0.074%, range 0–0.95%; with MPC, median 0.068%, range 0–0.91%) was comparable to that in blood samples of the same patients (without MPC, median 0.068%, range 0.02–0.32%; with MPC, median 0.064%, range 0.01–0.28%), although there was a significant (p <0.05) decrease in the absolute median numbers of mDCs/mL of blood in Chlamydia-infected women without MPC, as compared to controls. In cervical mDCs, significantly higher expression of CD83 and CD80 was observed in both Chlamydia-positive groups than in controls (p <0.05). Expression of CD86 was higher in both Chlamydia-positive groups, but the difference was significant (p <0.05) only in women with MPC (Table 2).

Table 1.   Absolute number of myeloid and plasmacytoid dendritic cells in cervical mucosa and blood samples
 Myeloid dendritic cellsPlasmacytoid dendritic cells
ControlCT-positiveControlCT-positive
Without MPCWith MPCWithout MPCWith MPC
  1. CT, Chlamydia trachomatis; MPC, mucopurulent cervicitis.

  2. The data represent median values for absolute numbers of myeloid and plasmacytoid dendritic cells (DCs) in cervical mucosa (DCs/cervical sample) and blood (DCs/mL blood). Ranges are given in parentheses..

  3. ap <0.05 of individual patient groups as compared to controls (Mann–Whitney U-test).

  4. bp <0.05 of the CT-positive group with MPC as compared to that without MCP (Mann–Whitney U-test).

Cervix36 (0–1256)1788 (0–40 170)a720 (0–10 444)a,b13 (0–1200)1750 (0–33 840)a3890 (1162–80 752)a
Blood17 572 (3254–55 718)1800 (458–13 786)a10 800 (1326–27 556)16 480 (3292–48 148)7216 (4637–15 430)a6240 (3341–22 382)a
Table 2.   Phenotypic characterization of dendritic cell subsets in cervical mucosa
Co-stimulatory moleculesPercentage expression on mDCsPercentage expression on pDCs
CT-positiveControlsCT-positiveControls
Without MPCWith MPCWithout MPCWith MPC
  1. CT, Chlamydia trachomatis; MPC, mucopurulent cervicitis; mDC, myeloid dendritic cell; pDC, plasmacytoid dendritic cell.

  2. The data represent co-stimulatory molecules on mDCs and pDCs in cervical mucosa.

  3. ap <0.05 of individual patient groups as compared to controls (Mann–Whitney U-test).

  4. bp <0.05 of the CT-positive group with MPC as compared to controls (Mann Whitney U-test).

CD8016.0 ± 2.8a26.0 ± 3.4a02.0 ± 0.703.9 ± 2.406.3 ± 3.0b02.5 ± 1.3
CD8324.0 ± 3.6a30.0 ± 4.2a11.0 ± 0.604.7 ± 0.907.1 ± 2.603.3 ± 1.2
CD8653.0 ± 4.265.0 ± 7.2b32.0 ± 5.102.2 ± 0.404.2 ± 2.101.7 ± 0.7

pDC population in cervical mucosa and peripheral blood

Five of the 28 healthy controls and three of the 23 Chlamydia-positive women without MPC did not have pDCs in their cervical samples. All the women with MPC had pDC in their cervical samples. The median and range of absolute numbers of pDCs/cervical sample are given in Table 1. Healthy controls had a significantly (p <0.001) lower number of pDCs in their cervical samples than Chlamydia-infected women. In comparison to cervical samples, there was a significant decrease in absolute numbers of pDCs/mL of blood in women infected with Chlamydia, as compared to controls. The decrease was more pronounced in women with MPC. Expression of all co-stimulatory molecules was higher in pDCs in Chlamydia-positive women than in controls, but was significant (p <0.05) only for CD80 (Table 2).

Concentration of cytokines in cervical washes and their correlation with DC populations

The median levels of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, TNF-α, IFN-α and IFN-γ in cervical washes of controls and Chlamydia-positive women, before and after therapy are shown in Table 3. The detection limit for all the cytokines was 2 pg/mL, as described by the manufacturers. Before therapy, significantly higher levels of IL-1β and IL-6 (p <0.05) were observed in Chlamydia-positive women with MPC than in those without MPC. IL-8 levels were significantly higher (p <0.05) in Chlamydia-positive women with MPC than in those without MPC and controls. IL-4 levels were below detection limits in all the samples. After therapy, IL-8 levels in women with MPC showed significant downregulation (p <0.01) (Table 3). No other cytokine showed any significant change in levels after therapy. Before therapy, a significant correlation (r = 0.65; p <0.05) was observed between the number of mDCs/cervical samples and levels of IL-12 in Chlamydia-positive women without MPC. The number of pDCs in Chlamydia-positive women with MPC showed a significant correlation with IL-8 levels (r = 0.58; p <0.05) and a less apparent correlation with IL-6 (r = 0.37).

Table 3.   Cytokine concentrations in cervical washes before and after therapy
 ControlCT-positiveCT-positive
With MPC (before therapy)Without MPC (before therapy)With MPC (after therapy)With MPC (after therapy)
  1. CT, Chlamydia trachomatis; MPC, mucopurulent cervicitis; UDL, under detection limit; IL, interleukin; IFN, interferon; TNF, tumour necrosis factor.

  2. The data represent median values. Ranges are given in parentheses.

  3. ap <0.05 of the CT-positive women with MCP as compared to CT-positive women without MCP (Mann–Whitney U-test).

  4. bp <0.01 of the CT-positive group with MPC as compared to both of the other groups (Mann–Whitney U-test).

IL-1β79.24 (UDL–350.14)37.62 (UDL–99.86)96.90 (8.70–298.0)a54.37 (UDL–296.45)90.25 (UDL–304.21)
IL-24.59 (UDL–30.35)3.37 (UDL–13.57)2.98 (UDL–16.42)3.47 (UDL–28.34)2.85 (UDL–15.93)
IL-629.46 (UDL–188.70)9.36 (UDL–39.63)70.96 (UDL–190.60)a15.41 (UDL–173.93)43.46 (UDL–201.48)a
IL-889.9 (UDL–247.57)137.4 (13.74–375.9)551.58 (32–1074.0)b112.31 (4.52–295.93)93.17 (13.69–231.94)
IL-107.10 (UDL–22.86)6.64 (UDL–22.96)11.68 (UDL–30.91)8.31 (UDL–24.61)9.28 (UDL–27.83)
IL-12p40157.35 (UDL–542.89)267.5 (24.85–727.56)166.25 (UDL–532.87)183.82 (19.27–562.37)159.74 (2.48–527.17)
IFN-γ145.78 (19.65–589.73)173.24 (7.33–566.6)136.96 (UDL–330.6)152.75 (5.38–478.59)141.72 (5.48–394.26)
TNF-α1.47 (UDL–6.61)1.45 (UDL–13.73)1.58 (UDL–6.87)1.48 (UDL–10.67)1.51 (UDL–5.85)
IFN-α20.42 (UDL–74.95)29.69 (UDL–92.57)38.57 (UDL–95.31)23.60 (UDL–77.39)26.86 (UDL–85.45)

After therapy, no correlation among mDC and pDC populations and cytokine levels was observed.

Correlation of mDC and pDC with chlamydial infectious load and CRP

The chlamydial infectious load was determined by inoculating cervical samples into HeLa  229 cells as described elsewhere [34], and IFUs/mL were quantified. In cervical mucosa, the number of mDCs/cervical sample showed a significant correlation with chlamydial IFUs/mL (r = 0.524; p <0.05) in Chlamydia-positive women without MPC. A positive, non-significant correlation was found between chlamydial IFUs/mL and mDCs/cervical sample in women with MPC (r = 0.312). In comparison, the number of pDCs/cervical sample showed a significant correlation with chlamydial IFUs/mL in women both with and without MPC (r = 0.853, p <0.001, and r = 0.724, p <0.01, respectively). The median CRP levels were found to be significantly higher in women with MPC than in women without MPC (data not shown), and showed a significant correlation with pDCs/cervical sample (r = 0.675, p <0.05). No correlation was found between the number of mDCs/cervical sample and CRP levels.

mDCs and pDCs in cervix and blood after resolution of chlamydial infection

All Chlamydia-positive women were advised to undergo full antibiotic therapy, and after 4–6 weeks, eight women from each Chlamydia-positive group who returned for follow-up were again enrolled. After treatment, none of the women in group II was Chlamydia-positive, as revealed by PCR and culture. One patient with MPC (CT18M) was found to be Chlamydia-positive even after therapy, although the number of PMNLs was less than ten, with no mucopurulent discharge. Comparison of paired measurements of mDCs and pDCs in cervical samples in only the patients with chlamydial infection who returned for follow-up evaluation revealed a significant increase in the number of mDCs after therapy, in both Chlamydia-infected groups (Fig. 3a,b). The median number of pDCs was lower in both the Chlamydia-infected groups after therapy. The pDC number was significantly lower in women with MPC after therapy (Fig. 3c,d). The median numbers of mDCs and pDCs in peripheral blood and cervical samples before and after therapy are given in Table 4. The relative frequency of mDCs in cervical samples significantly increased, from 0.074% before therapy to 0.19% after therapy in group II (p <0.05) and from 0.068% before therapy to 0.11% after therapy in group III (non significant). The relative frequency of pDCs in cervical samples of women with MPC significantly decreased from 0.18% before therapy to 0.04% after therapy (p <0.05).

image

Figure 3.  Change in number of dendritic cells (DCs) in cervical samples of Chlamydia-positive women without or with mucopurulent cervicitis (MCP) after resolution of chlamydial infection. (a, b) Myeloid DCs (mDCs) in cervical samples of eight women with or without MCP who returned for follow-up after resolution. (c, d) Similar data for plasmacytoid DCs (pDCs) in cervical samples.

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Table 4.   Absolute number of myeloid and plasmacytoid dendritic cells in cervical mucosa and blood samples before and after resolution of infection
 CT-positive without MPC
Before therapyAfter therapyBefore therapyAfter therapy
  1. CT, Chlamydia trachomatis; MPC, mucopurulent cervicitis; mDC, myeloid dendritic cell; pDC, plasmacytoid dendritic cell.

  2. The data represent median values for all cases before therapy and of the eight follow-ups after therapy.

  3. ap <0.05 as compared to paired measurements (Wilcoxon signed ranked test).

Cervix
 mDCs17885384a7203917a
 pDCs1750983a3890413a
Blood
 mDCs18005860a10 80018 560
 pDCs721613 277624017 472a

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

In women, chlamydial infections are often asymptomatic, and subsequent re-infection leads to inflammatory responses with pathological sequelae [36]. In the peripheral circulation, the LIN/DR+ DCs comprise 0.1–2% of the total peripheral blood mononuclear cells in healthy individuals, and the majority of these cells express either CD11c or CD123 markers [37]. The initiative to study the DC subsets during C. trachomatis infection was prompted by the currently incomplete knowledge concerning these subsets.

Women with and without MPC were enrolled for the study of differential recruitment of DC subsets during non-inflammatory and inflammatory conditions. The resulting data showed that, during C. trachomatis infection, both mDCs and pDCs are recruited to the site of infection. The mDC population in the cervix was more abundant during non-inflammatory conditions, whereas the pDC population was more abundant during inflammatory conditions.

This was supported by the fact that, in blood, the numbers of mDCs were significantly reduced, as compared to controls, in women without MPC but not in women with MPC, suggesting greater mobilization of mDCs to an infected but non-inflamed cervix. As compared to controls, in both of the Chlamydia-positive groups, significant upregulation of the CD83 and CD80 markers on mDCs was observed, with stronger upregulation in women with MPC, suggesting the presence of a larger number of matured mDCs with increased co-stimulatory molecule expression during inflammation of the cervix. The lower numbers of mDCs/cervical sample in women with MPC may be a possible explanation for the incomplete clearance of chlamydiae and, therefore, the pathogenesis of chlamydial infection.

The median chlamydial infectious load in women without MPC was found to be significantly lower than that in women with MPC (data not shown). pDCs, on the other hand, were more prevalent in an inflamed cervix than in a non-inflamed cervix, with significantly lower numbers in the blood of Chlamydia-positive women than in controls. pDCs were also significantly correlated with chlamydial infectious loads in both groups, with CRP levels in the serum of women with MPC, and with the number of PMNLs in women with MPC (data not shown).

No significant upregulation of the expression of any co-stimulatory molecule, except CD80, on pDCs of Chlamydia-positive women was observed, which suggests that such molecules may not be actually involved in initiating immune responses, but could have some role in the pathogenesis of chlamydial infection. However, this is hypothetical, as the expression of the reported maturation marker for pDCs [38], the inducible co-stimulatory ligand (ICOS-L), was not measured. No apparent correlation of mDCs was observed with the chlamydial infectious load in women with MPC, thus suggesting that C. trachomatis infection attracts more pDCs than mDCs to the cervix.

An inverse correlation of CRP levels with mDC numbers has been shown previously [25], but no correlation was found in the current study (data not shown). pDCs were also found to be more abundant in extensive coronary artery disease, according to authors who suggested a role for them in plaque progression [26]. A recent study by Brunham et al. [39] reported the persistence of Chlamydia inside DCs. This study, when compared with the current results, suggests that pDCs are not efficient in presenting chlamydial antigens to T-cells, and can be used by C. trachomatis for its survival and persistence, leading to inflammation.However, further studies are required to test this hypothesis.

The cervical cytokines in women were measured before therapy, and significantly higher levels of IL-1β, IL-6 and IL-8 were found in Chlamydia-positive women, with the levels of IL-1β and IL-8 being considerably higher in women with MPC. Following complete antibiotic therapy, the cytokine levels in both of the Chlamydia-positive groups showed little variation, as in the case of IL-1β, IL-2, IL-10, IL-12p40, IFN-γ, TNF-α and IFN-α. In contrast, IL-8 levels in women with MPC decreased significantly after therapy and tended to become equivalent to the levels in controls. Before therapy, the number of mDCs/cervical samples was significantly correlated with cervical IL-12 levels in women without MPC, and pDCs correlated with IL-8 levels in women with MPC. No such correlation was observed after therapy, which suggests that, during infection, recruitment of particular DC subset decides the cytokine profile in cervical mucosa.

As for cytokine production by these subsets, previous studies have shown production of IFN-α by pDCs upon viral challenge [40–42], and production of IL-12 by mDCs and IL-12, IL-10 and IFN-α by pDCs in response to fungal infections [19]. pDCs have been reported to express TLR9 and, upon stimulation with CpG motifs, they have been reported to express IFN-α, IL-6 [43] and IL-12p70 [44] in a mouse model, but the cytokine secretion pattern of pDCs upon bacterial challenge in humans is yet to be ascertained fully. Secretion of IL-12 by mDCs has been observed after incubation with mycobacteria [17], and secretion of IL-10 and IL-23 after incubation with Helicobacter pylori [45]. Few studies have been performed where these DCs were stimulated with lipopolysaccharide (LPS) in order to determine the cytokine secretion patterns [46]. These studies revealed that, upon LPS stimulation, mDCs produce large amounts of IL-8. This is in contrast to the current results, where a significant correlation between IL-8 and the number of pDCs is established. No significant increase in levels of IFN-α during chlamydial infection was found in the current study, which is somewhat in concordance with the results of Dai et al. [47], who have also shown an absence of induction of IFN-α production from pDCs when they are stimulated with LPS. The current results may shed new light on chlamydial pathogenesis, but the conclusions are still hypothetical, due to two limitations of the study: (i) the low cell numbers, making it impossible to separate the subsets in order to determine the expression of cytokines upon stimulation with C. trachomatis; and (ii) the fact that cytokines such as IL-12 are secreted by other cell populations, e.g. macrophages, which outnumber DCs in the cervix.

After resolution of infection, surprisingly, increased levels of mDCs were found in both the cervix and blood of Chlamydia-positive women. The levels of pDCs, on the other hand, were lower in both of the groups after infection was resolved, except for one patient (CT18M) in group III (MCP), in whom the infection was not resolved, confirming the hypothesis that C. trachomatis mobilizes pDCs in the cervix in accordance with its load. Many studies have been previously carried out concerning the population of DCs before and after therapy in blood samples, but very few have been undertaken to enumerate DCs at mucosal surfaces after therapy [14]. The increase in the number of DCs in blood can be explained by the fact that, because the infection is resolved, the DCs are not migrating from the blood to the cervix. We propose that lower numbers of mDCs in the cervix before therapy could be due to migration of these DCs to the lymph nodes for initiation of immune responses; however, after resolution of infection, these mature DCs return to the cervix, thereby increasing the numbers. A significant increase in the number of pDCs after therapy has been shown by many authors [40,48], but these results were obtained from analysis of blood. In the current study, the number of pDCs after therapy was lower in women without MPC, but the reduction was not significant by comparison with women with MPC, in whom the decrease in numbers after therapy was highly significant. This further confirms the hypothesis that pDCs are more involved in inflammation.

In conclusion, it has been shown here that both of the DC subsets are attracted to the site of chlamydial infection, with a greater prevalence of pDCs in inflammatory conditions. pDC numbers correlated with chlamydial load and, after resolution of infection, a sudden drop in these numbers was observed. This suggests a possible role of pDCs in the immunopathogenesis of chlamydial infection. A hypothesis that emerges from these results is that pDCs are not able to clear the infection, and somehow allow the persistence of Chlamydia inside them. However, further studies are required, as this study has the limitation that only a low number of cells was obtained from the cervix, which hindered the in vitro analysis of the two subsets. The uncertainty about whether the mobilization is due to chlamydial infection or other infections that also mobilize DCs to the cervix to the same extent was also a limitation of this study. Regardless of its limitations, this study will contribute to our understanding of the interplay between Chlamydia and DCs, which is of interest given the fact that a vaccine against C. trachomatis is still unavailable. As DCs are being targeted as an efficient adjuvant for delivery of chlamydial antigens, this study could provide insights facilitating the production of an efficient vaccine for Chlamydia.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

We thank M. Badhwar, A. Rani and R. Thomas for providing technical assistance.

Transparency Declaration

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

This study was supported by an Indo-US grant (BT/IN/USCRHR/AM/2002) from the Department of Biotechnology, Government of India. The University Grants Commission is acknowledged for providing assistance to T. Agrawal in the form of a fellowship. None of the authors have any potential financial conflict of interest related to this manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References