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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

Objective

To investigate the numerical and functional changes of CD4+CD25high regulatory T (Treg) cells during pregnancy and postpartum in patients with ankylosing spondylitis (AS).

Methods

The frequency of CD4+CD25high T cells was determined by flow cytometry in 10 pregnant and 5 nonpregnant patients with AS as well as in 14 pregnant and 4 nonpregnant healthy controls. Pregnant individuals were investigated at the third trimester and 8 weeks postpartum. Treg cells and CD4+CD25− effector T (Teff) cells separated by fluorescence-activated cell sorting were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies, alone or in coculture, to investigate proliferation and cytokine secretion.

Results

The frequency of CD4+CD25high Treg cells was significantly higher during pregnancy than postpartum in both healthy control subjects and patients with AS. In contrast to Treg cells in healthy pregnant women, Treg cells in pregnant women with AS secreted only small amounts of interleukin-10 and showed lower suppression of tumor necrosis factor α and interferon-γ secretion by CD4+CD25− Teff cells. At the postpartum time point, proinflammatory cytokine levels in the Treg/Teff cell cocultures and Teff cell monocultures were significantly higher in patients with AS than in healthy controls.

Conclusion

Pregnancy influenced the expansion and cytokine secretion of Treg cells in both patients with AS and control subjects. However, the Treg cells of pregnant patients with AS failed to support an antiinflammatory cytokine milieu, thereby possibly contributing to the persistent disease activity of AS during pregnancy.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

In contrast to the effect of pregnancy on rheumatoid arthritis (RA), pregnancy has no profound effect on the activity of ankylosing spondylitis (AS). In most patients, AS remains active during pregnancy, although a mitigation of symptoms is commonly seen in the third trimester, followed by postpartum aggravation (within 6–12 weeks postpartum) (1). A modulation of disease activity during pregnancy is observed in several autoimmune diseases. However, the effectors of this modulation are largely unknown.

Regulatory T (Treg) cells, which expand during gestation in mice and humans, play a key role in successful pregnancy outcomes (2, 3). This unique subpopulation of cells exhibits the suppression of antigen-specific effector T (Teff) cells. In contrast to thymically derived Treg cells that exert their function via cell-to-cell contact, Treg cells that are induced in the periphery use predominantly cytokine-based mechanisms (4).

A deficiency in the number and function of Treg cells is a feature of several autoimmune diseases, including RA (5). Recently, we showed that a population of inducible forkhead box P3 (FoxP3) expressing CD4+CD25high Treg cells that secreted interleukin-10 (IL-10) and provided enhanced suppression of proinflammatory cytokines increased during pregnancy in RA patients (6). Until now, studies of T cells in pregnant patients have not included those with AS. Few data are available about Treg cell function in nonpregnant patients with AS (7). Because, as a rule, AS disease activity is persistent during pregnancy, we investigated aspects of Treg cell function in a small cohort of pregnant patients with AS as compared with healthy pregnant women.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

Patients.

The study had a longitudinal, prospective design, required informed consent, and was approved by the ethics committee of the University of Bern. Included in the study were 15 AS patients (10 pregnant and 5 nonpregnant) as well as 18 age-matched healthy women (14 pregnant and 4 nonpregnant). AS patients fulfilled the modified New York criteria (8). Time points for clinical assessment and blood sampling were gestational weeks 30–34 and 8 weeks postpartum. Pregnant patients were clinically evaluated using the Bath Ankylosing Spondylitis Activity Index (BASDAI) (9). Serum C-reactive protein (CRP) levels were determined by a sensitive latex agglutination method (Sentinel Diagnostics, Milano, Italy). The clinical characteristics and drug therapy for the study population are shown in Table 1. Of those AS patients who were analyzed for functional changes in Treg cells during and after pregnancy, 3 were receiving acetaminophen at the third trimester, but none of the patients was receiving prednisone or any disease-modifying antirheumatic drug. At the postpartum time point, 3 of these AS patients were receiving nonsteroidal antiinflammatory drugs, and 2 were receiving acetaminophen.

Table 1. Patient characteristics*
 Ankylosing spondylitis (n = 15)Healthy controls (n = 18)
  • *

    Values are the median (range) or number. BASDAI = Bath Ankylosing Spondylitis Disease Activity Index; CRP = C-reactive protein; NSAIDs = nonsteroidal antiinflammatory drugs.

Age, years33 (24–40)34 (27–39)
Disease duration, years6 (1–19)
HLA–B27+15
BASDAI  
 Third trimester2.2 (0.5–7.0)
 8 weeks postpartum5.5 (1.3–7.2)
 Nonpregnant5.1 (2.4–7.1)
CRP, mg/dl  
 Third trimester5.9 (1.6–17.0)2.9 (0.9–3.8)
 8 weeks postpartum9.3 (5.6–32.2)1.2 (0.3–4.2)
 Nonpregnant6.2 (4.8–14.0)0.8 (0.6–0.9)
Medication  
 NSAIDs  
  Third trimester20
  8 weeks postpartum60
  Nonpregnant50
 Acetaminophen  
  Third trimester50
  8 weeks postpartum20
  Nonpregnant00
 Prednisone <10 mg/day  
  Third trimester10
  8 weeks postpartum10
  Nonpregnant00
 Sulfasalazine  
  Third trimester00
  8 weeks postpartum10
  Nonpregnant10
 Cyclosporine  
  Third trimester00
  8 weeks postpartum10
  Nonpregnant00

Cell isolation and sorting.

Peripheral blood mononuclear cells were stained with phycoerythrin (PE)–labeled anti-CD25 (clone 2A3; BD Biosciences, San Jose, CA) and anti-PE magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany), and CD25+ cells were enriched using the MidiMACS system (Miltenyi Biotec). CD25-enriched and CD25-depleted cell populations were stained with fluorescein isothiocyanate (FITC)–labeled anti-CD4 (clone RPA-T4; BD Biosciences) and sorted into CD4+CD25− and CD4+CD25high T cells on a FACSVantage SE flow cytometer (Becton Dickinson, Mountain View, CA). Cell analysis after sorting showed a purity of >98%.

Flow cytometric analysis.

For the frequency analysis of CD4+CD25high T cells, the following directly labeled monoclonal antibodies (mAb) were used: PE-conjugated anti-CD3 (clone HIT3a), peridinin chlorophyll A protein–conjugated anti-CD4 (clone SK3), and allophycocyanin-conjugated anti-CD25 (clone 2A3). All antibodies were obtained from BD Biosciences. Cells were incubated for 20 minutes at 4°C with each mAb. The number of CD4+CD25high T cells was always expressed as the percentage of CD4+ T cells.

Proliferation assay.

To assess proliferation, 2.5 × 104 purified CD4+CD25− and CD4+CD25high T cells separated by fluorescence-activated cell sorting were incubated alone or were cocultured at a 1:1 ratio in complete medium with 1 μg/ml plate-bound anti-CD3 (OKT-3; eBioscience, San Diego, CA) and 5 μg/ml soluble anti-CD28 (BD Biosciences) in 96-well U-bottomed plates. After 6 days of culture, cells were labeled with 1.0 μCi 3H-thymidine (Amersham Bioscience, Little Chalfont, UK) per well for the last 18 hours. Proliferation was measured by liquid scintillation counting and was expressed as counts per minute. The suppressive capacity of CD4+CD25high T cells was calculated as follows:

  • equation image

Multiplex cytokine assay.

For the analysis of cytokine production, isolated T cell populations were cultured alone or in a coculture setting, as mentioned previously. After 48 hours, the cytokines IL-10, interferon-γ (IFNγ), and tumor necrosis factor α (TNFα) in each cell culture supernatant were measured using a Bio-Plex suspension array system (Bio-Rad, Hercules, CA). TNFα and IFNγ suppression was calculated as follows:

  • equation image

Real-time reverse transcriptase–polymerase chain reaction.

Total RNA was extracted from CD4+CD25high cells isolated from 3 patients with AS and 3 healthy controls, using the RNeasy Mini Kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. Complementary DNA was prepared with random hexamers using Superscript II reverse transcriptase (Invitrogen, San Diego, CA). The expression of FoxP3 messenger RNA (mRNA) was determined using Assays-on-Demand reagents (Hs00203958-m1; Applied Biosystems). All reported mRNA levels were normalized to the expression of 18S ribosomal RNA and calculated relative to the expression of FoxP3 in Epstein-Barr virus–transfected B cells.

Statistical analysis.

Study groups were compared using the Mann-Whitney U test for unpaired data. To analyze the longitudinal changes, we performed Wilcoxon's test for paired samples. For all analyses, P values less than 0.05 (2-tailed) were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

At the third trimester of pregnancy, AS patients displayed moderate disease activity. However, a persistent active inflammatory process was reflected by elevated CRP levels (Table 1) that differed significantly from those of pregnant healthy controls (P < 0.05). At the postpartum time point, disease activity as measured by the BASDAI increased by 50% in 8 of 10 AS patients and remained stable in 2 AS patients.

The numerical changes in Treg cells at the third trimester and 8 weeks postpartum were analyzed in 10 pregnant and 5 nonpregnant AS patients as well as in 14 pregnant and 4 nonpregnant healthy controls. Both AS patients and healthy controls had a significantly higher frequency of CD4+CD25high Treg cells during the third trimester compared with the frequency at 8 weeks postpartum (for AS patients, median 4.4% [range 2.9–7.6%] versus 3.0% [range 2.1–4.7%] [P < 0.01]; for healthy controls, median 4.6% [range 2.2–7.0%] versus 3.5% [range 2.1–4.7%] [P < 0.01]). These longitudinal changes in the frequency of Treg cells did not differ between patients and healthy controls and therefore seemed to be a pregnancy-related phenomenon. In both nonpregnant AS patients and nonpregnant healthy controls, the number of Treg cells were comparable with those in postpartal AS patients and postpartal healthy controls (for AS median 2.7% [range 1.9–3.5%] versus 3.0% [range 2.1–4.7], for healthy controls median 3.2% [range 2.3–3.8%] versus 3.5% [range 2.1–4.7%]).

Freshly isolated CD4+CD25high Treg cells were further analyzed for the transcription factor FoxP3. The relative mRNA expression of FoxP3 was found to be lower in AS patients than in healthy controls (for healthy controls, median 3,983 [range 2,408–4,431] and 2,227 [range 1,824–2,805] at the third trimester and postpartum, respectively; for AS patients, median 1,678 [range 1,136–2,220] and 1,669 [range 1,513–1,824] at the third trimester and postpartum, respectively). In addition, elevated FoxP3 mRNA expression in Treg cells during the third trimester was detected only in healthy women.

To analyze the suppressive function of Treg cells, isolated CD4+CD25high cells were cocultured with CD4+CD25− T cells (Teff cells) from 6 pregnant and 5 nonpregnant AS patients as well as from 5 pregnant and 4 nonpregnant healthy controls. The capacity of CD4+CD25high T cells to suppress the proliferation of Teff cells was similar in pregnant patients and healthy women and was not significantly different between the third trimester and postpartum (for the third trimester, median 65.1% [range 64.2–74.6%] versus 74.33% [range 69.2–77.8%]; for postpartum, median 80.3% [range 74.9–85.6] versus 78.6% [range 61.3–98.1]). In contrast, Treg cells isolated from healthy women displayed significantly more intense TNFα and IFNγ suppression during pregnancy than postpartum (Figures 1A and B). This pregnancy-related effect was not detected in patients with AS. Moreover, Treg cells from pregnant, postpartum, or nonpregnant AS patients exhibited significantly weaker suppression of TNFα and IFNγ than those from healthy controls.

thumbnail image

Figure 1. CD4+ CD25high regulatory T (Treg) cells of pregnant patients with ankylosing spondylitis (AS) display an impaired suppression of tumor necrosis factor α (TNFα) and interferon-γ (IFNγ) as compared with healthy controls (HC). Isolated CD4+CD25high Treg of patients with AS (6 pregnant and 5 nonpregnant) and HC (5 pregnant and 4 nonpregnant) were cultured either alone or at a 1:1 ratio with autologous CD4+CD25– effector T cells. After 48 hours of stimulation with plate-bound anti-CD3 and soluble anti-CD28 culture, supernatants were harvested and analyzed to determine the secretion of TNFα (A) and IFNγ (B). All individuals were analyzed at the third trimester (lightly shaded boxes) and 8 weeks postpartum (darkly shaded boxes). Values were compared with those of nonpregnant controls (open boxes). Box plots represent the median (bold line) with 25th and 75th percentiles and whiskers represent the 10th and 90th percentiles. * P < 0.05, ** P < 0.01.

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IFNγ and TNFα secretion by CD4+CD25− Teff cells changed from low levels during pregnancy to high levels postpartum in patients and controls. For AS patients, the median levels of IFNγ were 49.4 pg/ml (range 39.1–60.3) in the third trimester and 121.3 pg/ml (range 70.6–191.1) postpartum, and the median levels of TNFα were 129.3 pg/ml (range 98.2–141.8) in the third trimester and 315.0 pg/ml (range 281.5–583.5) postpartum. For healthy control subjects, the median levels of IFNγ were 21.1pg/ml (range 19.2–24.9) in the third trimester and 58.6 pg/ml (range 33.1–68.0) postpartum, and the median levels of TNFα were 45.0 pg/ml (range 35.6–59.8) in the third trimester and 90.0 pg/ml (range 71.9–105.9) postpartum. However, at both time points, the levels of IFNγ and TNFα in the monocultures of CD4+CD25− Teff cells as well as in the Treg/Teff cocultures were significantly higher in AS patients compared with healthy controls (for IFNγ, P < 0.01 [third trimester] versus P < 0.01 [postpartum]; for TNFα, P < 0.01 [third trimester] versus P < 0.01 [postpartum]).

Interestingly, Treg cells isolated in the third trimester secreted the antiinflammatory cytokine IL-10 in both the monocultures (Figure 2) and the cocultures (data not shown). In contrast, significantly smaller amounts of IL-10 were observed in Treg cell cultures at the postpartum time point or in those of nonpregnant controls. Moreover, the Treg cells of pregnant AS patients secreted significantly less IL-10 compared with the Treg cells of healthy pregnant women.

thumbnail image

Figure 2. CD4+ CD25high regulatory T (Treg) cells of pregnant patients with ankylosing spondylitis (AS) secrete less interleukin-10 (IL-10) as compared with healthy controls (HC). Isolated CD4+CD25high Treg cells of patients with AS (6 pregnant and 5 nonpregnant) and HC (5 pregnant and 4 nonpregnant) were stimulated with plate-bound anti-CD3 and soluble anti-CD28. After 48 hours, culture supernatants were harvested for their IL-10 content. All individuals were analyzed at the third trimester (lightly shaded boxes) and 8 weeks postpartum (darkly shaded boxes). Values were compared with those of nonpregnant controls (open boxes). Box plots represent the median (bold line) with 25th and 75th percentiles and whiskers represent the 10th and 90th percentiles. * P < 0.05, ** P < 0.01.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

This study is the first to analyze the frequency and function of Treg cells in AS patients during the third trimester of pregnancy and postpartum. The numerical increase in Treg cells that has been described in healthy pregnant women was also observed in pregnant women with AS (2). The 2 main findings in AS patients were the failure of Treg cells to mount a substantial secretion of IL-10 during pregnancy, and the impaired ability of gestational Treg cells to augment the suppression of IFNγ and TNFα production by Teff cells. As opposed to the Treg cells of healthy controls, the Treg cells of pregnant AS patients failed to establish an effective antiinflammatory cytokine milieu.

The present results contrast with our findings in pregnant women with RA (6). In a previous study, we demonstrated that gestational Treg cells provided a more intense suppression of proinflammatory cytokines and secreted substantial amounts of IL-10. Remarkably, the stimulated Treg cells of pregnant women with AS secreted only 23% of the IL-10 amount that could be detected in healthy controls. However, IL-10–producing CD4+CD25high Treg cells were found to play a major role in the induction of tolerance in the context of pregnancy (10) as well as in the model of collagen-induced arthritis (11).

The CD4+CD25high Treg cells isolated from healthy pregnant women in the current study secreted substantial amounts of IL-10. This finding strongly suggests that these cells belong in the category of induced Treg cells (4, 10, 11). In contrast to natural Treg cells, induced Treg cells display unstable FoxP3 expression due to differences in epigenetic regulation (12). Our data show that pregnancy-induced Treg cells of healthy women expressed increased levels of FoxP3 in combination with augmentation of the suppressive function. A reversal of these changes appeared after the women gave birth. In contrast, a transient increase in FoxP3 was not observed in the pregnant women with AS, suggesting a functional deficiency in their pregnancy-induced Treg cells. These findings may reflect the persistent activity of inflammation that is present in most AS patients during pregnancy (1).

Moreover, the suppressive capacity of Treg cells was impaired even in nonpregnant AS patients. The continuous presence of proinflammatory cytokines, particularly TNFα, in our cohort as well as in other nonpregnant AS patients (7, 13, 14) could possibly contribute to the deficiency of Treg cell function. In this context, it is of interest that high levels of TNFα can inhibit Treg cell function in patients with active RA (15).

In conclusion, pregnancy is unable to fully revert the disease activity in AS. The impaired function of pregnancy-induced Treg cells in AS may play a role in the persistent proinflammatory milieu during gestation. However, further investigations are needed to analyze the exact pathogenetic aspects that account for the modified Treg cell function observed in pregnant and nonpregnant patients with AS.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

Dr. Förger had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Förger, Villiger, Østensen.

Acquisition of data. Förger, Østensen.

Analysis and interpretation of data. Förger, Villiger, Østensen.

Manuscript preparation. Förger, Villiger, Østensen.

Statistical analysis. Förger.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

We are grateful to all the patients and healthy women who participated in the study. We thank Tibor Schuster for expert statistical advice, Marianne Zwicker and Richard Kamgang for their technical assistance, and Bernadette Wider for excellent cell sorting.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES
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