ORIGINAL ARTICLE

Intravenous Immunoglobulin G Modulates Peripheral Blood Th17 and Foxp3+ Regulatory T Cells in Pregnant Women with Recurrent Pregnancy Loss Dong Jae Kim1, Sung Ki Lee1, Jee Yun Kim1, Baeg Ju Na2, Sung Eun Hur1, Millina Lee1, Joanne KwakKim3,4 1

Department Department 3 Department 4 Department USA 2

of of of of

Obstetrics and Gynecology, College of Medicine, Konyang University, Seo-gu, Daejeon, Korea; Preventive Medicine, College of Medicine, Konyang University, Seo-gu, Daejeon, Korea; Obstetrics and Gynecology, Chicago Medical School at Rosalind Franklin University of Medicine and Science, Vernon Hills, IL, USA; Microbiology and Immunology, Chicago Medical School at Rosalind Franklin University of Medicine and Science, North Chicago, IL,

Keywords IL-17, intravenous immunoglobulin, recurrent pregnancy loss, regulatory T cells, Th17 cells Correspondence Sung Ki Lee, Department of Obstetrics and Gynecology, College of Medicine, Konyang University, Gasoowon-dong, Seo-gu, Daejeon 302-718, Korea. E-mail: [email protected] Submission November 20, 2013; accepted January 5, 2014. Citation Kim DJ, Lee SK, Kim JY, Na BJ, Hur SE, Lee M, Kwak-Kim J. Intravenous immunoglobulin G modulates peripheral blood Th17 and Foxp3+ regulatory T cells in pregnant women with recurrent pregnancy loss. Am J Reprod Immunol 2014; 71: 441–450 doi:10.1111/aji.12208

Problem Th17 cells and Foxp3+ regulatory T (Treg) cells have been proposed as new risk factors for recurrent pregnancy loss (RPL). Intravenous immunoglobulin G (IVIG) was reported to modulate various immune cells. In this study, we investigated the effect of IVIG on the levels of Th17 and Treg cells and pregnancy outcome in women with RPL. Method of study Thirty-seven pregnant women with RPL were enrolled in this study. All had cellular immune abnormality in preconceptional evaluation. Blood was drawn on the day of IVIG treatment and 1 week later from the study subjects during early pregnancy. The proportions of IL-17+ and Foxp3+ T cells were analyzed using flow cytometry. Results Study population was divided into four groups (Q1–Q4) based on ascending order of the levels of Th17 and Foxp3+ T cells. IVIG down-regulated Th17 cells in the highest quartile, Q4 (P = 0.001), and up-regulated CD4+ Foxp3+ T cells in Q1 and Q2 (P = 0.025 and 0.029, respectively). In addition, Th17/CD4+ Foxp3+ T cell ratio decreased in Q4 (P = 0.040). We also found a positive trend between successful pregnancy outcome and CD8+ IL-17+ T cells before IVIG treatment (P = 0.05). Conclusion Intravenous immunoglobulin G treatment modulated imbalance of Th17 and Foxp3+ Treg cells in pregnant RPL women with cellular immune abnormality.

Introduction During pregnancy, maternal immune system accepts semi-allogeneic fetus, while it protects both mother and fetus against microbial infection.1 To this end, maternal immune system should be controlled by an American Journal of Reproductive Immunology 71 (2014) 441–450 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

elaborate mechanism to maintain a balance between pro and anti-inflammatory immune response. Proinflammatory immune response has been reported to lead pregnancy complications, including implantation failure, recurrent pregnancy loss (RPL), preeclampsia, and intrauterine fetal growth restriction. 441

KIM ET AL.

RPL, defined as two or more consecutive pregnancy losses prior to 20 weeks of gestation,2 seems to occur in 2–5% of reproductive age women. Significant portion of women with RPL have abnormal cellular and autoimmunity. Increased proportion and cytotoxicity of natural killer (NK) cells in the peripheral blood and the endometrium/decidua have been reported as poor prognostic factors to lead RPL3–5 and imbalance of Th1 and Th2 cytokine production of peripheral lymphocytes were found in idiopathic RPL women.6–8 Approximately 45% of women with idiopathic RPL had increased CD3 CD56+ NK cells, 38% had increased NK cell cytotoxicity, and 28% had increased Th1/Th2 cell ratios.9 T regulatory cells (Treg) are a subset of CD4+ T lymphocytes that regulate autoimmunity and express a unique transcriptional factor, Foxp3.10 IL-17-producing CD4+ T, namely T helper 17 (Th17) cells, was reported to induce inflammation via neutrophil recruitment and stimulation of proinflammatory cytokines such as IL-1, IL-6, IL-8, TNF-a, nitric oxide, matrix metalloproteinase, and receptor activator for nuclear factor jB ligand (RANKL).11,12 These Th17 cells play a role not only in defense against virus, fungi, and some bacteria, but also in development of chronic inflammatory disease such as autoimmune diseases and metabolic disorders.13 Recently, Treg and Th17 cells were investigated in women with reproductive failure. Foxp3 mRNA expression was decreased in the secretory endometrium of women with repeated in vitro fertilization failure, and idiopathic RPL women uniformly had decreased Treg cells and increased Th17 cells and Th17/Treg cell ratios in the peripheral blood or the deciduas as compared with those of fertile women.14–19 The imbalanced Th17/Treg cell ratio was suggested to negatively influence implantation and placental development.13 Therefore, we speculate that regulation of Th17/Treg cell ratio may lead to successful pregnancy outcome in women with idiopathic RPL. Intravenous immunoglobulin G (IVIG) has been applied to the immunotherapy of idiopathic RPL women.20–24 Although the therapeutic effect of IVIG is controversial in idiopathic RPL, most positive results were obtained from the trials in RPL women with cellular immune abnormality, such as increased NK cell level and/or cytotoxicity, and T cell abnormality.25–29 However, to the best of our knowledge, there is no report dealing with IVIG effect on Th17 and Treg cell ratio in women with RPL. Thus, we investigated the effect of IVIG on Th17 or Treg cells 442

in RPL women with cellular immune abnormalities and evaluated pregnancy outcome of these women following IVIG treatment. Materials and methods Study Population This study was performed as a prospective observational study. Study subjects were recruited at department of Obstetrics and Gynecology, Konyang University Hospital. The study was approved by the local institutional review board (IRB), and participants signed an informed consent before the enrollment. Totally 37 pregnant women with a history of idiopathic RPL were consecutively enrolled in this study between January 1, 2010 and April 30, 2013. Investigation of RPL women was performed prior to the index pregnancy. None had anatomical, infectious, endocrine, or genetic etiologies for pregnancy losses. All subjects had one or more cellular immune abnormalities prior to index pregnancy including CD3 CD56+ NK cell levels (17/37, 46%), NK cell cytotoxicity assay (24/37, 65%), and Th1/Th2 cytokineproducing T cell ratio (15/37, 41%), which are clinical immune markers previously reported.9 Age and obstetrical history of study populations are listed in Table I. Study Design Blood was drawn at the time of positive pregnancy test. Th17 and Treg cells were investigated along with cellular immune tests that were abnormal in preconceptional evaluation. On the same day, IVIG 400 mg/ kg was administrated intravenously to the study subject. One week after IVIG administration, peripheral blood was drawn again for Th17 and Treg cell study. IVIG treatment was repeated every 3 or 4 weeks until

Table I Age and Obstetrical History of Pregnant Women with Idiopathic Recurrent Pregnancy Losses (n = 37)

Age (year) Parity (number) SAB (number) G.A (weeks)a

Mean  S.D.

Median

Range

   

32 0 3 5

26–38 0–2 2–6 4–9

32.5 0.2 3.2 5.1

3.0 0.5 1.0 1.1

SAB, spontaneous abortion. a G.A (weeks), weeks of gestation at intravenous immunoglobulin G administration.

American Journal of Reproductive Immunology 71 (2014) 441–450 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

IVIG EFFECT ON TH17 AND TREG CELLS IN RPL WOMEN

28–30 weeks of gestation for treatment of cellular immune abnormality. Successful outcome of IVIG treatment was considered if index pregnancy was ongoing beyond 28 weeks of gestation. Separation of Peripheral Blood Mononuclear Cells Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll–Hypaque (Biotech, Uppsala, Sweden) density centrifugation with LeucosepTM tubes (Greinner bio-one, Frickenhausen, Germany). After washing with Hanks’ balanced salt solution (Gibco, Grand Island, NY, USA), the cells were adjusted to a final concentration of 5 9 106 cells/mL in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1 mL of antibioticantimycotic solution (1009; Gibco). Prepared PBMCs were stored at 4°C in the dark. Laboratory Evaluation CD3 CD56+ NK cell level, NK cell cytotoxicity, and Th1/ Th2 cytokine study CD3 CD56+ NK cell level, NK cell cytotoxicity, and Th1/Th2 cytokine-producing T cell analyses were performed using flow cytometric analysis as previously reported.30 Briefly, for immunophenotyping, twocolor direct immunofluorescence reagent kits (BD SimultestTM IMK-Lymphocyte; BD Biosciences, San Jose, CA, USA) were utilized to enumerate percentages of the following human leukocyte subsets in erythrocyte-lysed whole blood: CD3+ T, CD3+ CD4+ T, CD3+ CD8+ T, CD19+ B, CD3 CD56+ NK, and CD3+ CD56+ NKT cells. In NK cell cytotoxicity assay, NK cell cytotoxicity was determined at effector to target cell (E:T) ratios of 50:1, 25:1 and 12.5:1. Target cells were prepared by culturing the cell line K562, an NK cell-sensitive human erythromyelocytic leukaemia cell line. After 2-hr co-culture of PBMC and K562 cells, 0.2 lL propidium iodide (Sigma, St-Louis, MO, USA) solution (10 mg/mL) was added and the cells were vortexed. NK cell cytotoxicity was expressed as the percentage of dead K562 cells containing propidium iodide. For Th1/Th2 cytokine study, PBMCs were activated with 25 ng/mL phorbol myristate acetate (PMA; Sigma) and 1 lM ionomycin (Sigma). A 0.2 lM of Golgiplug (BD Biosciences, Franklin Lakes, NJ, USA) was also applied to inhibit cytokine secretion. Cell staining procedure was carried out according to the manufacturer’s instructions with the Cytofix/Cytoperm kit (BD Biosciences). Anti-CD3-peridinin American Journal of Reproductive Immunology 71 (2014) 441–450 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

chlorophyll (PerCP; BD Biosciences) and anti-CD8fluorescein isothiocyanate (FITC; BD Biosciences) were used to identify T cell populations. To detect intracellular cytokines, 0.2 lg of monoclonal antibodies (phycoerythrin, PE; BD Biosciences) for tumor necrosis factor (TNF)-a, and interleukin (IL)-10 were applied. For TNF-a/IL-10 cytokine-producing CD4+ T cell ratio, TNF-a-producing CD4+ T cell levels were divided by IL-10-producing CD4+ T cell levels. IL-17-producing T and Foxp3-expressing T cell study The levels of IL-17+ T cells and Foxp3+ T cells were determined by flow cytometry as described previously.9,16 To activate the PBMCs, 1 mL of 5 9 106/ ml of cell suspension was incubated with 10 ng/mL PMA (Sigma) and 0.5 lM ionomycin (Sigma) for 5 hr at 37°C in a 5% CO2 humidified incubator. One microliter of Monensin (91000 solution; eBioscience, San Diego, CA, USA) was also applied to enhance intracellular cytokine staining. After incubation, PBMCs were washed in phosphate-buffered saline (PBS) with 0.09% w/v sodium azide (Sigma) twice followed by staining with the anti-CD3-PerCP (BD Biosciences) and anti-CD8-FITC (BD Biosciences) and then incubated for 15 min at 4°C. The cells were washed in PBS and fixed with fixation buffer (eBioscience). The cells were washed twice with 19 permeabilization buffer (eBioscience) and incubated with 0.25 lg conjugated anti-human IL-17A-PE (eBioscience) at room temperature for 20 min. After intracellular staining, the cells were washed with 19 permeabilization buffer and resuspended in 0.5 mL of permeabilization buffer. The proportion of IL-17producing T cells in peripheral blood lymphocytes was enumerated by flow cytometry. To identify Treg cells, PBMCs were stained with anti-CD4-FITC monoclonal antibody (BD Biosciences) for surface antigens and anti-Foxp3-PE monoclonal antibody (eBioscience) for intracellular molecules according to the manufacturer’s instruction. PBMC (1 9 106 cells) was washed twice in PBS following staining with the fluorochrome-conjugated antibodies specific for cell surface antigen markers for 20 min in the dark at 4°C. Then, cells were washed twice with PBS followed by fixation of the surface markers. To stain an intracellular molecule, Foxp3, cells were permeabilized with permeabilization/fixation buffer and stained with anti-Foxp3 antibody following the surface staining. PE-rat IgG2a was used as an isotype control for anti-Foxp3-PE antibody. Cells were resuspended in 0.5 mL of stain443

KIM ET AL.

ing buffer for subsequent flow cytometric analysis. For IL-17+ T/Foxp3+ T cell ratio calculation, percentage of IL-17+ T cells was divided by percentage of Foxp3+ T cells. The prepared staining cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences). CellQuest Pro software (BD Biosciences) was used for data analysis. A total of 10,000 cells were counted. Viable lymphocytes were gated based on their forward and side scatter profile. Statistical Analysis Statistical analysis was performed using SPSS PC Statistics (version 15.0; SPSS Inc., Chicago, IL, USA). To compare the results of immunologic studies before and after IVIG treatment, paired t-test was applied. Linear-by-linear association chi-square test was performed in quartile analysis to test linear trend among the quartile groups of immune variables, response rate (defined as the percentage of cases who decreased in IL-17+ T cells or IL-17+/Foxp3+ T cell ratio and who increased in Foxp3+ T cells following IVIG treatment) of the variables, and pregnancy outcome. Pearson’s correlation analysis was used to test correlation between IL-17+ T/Treg cell balance and other immune markers. P-values < 0.05 were reported to be statistically significant. Results Prevalence of Cellular Immune Abnormality in the Study Subjects On the day of first IVIG treatment, we evaluated cellular immune status of pregnant RPL women who had immune abnormality in preconceptional cellular immune tests. Significant proportion of tested pregnant RPL women showed high level of NK cells (69%), increased NK cell cytotoxicity (64%), and TNF-a/IL-10-producing CD4+ T cell ratio (40%). Six RPL women (16%) who had cellular immune abnormality prior to index pregnancy showed no detectable immune abnormality (Fig. 1). Effects of IVIG Treatment on IL-17+ and Foxp3+ T Cells in Pregnant RPL Women with Cellular Immune Abnormality In comparison with the levels of IL-17+ T and Foxp3+ T cells before and after IVIG treatment, IVIG treatment to all RPL women (n = 37) did not result 444

Fig. 1 Prevalence of cellular immune abnormality in study subjects. On the day of first IVIG treatment, cellular immune abnormalities of pregnant women with recurrent pregnancy loss who had cellular immune abnormalities prior to pregnancy were evaluated. IVIG, intravenous immunoglobulin G.

in significant changes on levels of IL-17+ T cells, Foxp3+ T cells, and IL-17+/Foxp3+ T cell ratios (Table II). Next, the study subjects were divided into four groups based on ascending order of the levels or ratio of IL-17+ T cells and Foxp3+ T cells: the lowest quartile [quartile 1 (Q1), 1st to