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Vitamin D deficiency in pregnant women impairs regulatory T cell function
Journal of Steroid Biochemistry & Molecular Biology 147 (2015) 48–55
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Vitamin D deficiency in pregnant women impairs regulatory T cell function A. Vijayendra Chary a , R. Hemalatha a, * , M. Seshacharyulu a , M. Vasudeva Murali b , D. Jayaprakash c , B. Dinesh Kumar d a
Department of clinical microbiology and Immunology, National Institute of Nutrition (NIN), ICMR, Hyderabad, Telangana 500007, India Department of Paediatrics, Gandhi Hospital, Hyderabad, India Department of Technology, Osmania University, Hyderabad, India d Food and Drug Toxicology Division, NIN, Hyderabad, India b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 November 2014 Accepted 19 November 2014 Available online 21 November 2014
Regulatory T cells and IgE receptors (CD23 and CD21) on B cells were assessed in vitamin D deficient pregnant women. For this, 153 pregnant women were recruited from a government hospital and were categorized into three groups based on 25-hydroxyvitamin D3 (25(OH)D3) status. Regulatory T cell population (Treg cells) and CD23/CD21 expression on B cells were quantified by FACS ARIA II in maternal blood at third trimester; and the same parameters were evaluated in cord blood soon after delivery. In addition, TGF b and IL-10 were quantified in maternal and cord blood by using Milliplex kits. In a representative sample of eight women from each group (vitamin D sufficient, insufficient and deficient), placental tissues were processed for mRNA expressions of vitamin D receptor (VDR), retinoic acid receptor (RXR), vitamin D binding protein (VDBP) and vitamin D regulating enzymes. Of the 153 pregnant women, 18 were sufficient (30 ng/mL), 55 were insufficient (20–29 ng/mL) and 80 were deficient (19 ng/mL) for 25(OH)D3 status. The maternal blood Treg cell population (mean (%) SE) was lower (p < 0.05) in 25(OH)D3 deficient (0.2 0.01) pregnant women compared to insufficient (0.34 0.01) and sufficient (0.45 0.02) pregnant women. Similarly, cord blood Treg cell population (mean (%) SE) was also lower (p < 0.05) in 25(OH)D3 deficient (0.63 0.03) pregnant women when compared to insufficient (1.05 0.04) and sufficient (1.75 0.02) pregnant women. Mean (%) SE of B cells with CD23 and CD21 in maternal blood was higher (p < 0.05) in 25(OH)D3 deficient pregnant women (0.35 0.02; 1.65 0.04) when compared to insufficient (0.22 0.02; 0.55 0.05) and sufficient (0.15 0.02; 0.21 0.01) pregnant women. Similarly, mean (%) SE of B cell population with CD23 and CD21 in cord blood was also higher (p < 0.05) in 25(OH)D3 deficient (0.41 0.02; 1.2 0.03) when compared to insufficient (0.32 0.01; 0.6 0.05) and sufficient (0.2 0.01; 0.4 0.02) pregnant women. Regulatory cytokines, TGF b and IL-10 were lower (p < 0.05) in 25(OH)D3 insufficient and deficient subjects. In the placenta tissue of women with 25(OH)D3 deficiency, the regulatory T cell transcription factor FOXP3, vitamin D receptor (VDR) and retinoic acid receptor (RXR) expressions were downregulated. In contrast, CD23, CD21 and VDBP expressions were upregulated in 25(OH) D3 deficient and insufficient women. Vitamin D regulating enzymes (CYP24A1, CYP2R1 and CYP27B1) expression were also altered in women with 25(OH)D3 deficiency. The current study shows that impaired maternal 25(OH)D3 during pregnancy influences the spectrum of immune cells such as regulatory T cells and B cells with IgE receptors and this in turn may be linked to allergy and asthma in neonates. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Vitamin D VDR Regulatory T cells FOXP3 CD23 CD21 Pregnancy
Abbreviations: FOXP3, forkhead box P3; IgE, immunoglobulin E; CD, cluster of differentiation; HPLC, high-performance liquid chromatography; FACS, fluorescence activated cell sorter; TGF b, transforming growth factor beta; IL-10, interleukin-10; RXR, retinoic acid receptor; VDBP, vitamin D binding protein; VDR, vitamin D receptor; CYP24A1, cytochrome p24 A1 or 1,25-dihydroxyvitamin D3 24-hydroxylase; CYP2R1, vitamin D3 25-hydroxylase; CYP27B1, 25-hydroxyvitamin D3 1a-hydroxylase; 1,25 (OH)2D3, 1,25-dihydroxyvitamin D3; RBC, red blood cells; RQ, relative quantification. * Corresponding author. Tel.: +9140 27197297; fax: +9140 27019074. E-mail address: [email protected] (R. Hemalatha). http://dx.doi.org/10.1016/j.jsbmb.2014.11.020 0960-0760/ ã 2014 Elsevier Ltd. All rights reserved.
A. Vijayendra Chary et al. / Journal of Steroid Biochemistry & Molecular Biology 147 (2015) 48–55
1. Introduction Vitamin D deficiency in pregnant women has been linked with complications such as preeclampsia, insulin resistance and gestational diabetes [1–3]. More recently, low vitamin D has been associated with lung function and increased risk of atopic allergy in offspring [4]. Vitamin D receptor agonists have also been shown to induce tolerance, suppress allergen-specific IgE synthesis and promote regulatory T cell function (CD4+/CD25 +/CD127 /FOXP3 cells or Treg cells) [5,6]. Beneficial effects of vitamin D on atopic allergic responses have been suggested to be mediated through Treg cell induction [7]. Apart from allergen specific responses such as suppression of IgE mediated reactions and induction of regulatory cytokine [transforming growth factor beta (TGF b) and interleukin-10 (IL-10)], Treg cells are known to play a significant role in the maintenance of maternal tolerance to the fetus and are detected in the human decidua and peripheral blood throughout pregnancy [8,9]. Women with impaired Treg cell concentration suffer from spontaneous abortion and infertility, and therefore expression of Treg cells has been considered to be essential for successful pregnancy outcome [9,10]. Vitamin D deficiency is prevalent among 84% of pregnant women and 66.7% of infants in India; and therefore, we felt that it is important to understand the effect of vitamin D deficiency on Treg cell induction in pregnant women [11,12]. CD23 and CD21 are substrates on the B lymphocytes that act as receptors for IgE antibody and are further linked to asthma/allergy in children as well as in adults [13,14]. B cells can be identified by their characteristic presence of CD19 receptor, which is expressed during all stages of B-cell differentiation, maturation and proliferation [15,16]. The expression of CD23 and CD21 has been shown to have an important role in IgE production during fetal development [17]. Vitamin D3 is activated by two enzymatic steps. After conversion of 7-dehydrocholesterol to cholecalciferol (vitamin D3) by UVB radiation, vitamin D3 is metabolized to 25-hydroxycholecalciferol (calcidiol or 25-hydrxyvitamin D3 or 25(OH)D3) by the hepatic 25-hydroxylases (CYP2R1 or CYP27A1) in the liver. Subsequently, 25 (OH)D3 is converted to 1, 25-dihydroxyvitamin D3 (calcitriol or 1,25 (OH)2D3) by the enzyme, 1a-hydroxylase (CYP27B1) in the kidney. The active form of vitamin D, calcitriol, complexes with vitamin D receptor (VDR) and forms a heterodimer with 9-cis-retinoic acid receptor (RXR) and interacts with specific DNA sequences of target genes [18]. Calcitriol is produced in the cells of the proximal tubule of the nephron in the kidneys by the action of CYP27B1. Several other cell types, including dermal, intestinal epithelial cells, lymph nodes, monocytes and placenta, express CYP27B1 enzyme, that enables extra renal activation of 25(OH)D3 to the active hormonal form 1,25(OH)2D3 [18]. Vitamin D, VDR, RXR and vitamin D regulating enzymes are now known to be responsible for inducing several genes that are important in pregnant women [19]. Therefore, in the current study, apart from examining the regulatory T cell population and the B cell IgE receptors (CD23 and CD21) in maternal and cord blood of women with vitamin D deficiency, the vitamin D metabolizing enzymes (CYP2R1, CYP27B1 and CYP24A1), the VDR and the RXR were explored in the placenta of women to study their link with vitamin D deficiency. 2. Materials and methods 2.1. Ethical information This study was conducted according to the prescribed guidelines for human research. The study was approved by both the
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Institutional Ethics Committees from Gandhi hospital and National Institute of Nutrition, ICMR, Hyderabad. 2.2. Enrollment of study subjects One hundred and fifty three pregnant women, with gestational age of 36–39 weeks, singleton and with a BMI of 19–26 kg/m2, visiting Gandhi hospital (a tertiary care state government hospital) for their antenatal check-up, were included in the study. Women with gestational diabetes, pre-eclampsia, chronic hypertension, fetal anomaly, rheumatoid arthritis, parathyroid disorders, hepatic or renal diseases were excluded from the study. After collecting anthropometric data, a blood sample was collected and all the women were followed till delivery to collect cord blood, placenta and new-born anthropometry data. Maternal and cord blood sample 25(OH)D3 was quantified by high-performance liquid chromatography (HPLC). Treg cells, CD23 and CD21 on B cells were determined by fluorescence activated cell sorter (FACS ARIA II). All the 153 women were categorized into three groups based on their (25(OH)D3) status; such as vitamin D sufficient (25(OH)D3 level, 30 ng/mL), insufficient (25(OH)D3 level, 20–29 ng/mL) and deficient (25(OH)D3 level, 19 ng/mL) groups. In a representative sample of eight women from each group (vitamin D sufficient, insufficient and deficient) placenta tissues were processed for FOXP3, CD23 and CD21 gene expression. In addition, mRNA expression of VDR, RXR, vitamin D binding protein (VDBP) and vitamin D regulating enzyme (CYP2R1, CYP27B1 and CYP24A1) genes were evaluated by real time polymerase chain reaction (RT-PCR). 2.3. Sample collection Maternal blood in the third trimester of pregnancy and cord blood (soon after delivery) were collected in commercial containers containing EDTA. Placenta tissues were obtained immediately after delivery. Placental tissue pieces were dissected midway between the chorionic and basal plates. Two samples per placenta were taken free of visible infarction, calcification or hematoma. The placenta tissue samples were immediately washed with ice-cold saline to remove blood. Specimens of placenta were snap-frozen in liquid nitrogen and stored at 80 C until further processing. 2.4. Biochemical analysis 2.4.1. 25(OH)D3 quantification by HPLC in maternal and cord blood 25(OH)D3 was estimated in all maternal and cord blood samples by HPLC. Maternal and cord blood was allowed to clot, and serum was separated. 500 mL serum was used for this assay. Serum samples were extracted with n-hexane on Schimadzu system with a quaternary pump. Separation was done in SUPELCOSIL column (4.6 mm 25 cm; 5 m particle size; Sigma–Aldrich) and was maintained at 4 C. The mobile phase was methanol and water in 85:15 ratio with a flow rate of 2 mL/min. 25(OH)D3 was detected at 265 nm. 2.4.2. Regulatory T cell (CD4+/CD25+/CD127-/FOXP3) population in maternal and cord blood The proportion of Tregs was estimated in maternal and cord blood by the method described by Hrdy et al. [20]. Whole blood (100 mL) was stained for 20 min at room temperature with 20 mL of human regulatory T cell cocktail, which contained FITC anti-human CD4 antibody, PE-Cy7 anti-human CD25 antibody and Alexa Fluor1 647 anti-human CD127 antibody (Cat. No. 560249, BD Sciences, CA, USA). After 20 min the erythrocytes were lysed with 2 mL of BD FACS red blood cells (RBC) lysing buffer (Cat.
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No. 349202) and was centrifuged. After centrifugation, the supernatant was discarded and the cells were washed using 2 mL of BD Pharmingen stain buffer (Cat. No. 554656). For fixation of the cells, 2 mL of 1X working solution and human FOXP3 buffer A were added and incubated for 10 min at room temperature. The cells were then washed and centrifuged at 500 g for 10 min and the supernatant was discarded. To permeabilize the cells, 0.5 mL 1X human FOXP3 buffer C was added and was incubated for 30 min in dark room. The cells were again washed and were stained using the marker PE mouse anti-human FOXP3 (Cat. No. 560082) for 30 min in dark room. The mixture was washed with 2 mL of stain buffer and the pellet was suspended in 100 mL of flow staining buffer. 50,000 total events were acquired per sample in BD FACS ARIA II. The lymphocyte cells were first gated based on forward and side scatter to exclude dead cells and cell debris. CD4+ T cells were then gated based on the expression of CD4 marker. The CD4+ T cells were further gated for CD25+ and CD127 cells. CD4 +/CD25+/CD127 cells were further gated for FOXP3+ cells (CD4+ CD25+/CD127 /FOXP3+ or Treg cells). The data were obtained by FACS DIVA software and were analysed by FCS Express software.
Table 1 Primers used in the RT-PCR analysis.
2.4.3. B cells with CD23/CD21 receptors in maternal and cord blood The proportions of B cells with CD23 and CD21 receptors were estimated in maternal and cord blood. B cells were identified by their characteristic CD19 positivity. 100 mL whole blood was stained with 20 mL Alexa Fluor1 700 mouse anti-human CD19 antibody (Cat. No. 557921), 20 mL FITC mouse anti-human CD23 antibody (Cat. No. 561146) and 5 mL APC mouse anti-human CD21 (Cat. No. 559867), for 20 min at room temperature according to the manufacturer's instructions (BD Sciences, CA, USA). The RBCs were lysed by BD FACS RBC lysing solution (Cat. No. 349202) and after washing with 2 mL of flow staining buffer, the pellet was suspended in 100 mL of flow staining buffer. 50,000 events were acquired per sample on FACS ARIA II system. The B cells were gated based on CD19 marker and were further gated for CD 19+/CD 23+ and CD19+/CD21+ cells. The data were obtained by FACS DIVA software and were analysed by FCS Express software.
Geneiou's pro software (version; 5.4.6, New Zealand) and synthesized by Imperial Life Sciences, New Delhi, India (Table 1). GAPDH was used as internal control. In order to quantify gene expression of target genes like VDR, CD23, CD21 and FOXP3, the ABI step one plus system was used with program of 95 C for 10 min followed by 40 cycles of 95 C for 15 s and 60 C for one minute. The difference in cycle threshold (Ct) value between the control gene (GAPDH) and the target genes (CD23, CD21, FOXP3, VDR, CYP2R1, CYP27B1, CYP24A1, VDBP and RXR) was obtained for each sample. The critical threshold cycle (CT) value was determined using the ABI system and Step One plus software and was normalized with GAPDH. The difference in CT values (DCT) between the target genes and GAPDH was normalized to the corresponding DCT of the calibrator (DDCT) and was expressed in fold expression (2 (DDCT)) or relative quantification (RQ) to assess the relative difference in mRNA for each gene.
2.4.4. Cytokines, calcium and hemoglobin assays in maternal and cord blood Regulatory cytokines (TGF b and IL-10) assay was performed using the Milliplex xMAP technology according to the manufacturer instructions (Merck Millipore, USA). The intra and inter assay variability was less than 10%. Serum calcium was quantified by spectrophotometer using ACCUCARE lab kit. Hemoglobin was estimated by the Drabkin's (cyanmethemoglobin) method.
2.6. Data analysis and statistics
2.5. FOXP3, CD23, CD21, VDR, CYP2R1, CYP27B1, CYP24A1, VDBP and RXR gene expression in the placenta tissue by real-time PCR 2.5.1. Total RNA extraction from placenta tissue The placenta tissue was processed for total RNA extraction using Chomzinsky and Sacchi method. Total RNA was treated by DNase I (Ambion) according to manufacturer protocol. The quality and the yield of total RNA were checked using Agilent Bioanalyzer 2100 (Agilent Technologies) and Nanodrop 1000 (Thermo Scientific). Electrophoresis was employed to check integrity and purity of the RNA for cDNA synthesis by Masek et al. procedure [21]. 2.5.2. cDNA synthesis and gene expression from mRNA Five micrograms of total RNA were transcribed to cDNA using RevertAid first strand cDNA synthesis kit (Thermo Scientific). The cDNA thus obtained was aliquoted and stored at –20 C. For the RT-PCR assay, primers were designed by
Gene
Primer sequence
GAPDH
Forward: 51-TCGACAGTCAGCCGCATCTTCTTT-31 Reverse: 51-ACCAAATCCGTTGACTCCGACCTT-31 Forward:51-TGTAATCCCAGCAGTTTGGGAGGT-31 Reverse: 51-AGGGTTTCTCCATGTTGGTCAGGT-31 Forward: 51AGATCTACCACTGGTTCACACGCA-31 Reverse: 51-GCACAAAGCACTTGTGCAGACTCA-31 Forward: 51-GGAATTGAACGAGAGGAACGAAG-31 Reverse: 51-AAAGCCGCTGGACACCTG-31 Forward: 51-CCCATAGTACCAGGAGGATACA-31 Reverse: 51-CCGTTCATGGAGAAGTTGGT-31 Forward: 51-CCATGTGGCAGAAGGGATAA-31 Reverse: 51-AAACCGTAAACCAGGCTAGG-31 Forward: 51-GACAGACCATGCCTTCCTTTA-31 Reverse: 51-ATCGTCTGTGATCAACCCATC-31 Forward: 51-CGCCTCAGATGGTGGTATTT-31 Reverse: 51-AGCAGTGAACCCTGTAGAATG-31 Forward: 51-GGACCCTCCTTTGGTGAAAT-31 Reverse: 51-AGGATTGGGAACGGCTAAAG-31 Forward: 51-GGTACTTGAGCCAACCCTAAA-31 Reverse: 51-GTAGAGGGCCCTTAGCATTAAA-31
VDR FOXP3 CD23 CD21 CYP27B1 CYP2R1 CYP24A1 RXR VDBP
Base pairs 94 88 105 97 100 100 94 92 105 86
For descriptive data, mean values were calculated and differences between groups were assessed by Kruskal–Wallis test using SPSS software (version. 18.0). Graphical analysis was performed in SigmaPlotTM 12.3 (Systat Software, USA). Values are expressed in mean SE. The mean differences were considered significant when the p values were less than 0.05. Correlations of various parameters were analysed by Spearman's rank correlation coefficient. 3. Results One hundred and fifty three pregnant women visiting the hospital from rural and urban areas for their antenatal check-up were the subjects. Most women completed higher secondary schooling and a few were illiterate, and were either daily laborer or homemakers (Table 2). Majority of the women recruited in the study had insufficient milk intake and had less exposure to sunlight (Table 3). The mean SD of age and gestational age was 24.5 2.6 years and 39 1.5 weeks, respectively. The newborn birth weight mean SD was 2.59 0.40 kg (Table 3). The maternal serum calcium concentrations were comparable among 25(OH) D3 sufficient (9.11 2.1 mg/dL), insufficient (9.1 1.8 mg/dL) and deficient (8.91 mg/dL) pregnant women. Similarly, the hemoglobin concentrations were not different in 25(OH)D3 deficient (10.02 0.9 gm/dL), insufficient (10.72 0.7 gm/dL) and sufficient (10.71 0.5 gm/dL) pregnant women.
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Table 2 Socioeconomic status of pregnant women (153). Socio economic variables
Proportion (number)
Locality Urban Rural
37.90 (58) 62.09 (95)
Religion Hindu Muslim
75.16 (115) 24.84 (38)
Education Illiterate Primary School High School Post high school
12.41 (19) 34.64 (53) 31.37 (48) 21.56 (33)
Occupation Not working (house wife) Agriculture Other labour work
44.44 (68) 24.18 (37) 31.37 (48)
Socio economic status Upper Upper middle Middle/lower middle Lower/upper lower Lower
3.92 (6) 18.30 (28) 33.98 (52) 14.37 (22) 29.41 (45)
Values are given in percent. Parenthesis indicate number of subjects.
Table 3 Demographic and clinical profile of pregnant women (153). Characteristic
Values
Mean age (yrs) Gestational age (weeks) Hb (gm/dL) BMI Birth weight (kg) Head circumference (cm) Birth length (cm) Sunlight exposure duration (%) <60 min >60 min Milk consumption (%) <500 mL >500 mL
24.5 2.6 39.0 1.5 10.4 3.7 21.5 1.4 2.59 0.4 34.2 2.2 38.4 4.5 82.0 18.0 76.0 24.0
Values are mean SD, otherwise % of subjects.
3.1. 25(OH)D3 in maternal and cord blood Of the 153 pregnant women, 18 were 25(OH)D3 sufficient (30 ng/mL), 55 were 25(OH)D3 insufficient (20–29 ng/mL) and 80 were 25(OH)D3 deficient (19 ng/mL). The mean SD values of maternal and cord blood 25(OH)D3 are given in Table 4. Maternal 25(OH)D3 concentrations among all groups were significantly higher than cord blood and were directly correlated (r = 0.68; p = 0.001) (Fig. 1).
Fig. 1. Spearman's rank correlation between maternal and cord blood 25(OH)D3 (r = 0.68; p = 0.001).
3.2. TGF b and IL-10 regulatory cytokines in maternal and cord blood The mean SE of TGF b (pg/mL) in maternal blood was lower (p < 0.05) in 25(OH)D3 deficient (164 1.92) than in insufficient (197 3.4) and sufficient (236 4.2) pregnant women. Similarly, in cord blood the TGF b (pg/mL) was lower (p < 0.05) in 25(OH)D3 deficient (113 2.11) than in insufficient (136 3.77) and sufficient (176 5.4) groups (Fig. 2A). The mean SE of IL-10 (pg/mL) was also lower (p < 0.05) in 25(OH) D3 deficient (maternal, 16 0.3; cord, 12 0.39) than in insufficient (maternal, 20 0.48; cord, 16 0.36) and sufficient (maternal, 23 0.71; cord, 17 0.57) pregnant women (Fig. 2B). 3.3. Regulatory T cell (CD+/CD25+/CD127-/FOXP3) population in maternal and cord blood and FOXP3 gene expression in placenta tissue The maternal Treg cell population ranged from 0.1 to 0.7%; while, cord blood Treg cell population ranged from 0.1 to 2.8%. The Treg cell population (mean (%) SE) was significantly (p < 0.05) lower in 25(OH)D3 deficient (0.2 0.01) when compared to insufficient (0.34 0.01) and sufficient (0.45 0.02) pregnant women. Similarly, in cord blood the mean (%) SE of Treg cell population was lower (p < 0.05) in 25(OH)D3 deficient (0.63 0.03) when compared to insufficient (1.05 0.04) and sufficient (1.75 0.02) pregnant women (Fig. 3A). Maternal 25 (OH)D3 was directly correlated with maternal Treg cell population (r = 0.463; p = 0.001) (Fig. 3B). Similarly, cord blood 25(OH)D3 was directly correlated with cord blood Treg cell population (r = 0.512; p = 0.001) (Fig. 3C). In line with the above results, the FOXP3 mRNA expression in the placenta tissues was downregulated in 25(OH)D3 insufficient and deficient pregnant women and was significantly (p < 0.05) different from the sufficient subjects (Fig. 4).
Table 4 25(OH)D3 concentrations in maternal and cord blood. Subjects
Percentage (%) of subjects (153)
Maternal blood 25(OH)D3 (ng/mL)
Cord blood 25(OH)D3 (ng/mL)
p value
25(OH)D3 sufficient (18) 25(OH)D3 insufficient (55) 25(OH)D3 deficient (80)
11.7 35.9 52.2
37.04 2.74 23.16 3.05 9.5 5.19
16.12 3.01 12.58 3.7 6.8 5.2
p < 0.05 p < 0.05 p < 0.05
Values are expressed as mean SD. 30 ng/mL considered as 25(OH)D3 sufficient. 20–29 ng/mL considered as 25(OH)D3 insufficient. 19 ng/mL considered as 25(OH) D3 deficient.
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Fig. 2. (2A) TGF b concentration (pg/mL) in maternal and cord blood. (2B) IL-10 concentration (pg/mL) in maternal and cord blood. Different superscripts indicate significant difference at p < 0.05. Values are expressed as mean SE.
Fig. 3. (3A) Treg cell population in maternal and cord blood. Different superscripts indicate significant difference at p < 0.05. Values are expressed as mean SE. (3B) Spearman's rank correlation between maternal 25(OH)D3 and Treg cell population (r = 0.463; p = 0.001). (3C) Spearman's rank correlation between cord blood 25(OH)D3 and Treg cell population (r = 0.512; p = 0.001).
3.4. CD23 and CD21 receptors on B cells in maternal and cord blood and their gene's expressions in placenta tissue The maternal and cord blood B cells with CD23 expression ranged from 0.1 to 0.8% and 0.2 to 0.8%, respectively. In maternal blood, the proportions of B cells with CD23 expression (mean (%) SE) were significantly (p < 0.05) higher in 25(OH)D3 deficient (0.35 0.02) when compared to insufficient (0.22 0.02) and sufficient (0.15 0.02) pregnant women. Similarly, in cord blood the proportion of B cells with CD23 expression (mean (%) SE) was higher (p < 0.05) in 25(OH)D3 deficient (0.41 0.02) when
compared to insufficient (0.32 0.01) and sufficient (0.2 0.01) pregnant women (Fig. 5A). The maternal and cord blood B cells with CD21 expression ranged from 0.1 to 2.9% and 0.2 to 2.1%, respectively. Similar to B cells with CD23 expression, B cells with CD21 expression (mean (%) SE) in maternal blood were significantly higher (p < 0.05) in 25(OH)D3 deficient (1.65 0.04) than in insufficient (0.55 0.05) and sufficient pregnant women (0.21 0.01). In cord blood, the proportion of B cells with CD21 expression (mean (%) SE) was higher (p < 0.05) in 25 (OH)D3 deficient (1.2 0.03) than in insufficient (0.6 0.05) and sufficient pregnant women (0.4 0.02) (Fig. 5B). Maternal 25(OH)
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Fig. 4. mRNA expression (RQ) of CD23, CD21 and FOXP3 genes in placenta tissue of women with 25(OH)D3 deficiency. 25(OH)D3 sufficient group was taken as calibrator and GAPDH was used as internal control. Different superscripts indicate significant difference at p < 0.05. Values are expressed as mean SE.
D3 was inversely correlated with maternal B cells with CD23 (r = 0.389; p = 0.001) and CD21 (r = 0.783; p = 0.001) expression (data not shown); and likewise, maternal blood Treg cell population was inversely correlated with maternal B cells with CD23 (r = 0.298; p = 0.001) and CD21 expression (r = 0.538; p = 0.001) (data not shown). As expected, the placenta tissues CD23 and CD21 mRNA gene expressions were upregulated in 25 (OH)D3 insufficient and deficient women (Fig. 4). 3.5. Vitamin D regulating enzymes and receptor genes Placenta tissue mRNA expressions are expressed in relative quantification (RQ) values and shown in Fig. 6. The VDR was downregulated (p < 0.05) in the placenta tissue of women with 25 (OH)D3 deficiency, but was comparable between 25(OH) D3 sufficient and insufficient pregnant women. The nuclear receptor RXR expression was significantly (p < 0.05) downregulated in 25(OH)D3 deficient and insufficient women. As for the vitamin D activating enzymes, the CYP27B1 was downregulated, while CYP2R1 was upregulated in women with 25(OH) D3 insufficiency and deficiency and were significantly different (p < 0.05) from sufficient women. The vitamin D inactivating
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Fig. 6. mRNA expression (RQ) of VDR, CYP27B1CYP2R1, CYP24A1, VDBP and RXR genes in the placenta tissue of women. Vitamin D sufficient group was taken as calibrator and GAPDH was taken as internal control. Different superscripts indicate significant difference at p < 0.05. Values are expressed as mean SE.
enzyme, CYP24A1 and the VDBP were upregulated in the placenta of women with 25(OH)D3 insufficiency and deficiency (Fig. 6). 4. Discussion The results from the current study show that impaired maternal 25(OH)D3 during pregnancy influences the spectrum of immune cells such as regulatory T cells and B cells with IgE receptors in the maternal and cord blood, which in turn may be important in maintaining optimal fetal health and pregnancy outcomes. Under-nutrition is commonly prevalent in India, where 30% of pregnant women are chronically undernourished (body mass index (BMI), <18.5) and 84.9% are anemic. About 30% of new-borns in India are low birth weight (LBW) babies and 13% of deliveries are preterm [22,23]. The average hemoglobin concentration of pregnant women in India is 10.8 gm/dL, and the mean gestational age, birth weight and hemoglobin concentration observed in this study are close to the national average [23]. The subjects studied here represent the majority of pregnant women population in India and therefore the results can be generalised. As for vitamin D, a few studies have shown that about 84% of pregnant women have inadequate vitamin D concentration [11].
Fig. 5. (5A) B cells with CD23 expression in maternal and cord blood. (5B) B cells with CD21 expression in maternal and cord blood. Different superscripts indicate significant difference at p < 0.05. Values are expressed as mean SE.
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Though India is a tropical country with immense sun shine, only 18 out of 153 women had sufficient 25(OH)D3 concentration in our studies [11,12]. The cultural practice of confining pregnant women to indoors and thus keeping them away from daily activity in agricultural fields or outside homes may be one strong reason for less sunlight exposure and thus vitamin D deficiency. Moreover, significant proportions of pregnant women in our study were Muslims, who are usually covered from head to toe and thus are not exposed to sunshine. So far, human transplacental transport of 1,25(OH)2D3 has not been studied. In rats, however, transport of inactive forms of vitamin D (25(OH)2D3 and 24,25(OH)2D3) but not active form of vitamin D (1,25(OH)2D3) was shown through the placenta [24]. In our study, correlation of the maternal with the cord blood 25(OH) D3 suggests that higher serum concentrations of 25(OH)D3 in maternal blood would facilitate passage of 25(OH)D3 from the mother to the fetus. The hydroxylation of 25(OH)D3 to the active form 1,25 (OH)2D3 is performed by CYP27B1, whereas, CYP24A1 is central for 1,25(OH)2D3 catabolism and degradation [25]. Conversely, CYP2R1 has been suggested to be active in the hydroxylation of both ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3) [25,26]. The CYP27B1 enzyme has been shown to generate a large amount of 1,25(OH)2D3 in the decidua and the placenta. Thus, both CYP27B1 and CYP24A1 play an important role in governing tissue vitamin D concentrations [27–29]. In the current study, increased expression of CYP2R1 on the background of low CYP27B1, and high catabolic CYP24A1 enzyme may be presumed as an attempt to increase the availability of the active form of vitamin D in the vitamin D deficient pregnant women. The observed altered expressions of vitamin D metabolising enzymes suggest altered vitamin D homeostasis in pregnant women with vitamin D deficiency, which is likely to play an important role in the fetal immune development and overall fetal developmentas well. In contrast to our finding, vitamin D deficiency is usually associated with low circulating VDBP, a key carrier binding protein for 25(OH)D3 and 1,25(OH)2D3 [30]. The majority of circulating 25 (OH)D3 is bound to VDBP, and its uptake into cells occurs in both bound and unbound forms [31]. Low VDBP has been suggested to have a protective role against the symptoms associated with lower vitamin D status. Conversely, in the current study we registered an increase in VDBP gene expression, the reason for which is unclear [32]. VDR acts primarily as a heterodimer with the retinoid X receptor (RXR) on vitamin D response elements (VDREs) [33]. VDR is abundantly present in human placenta and plays a critical role in transportation of calcium from mother to fetus. In addition, VDR expression regulates up to 500 vitamin D responsive genes, which influence mineral metabolism, differentiation and proliferation of various cells [34]. Hence, low VDR and RXR expressions in the vitamin D deficient pregnant women as observed in the present study must be viewed critically. Both prenatal and perinatal vitamin D deficiency have been shown to influence early life respiratory morbidity and have been suggested to be vital for lung development and growth. Moreover, low cord blood 25(OH)D3 levels are associated with respiratory syncytial viral bronchiolitis in the first year with a relative risk of 6.2 [35]. Furthermore, some studies have demonstrated prevention of exaggerated autoimmune response in diabetes mellitus upon supplementation of cholecalciferol, which was associated with increased Treg cells percent [36,37]. Vitamin D may play an important role in the maintenance of B cell homeostasis and may be useful in the treatment of B cell-mediated autoimmune disorders [38]. In this study, low Treg cells and increased CD21 and CD23 expression on B cells may have a key role as receptors of IgE antibody and conceivably are linked with increased respiratory morbidity among vitamin D deficient
subjects. From the results of the present study, it may be speculated that vitamin D deficiency may increase early life respiratory morbidity by impairing T cell regulatory function and by increasing B cells with IgE receptors. Treg cell population increases right from the very early stages of pregnancy. Women with impaired Treg cell concentration suffer from spontaneous abortion and infertility and therefore expression of Treg cells has been considered to be essential for successful pregnancy outcome [39]. The migration mechanisms of Treg cells to the pregnant uterus are still unclear, however, human chorionic gonadotropin (hCG), secreted by the trophoblasts after fertilization has been shown to attract Treg cells to the fetal–maternal interface [40]. In pregnant women Treg cell population that comprises less than 1% of peripheral CD4+ cells has the ability to suppress both Th1 and Th2 immunity against paternal/fetal alloantigen, a process, critical for immune regulation and continuation of pregnancy [41]. This is the first study to demonstrate low circulating Treg cells in both maternal and cord blood and impaired FOXP3 gene expression in placenta of vitamin D deficient pregnant women. However, Treg cell response to vitamin D supplementation would have delineated the causal role of vitamin D on Treg cell function. Nevertheless, in view of the proposed link between vitamin D deficiency and adverse events in pregnancy, the results of the current study become more interesting. The study shows that impaired maternal vitamin D during pregnancy may influence a spectrum of immune cells in the mother and placenta, which in turn may be linked to allergy and asthma in neonates. The role of vitamin D in immune function and inflammation in pregnant women must not be underestimated and in light of the existing literature, estimation of serum levels of vitamin D and supplementation in pregnant women must be considered. This is needed especially keeping in view the extent to which low vitamin D status and impaired Treg cell function in utero may lead to the development of chronic diseases in adult life. Conflict of interest The authors report no conflict of interest. Acknowledgments The authors sincerely acknowledge the Golden Triangle Programme (funded by ICMR,CSIR and ICAR) for providing financial support and the Director, National Institute of Nutrition for providing facilities to conduct the study. The authors greatly acknowledge the CSIR-HRDG for the SRF fellowship to Mr. Vijayendra Chary. The authors are thankful to the superintendent of Gandhi Hospital and the nursing staff especially Mrs. Aruna Reddy for their immense support in the recruitment of subjects and collection of samples. References [1] L.M. Bodnar, J.M. Catov, H.N. Simhan, M.F. Holick, R.W. Powers, J.M. Roberts, Maternal vitamin D deficiency increases the risk of preeclampsia, J. Clin. Endocrinol. Metab. 92 (2007) 3517–3522. [2] K.C. Chiu, A. Chu, V.L. Go, Md.F. Saad, Hypovitaminosis D is associated with insulin resistance and beta cell dysfunction, Am. J. Clin. Nutr. 79 (5) (2004) 820–825. [3] A.G. Pittas, J. Lau, F.B. Hu, B. Dawson-Hughes, The role of vitamin D and calcium in type 2 diabetes. A systematic review and meta-analysis, J. Clin. Endocrinol. Metab. 92 (6) (2007) 2017–2029. [4] M. Erkkola, B.I. Nwaru, H.T. Viljakainen, Maternal vitamin D during pregnancy and its relation to immune-mediated diseases in the offspring, Vitam. Horm. 86 (2011) 239–260. [5] S. Dimeloe, A. Nanzer, K. Ryanna, C. Hawrylowicz, Regulatory T cells, inflammation and the allergic response: the role of glucocorticoids and vitamin D, J. Steroid Biochem. Mol. Biol. 120 (2010) 86–95.
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Vitamin D deficiency in pregnant women impairs regulatory T cell function A. Vijayendra Chary a , R. Hemalatha a, * , M. Seshacharyulu a , M. Vasudeva Murali b , D. Jayaprakash c , B. Dinesh Kumar d a
Department of clinical microbiology and Immunology, National Institute of Nutrition (NIN), ICMR, Hyderabad, Telangana 500007, India Department of Paediatrics, Gandhi Hospital, Hyderabad, India Department of Technology, Osmania University, Hyderabad, India d Food and Drug Toxicology Division, NIN, Hyderabad, India b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 November 2014 Accepted 19 November 2014 Available online 21 November 2014
Regulatory T cells and IgE receptors (CD23 and CD21) on B cells were assessed in vitamin D deficient pregnant women. For this, 153 pregnant women were recruited from a government hospital and were categorized into three groups based on 25-hydroxyvitamin D3 (25(OH)D3) status. Regulatory T cell population (Treg cells) and CD23/CD21 expression on B cells were quantified by FACS ARIA II in maternal blood at third trimester; and the same parameters were evaluated in cord blood soon after delivery. In addition, TGF b and IL-10 were quantified in maternal and cord blood by using Milliplex kits. In a representative sample of eight women from each group (vitamin D sufficient, insufficient and deficient), placental tissues were processed for mRNA expressions of vitamin D receptor (VDR), retinoic acid receptor (RXR), vitamin D binding protein (VDBP) and vitamin D regulating enzymes. Of the 153 pregnant women, 18 were sufficient (30 ng/mL), 55 were insufficient (20–29 ng/mL) and 80 were deficient (19 ng/mL) for 25(OH)D3 status. The maternal blood Treg cell population (mean (%) SE) was lower (p < 0.05) in 25(OH)D3 deficient (0.2 0.01) pregnant women compared to insufficient (0.34 0.01) and sufficient (0.45 0.02) pregnant women. Similarly, cord blood Treg cell population (mean (%) SE) was also lower (p < 0.05) in 25(OH)D3 deficient (0.63 0.03) pregnant women when compared to insufficient (1.05 0.04) and sufficient (1.75 0.02) pregnant women. Mean (%) SE of B cells with CD23 and CD21 in maternal blood was higher (p < 0.05) in 25(OH)D3 deficient pregnant women (0.35 0.02; 1.65 0.04) when compared to insufficient (0.22 0.02; 0.55 0.05) and sufficient (0.15 0.02; 0.21 0.01) pregnant women. Similarly, mean (%) SE of B cell population with CD23 and CD21 in cord blood was also higher (p < 0.05) in 25(OH)D3 deficient (0.41 0.02; 1.2 0.03) when compared to insufficient (0.32 0.01; 0.6 0.05) and sufficient (0.2 0.01; 0.4 0.02) pregnant women. Regulatory cytokines, TGF b and IL-10 were lower (p < 0.05) in 25(OH)D3 insufficient and deficient subjects. In the placenta tissue of women with 25(OH)D3 deficiency, the regulatory T cell transcription factor FOXP3, vitamin D receptor (VDR) and retinoic acid receptor (RXR) expressions were downregulated. In contrast, CD23, CD21 and VDBP expressions were upregulated in 25(OH) D3 deficient and insufficient women. Vitamin D regulating enzymes (CYP24A1, CYP2R1 and CYP27B1) expression were also altered in women with 25(OH)D3 deficiency. The current study shows that impaired maternal 25(OH)D3 during pregnancy influences the spectrum of immune cells such as regulatory T cells and B cells with IgE receptors and this in turn may be linked to allergy and asthma in neonates. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Vitamin D VDR Regulatory T cells FOXP3 CD23 CD21 Pregnancy
Abbreviations: FOXP3, forkhead box P3; IgE, immunoglobulin E; CD, cluster of differentiation; HPLC, high-performance liquid chromatography; FACS, fluorescence activated cell sorter; TGF b, transforming growth factor beta; IL-10, interleukin-10; RXR, retinoic acid receptor; VDBP, vitamin D binding protein; VDR, vitamin D receptor; CYP24A1, cytochrome p24 A1 or 1,25-dihydroxyvitamin D3 24-hydroxylase; CYP2R1, vitamin D3 25-hydroxylase; CYP27B1, 25-hydroxyvitamin D3 1a-hydroxylase; 1,25 (OH)2D3, 1,25-dihydroxyvitamin D3; RBC, red blood cells; RQ, relative quantification. * Corresponding author. Tel.: +9140 27197297; fax: +9140 27019074. E-mail address: [email protected] (R. Hemalatha). http://dx.doi.org/10.1016/j.jsbmb.2014.11.020 0960-0760/ ã 2014 Elsevier Ltd. All rights reserved.
A. Vijayendra Chary et al. / Journal of Steroid Biochemistry & Molecular Biology 147 (2015) 48–55
1. Introduction Vitamin D deficiency in pregnant women has been linked with complications such as preeclampsia, insulin resistance and gestational diabetes [1–3]. More recently, low vitamin D has been associated with lung function and increased risk of atopic allergy in offspring [4]. Vitamin D receptor agonists have also been shown to induce tolerance, suppress allergen-specific IgE synthesis and promote regulatory T cell function (CD4+/CD25 +/CD127 /FOXP3 cells or Treg cells) [5,6]. Beneficial effects of vitamin D on atopic allergic responses have been suggested to be mediated through Treg cell induction [7]. Apart from allergen specific responses such as suppression of IgE mediated reactions and induction of regulatory cytokine [transforming growth factor beta (TGF b) and interleukin-10 (IL-10)], Treg cells are known to play a significant role in the maintenance of maternal tolerance to the fetus and are detected in the human decidua and peripheral blood throughout pregnancy [8,9]. Women with impaired Treg cell concentration suffer from spontaneous abortion and infertility, and therefore expression of Treg cells has been considered to be essential for successful pregnancy outcome [9,10]. Vitamin D deficiency is prevalent among 84% of pregnant women and 66.7% of infants in India; and therefore, we felt that it is important to understand the effect of vitamin D deficiency on Treg cell induction in pregnant women [11,12]. CD23 and CD21 are substrates on the B lymphocytes that act as receptors for IgE antibody and are further linked to asthma/allergy in children as well as in adults [13,14]. B cells can be identified by their characteristic presence of CD19 receptor, which is expressed during all stages of B-cell differentiation, maturation and proliferation [15,16]. The expression of CD23 and CD21 has been shown to have an important role in IgE production during fetal development [17]. Vitamin D3 is activated by two enzymatic steps. After conversion of 7-dehydrocholesterol to cholecalciferol (vitamin D3) by UVB radiation, vitamin D3 is metabolized to 25-hydroxycholecalciferol (calcidiol or 25-hydrxyvitamin D3 or 25(OH)D3) by the hepatic 25-hydroxylases (CYP2R1 or CYP27A1) in the liver. Subsequently, 25 (OH)D3 is converted to 1, 25-dihydroxyvitamin D3 (calcitriol or 1,25 (OH)2D3) by the enzyme, 1a-hydroxylase (CYP27B1) in the kidney. The active form of vitamin D, calcitriol, complexes with vitamin D receptor (VDR) and forms a heterodimer with 9-cis-retinoic acid receptor (RXR) and interacts with specific DNA sequences of target genes [18]. Calcitriol is produced in the cells of the proximal tubule of the nephron in the kidneys by the action of CYP27B1. Several other cell types, including dermal, intestinal epithelial cells, lymph nodes, monocytes and placenta, express CYP27B1 enzyme, that enables extra renal activation of 25(OH)D3 to the active hormonal form 1,25(OH)2D3 [18]. Vitamin D, VDR, RXR and vitamin D regulating enzymes are now known to be responsible for inducing several genes that are important in pregnant women [19]. Therefore, in the current study, apart from examining the regulatory T cell population and the B cell IgE receptors (CD23 and CD21) in maternal and cord blood of women with vitamin D deficiency, the vitamin D metabolizing enzymes (CYP2R1, CYP27B1 and CYP24A1), the VDR and the RXR were explored in the placenta of women to study their link with vitamin D deficiency. 2. Materials and methods 2.1. Ethical information This study was conducted according to the prescribed guidelines for human research. The study was approved by both the
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Institutional Ethics Committees from Gandhi hospital and National Institute of Nutrition, ICMR, Hyderabad. 2.2. Enrollment of study subjects One hundred and fifty three pregnant women, with gestational age of 36–39 weeks, singleton and with a BMI of 19–26 kg/m2, visiting Gandhi hospital (a tertiary care state government hospital) for their antenatal check-up, were included in the study. Women with gestational diabetes, pre-eclampsia, chronic hypertension, fetal anomaly, rheumatoid arthritis, parathyroid disorders, hepatic or renal diseases were excluded from the study. After collecting anthropometric data, a blood sample was collected and all the women were followed till delivery to collect cord blood, placenta and new-born anthropometry data. Maternal and cord blood sample 25(OH)D3 was quantified by high-performance liquid chromatography (HPLC). Treg cells, CD23 and CD21 on B cells were determined by fluorescence activated cell sorter (FACS ARIA II). All the 153 women were categorized into three groups based on their (25(OH)D3) status; such as vitamin D sufficient (25(OH)D3 level, 30 ng/mL), insufficient (25(OH)D3 level, 20–29 ng/mL) and deficient (25(OH)D3 level, 19 ng/mL) groups. In a representative sample of eight women from each group (vitamin D sufficient, insufficient and deficient) placenta tissues were processed for FOXP3, CD23 and CD21 gene expression. In addition, mRNA expression of VDR, RXR, vitamin D binding protein (VDBP) and vitamin D regulating enzyme (CYP2R1, CYP27B1 and CYP24A1) genes were evaluated by real time polymerase chain reaction (RT-PCR). 2.3. Sample collection Maternal blood in the third trimester of pregnancy and cord blood (soon after delivery) were collected in commercial containers containing EDTA. Placenta tissues were obtained immediately after delivery. Placental tissue pieces were dissected midway between the chorionic and basal plates. Two samples per placenta were taken free of visible infarction, calcification or hematoma. The placenta tissue samples were immediately washed with ice-cold saline to remove blood. Specimens of placenta were snap-frozen in liquid nitrogen and stored at 80 C until further processing. 2.4. Biochemical analysis 2.4.1. 25(OH)D3 quantification by HPLC in maternal and cord blood 25(OH)D3 was estimated in all maternal and cord blood samples by HPLC. Maternal and cord blood was allowed to clot, and serum was separated. 500 mL serum was used for this assay. Serum samples were extracted with n-hexane on Schimadzu system with a quaternary pump. Separation was done in SUPELCOSIL column (4.6 mm 25 cm; 5 m particle size; Sigma–Aldrich) and was maintained at 4 C. The mobile phase was methanol and water in 85:15 ratio with a flow rate of 2 mL/min. 25(OH)D3 was detected at 265 nm. 2.4.2. Regulatory T cell (CD4+/CD25+/CD127-/FOXP3) population in maternal and cord blood The proportion of Tregs was estimated in maternal and cord blood by the method described by Hrdy et al. [20]. Whole blood (100 mL) was stained for 20 min at room temperature with 20 mL of human regulatory T cell cocktail, which contained FITC anti-human CD4 antibody, PE-Cy7 anti-human CD25 antibody and Alexa Fluor1 647 anti-human CD127 antibody (Cat. No. 560249, BD Sciences, CA, USA). After 20 min the erythrocytes were lysed with 2 mL of BD FACS red blood cells (RBC) lysing buffer (Cat.
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No. 349202) and was centrifuged. After centrifugation, the supernatant was discarded and the cells were washed using 2 mL of BD Pharmingen stain buffer (Cat. No. 554656). For fixation of the cells, 2 mL of 1X working solution and human FOXP3 buffer A were added and incubated for 10 min at room temperature. The cells were then washed and centrifuged at 500 g for 10 min and the supernatant was discarded. To permeabilize the cells, 0.5 mL 1X human FOXP3 buffer C was added and was incubated for 30 min in dark room. The cells were again washed and were stained using the marker PE mouse anti-human FOXP3 (Cat. No. 560082) for 30 min in dark room. The mixture was washed with 2 mL of stain buffer and the pellet was suspended in 100 mL of flow staining buffer. 50,000 total events were acquired per sample in BD FACS ARIA II. The lymphocyte cells were first gated based on forward and side scatter to exclude dead cells and cell debris. CD4+ T cells were then gated based on the expression of CD4 marker. The CD4+ T cells were further gated for CD25+ and CD127 cells. CD4 +/CD25+/CD127 cells were further gated for FOXP3+ cells (CD4+ CD25+/CD127 /FOXP3+ or Treg cells). The data were obtained by FACS DIVA software and were analysed by FCS Express software.
Table 1 Primers used in the RT-PCR analysis.
2.4.3. B cells with CD23/CD21 receptors in maternal and cord blood The proportions of B cells with CD23 and CD21 receptors were estimated in maternal and cord blood. B cells were identified by their characteristic CD19 positivity. 100 mL whole blood was stained with 20 mL Alexa Fluor1 700 mouse anti-human CD19 antibody (Cat. No. 557921), 20 mL FITC mouse anti-human CD23 antibody (Cat. No. 561146) and 5 mL APC mouse anti-human CD21 (Cat. No. 559867), for 20 min at room temperature according to the manufacturer's instructions (BD Sciences, CA, USA). The RBCs were lysed by BD FACS RBC lysing solution (Cat. No. 349202) and after washing with 2 mL of flow staining buffer, the pellet was suspended in 100 mL of flow staining buffer. 50,000 events were acquired per sample on FACS ARIA II system. The B cells were gated based on CD19 marker and were further gated for CD 19+/CD 23+ and CD19+/CD21+ cells. The data were obtained by FACS DIVA software and were analysed by FCS Express software.
Geneiou's pro software (version; 5.4.6, New Zealand) and synthesized by Imperial Life Sciences, New Delhi, India (Table 1). GAPDH was used as internal control. In order to quantify gene expression of target genes like VDR, CD23, CD21 and FOXP3, the ABI step one plus system was used with program of 95 C for 10 min followed by 40 cycles of 95 C for 15 s and 60 C for one minute. The difference in cycle threshold (Ct) value between the control gene (GAPDH) and the target genes (CD23, CD21, FOXP3, VDR, CYP2R1, CYP27B1, CYP24A1, VDBP and RXR) was obtained for each sample. The critical threshold cycle (CT) value was determined using the ABI system and Step One plus software and was normalized with GAPDH. The difference in CT values (DCT) between the target genes and GAPDH was normalized to the corresponding DCT of the calibrator (DDCT) and was expressed in fold expression (2 (DDCT)) or relative quantification (RQ) to assess the relative difference in mRNA for each gene.
2.4.4. Cytokines, calcium and hemoglobin assays in maternal and cord blood Regulatory cytokines (TGF b and IL-10) assay was performed using the Milliplex xMAP technology according to the manufacturer instructions (Merck Millipore, USA). The intra and inter assay variability was less than 10%. Serum calcium was quantified by spectrophotometer using ACCUCARE lab kit. Hemoglobin was estimated by the Drabkin's (cyanmethemoglobin) method.
2.6. Data analysis and statistics
2.5. FOXP3, CD23, CD21, VDR, CYP2R1, CYP27B1, CYP24A1, VDBP and RXR gene expression in the placenta tissue by real-time PCR 2.5.1. Total RNA extraction from placenta tissue The placenta tissue was processed for total RNA extraction using Chomzinsky and Sacchi method. Total RNA was treated by DNase I (Ambion) according to manufacturer protocol. The quality and the yield of total RNA were checked using Agilent Bioanalyzer 2100 (Agilent Technologies) and Nanodrop 1000 (Thermo Scientific). Electrophoresis was employed to check integrity and purity of the RNA for cDNA synthesis by Masek et al. procedure [21]. 2.5.2. cDNA synthesis and gene expression from mRNA Five micrograms of total RNA were transcribed to cDNA using RevertAid first strand cDNA synthesis kit (Thermo Scientific). The cDNA thus obtained was aliquoted and stored at –20 C. For the RT-PCR assay, primers were designed by
Gene
Primer sequence
GAPDH
Forward: 51-TCGACAGTCAGCCGCATCTTCTTT-31 Reverse: 51-ACCAAATCCGTTGACTCCGACCTT-31 Forward:51-TGTAATCCCAGCAGTTTGGGAGGT-31 Reverse: 51-AGGGTTTCTCCATGTTGGTCAGGT-31 Forward: 51AGATCTACCACTGGTTCACACGCA-31 Reverse: 51-GCACAAAGCACTTGTGCAGACTCA-31 Forward: 51-GGAATTGAACGAGAGGAACGAAG-31 Reverse: 51-AAAGCCGCTGGACACCTG-31 Forward: 51-CCCATAGTACCAGGAGGATACA-31 Reverse: 51-CCGTTCATGGAGAAGTTGGT-31 Forward: 51-CCATGTGGCAGAAGGGATAA-31 Reverse: 51-AAACCGTAAACCAGGCTAGG-31 Forward: 51-GACAGACCATGCCTTCCTTTA-31 Reverse: 51-ATCGTCTGTGATCAACCCATC-31 Forward: 51-CGCCTCAGATGGTGGTATTT-31 Reverse: 51-AGCAGTGAACCCTGTAGAATG-31 Forward: 51-GGACCCTCCTTTGGTGAAAT-31 Reverse: 51-AGGATTGGGAACGGCTAAAG-31 Forward: 51-GGTACTTGAGCCAACCCTAAA-31 Reverse: 51-GTAGAGGGCCCTTAGCATTAAA-31
VDR FOXP3 CD23 CD21 CYP27B1 CYP2R1 CYP24A1 RXR VDBP
Base pairs 94 88 105 97 100 100 94 92 105 86
For descriptive data, mean values were calculated and differences between groups were assessed by Kruskal–Wallis test using SPSS software (version. 18.0). Graphical analysis was performed in SigmaPlotTM 12.3 (Systat Software, USA). Values are expressed in mean SE. The mean differences were considered significant when the p values were less than 0.05. Correlations of various parameters were analysed by Spearman's rank correlation coefficient. 3. Results One hundred and fifty three pregnant women visiting the hospital from rural and urban areas for their antenatal check-up were the subjects. Most women completed higher secondary schooling and a few were illiterate, and were either daily laborer or homemakers (Table 2). Majority of the women recruited in the study had insufficient milk intake and had less exposure to sunlight (Table 3). The mean SD of age and gestational age was 24.5 2.6 years and 39 1.5 weeks, respectively. The newborn birth weight mean SD was 2.59 0.40 kg (Table 3). The maternal serum calcium concentrations were comparable among 25(OH) D3 sufficient (9.11 2.1 mg/dL), insufficient (9.1 1.8 mg/dL) and deficient (8.91 mg/dL) pregnant women. Similarly, the hemoglobin concentrations were not different in 25(OH)D3 deficient (10.02 0.9 gm/dL), insufficient (10.72 0.7 gm/dL) and sufficient (10.71 0.5 gm/dL) pregnant women.
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Table 2 Socioeconomic status of pregnant women (153). Socio economic variables
Proportion (number)
Locality Urban Rural
37.90 (58) 62.09 (95)
Religion Hindu Muslim
75.16 (115) 24.84 (38)
Education Illiterate Primary School High School Post high school
12.41 (19) 34.64 (53) 31.37 (48) 21.56 (33)
Occupation Not working (house wife) Agriculture Other labour work
44.44 (68) 24.18 (37) 31.37 (48)
Socio economic status Upper Upper middle Middle/lower middle Lower/upper lower Lower
3.92 (6) 18.30 (28) 33.98 (52) 14.37 (22) 29.41 (45)
Values are given in percent. Parenthesis indicate number of subjects.
Table 3 Demographic and clinical profile of pregnant women (153). Characteristic
Values
Mean age (yrs) Gestational age (weeks) Hb (gm/dL) BMI Birth weight (kg) Head circumference (cm) Birth length (cm) Sunlight exposure duration (%) <60 min >60 min Milk consumption (%) <500 mL >500 mL
24.5 2.6 39.0 1.5 10.4 3.7 21.5 1.4 2.59 0.4 34.2 2.2 38.4 4.5 82.0 18.0 76.0 24.0
Values are mean SD, otherwise % of subjects.
3.1. 25(OH)D3 in maternal and cord blood Of the 153 pregnant women, 18 were 25(OH)D3 sufficient (30 ng/mL), 55 were 25(OH)D3 insufficient (20–29 ng/mL) and 80 were 25(OH)D3 deficient (19 ng/mL). The mean SD values of maternal and cord blood 25(OH)D3 are given in Table 4. Maternal 25(OH)D3 concentrations among all groups were significantly higher than cord blood and were directly correlated (r = 0.68; p = 0.001) (Fig. 1).
Fig. 1. Spearman's rank correlation between maternal and cord blood 25(OH)D3 (r = 0.68; p = 0.001).
3.2. TGF b and IL-10 regulatory cytokines in maternal and cord blood The mean SE of TGF b (pg/mL) in maternal blood was lower (p < 0.05) in 25(OH)D3 deficient (164 1.92) than in insufficient (197 3.4) and sufficient (236 4.2) pregnant women. Similarly, in cord blood the TGF b (pg/mL) was lower (p < 0.05) in 25(OH)D3 deficient (113 2.11) than in insufficient (136 3.77) and sufficient (176 5.4) groups (Fig. 2A). The mean SE of IL-10 (pg/mL) was also lower (p < 0.05) in 25(OH) D3 deficient (maternal, 16 0.3; cord, 12 0.39) than in insufficient (maternal, 20 0.48; cord, 16 0.36) and sufficient (maternal, 23 0.71; cord, 17 0.57) pregnant women (Fig. 2B). 3.3. Regulatory T cell (CD+/CD25+/CD127-/FOXP3) population in maternal and cord blood and FOXP3 gene expression in placenta tissue The maternal Treg cell population ranged from 0.1 to 0.7%; while, cord blood Treg cell population ranged from 0.1 to 2.8%. The Treg cell population (mean (%) SE) was significantly (p < 0.05) lower in 25(OH)D3 deficient (0.2 0.01) when compared to insufficient (0.34 0.01) and sufficient (0.45 0.02) pregnant women. Similarly, in cord blood the mean (%) SE of Treg cell population was lower (p < 0.05) in 25(OH)D3 deficient (0.63 0.03) when compared to insufficient (1.05 0.04) and sufficient (1.75 0.02) pregnant women (Fig. 3A). Maternal 25 (OH)D3 was directly correlated with maternal Treg cell population (r = 0.463; p = 0.001) (Fig. 3B). Similarly, cord blood 25(OH)D3 was directly correlated with cord blood Treg cell population (r = 0.512; p = 0.001) (Fig. 3C). In line with the above results, the FOXP3 mRNA expression in the placenta tissues was downregulated in 25(OH)D3 insufficient and deficient pregnant women and was significantly (p < 0.05) different from the sufficient subjects (Fig. 4).
Table 4 25(OH)D3 concentrations in maternal and cord blood. Subjects
Percentage (%) of subjects (153)
Maternal blood 25(OH)D3 (ng/mL)
Cord blood 25(OH)D3 (ng/mL)
p value
25(OH)D3 sufficient (18) 25(OH)D3 insufficient (55) 25(OH)D3 deficient (80)
11.7 35.9 52.2
37.04 2.74 23.16 3.05 9.5 5.19
16.12 3.01 12.58 3.7 6.8 5.2
p < 0.05 p < 0.05 p < 0.05
Values are expressed as mean SD. 30 ng/mL considered as 25(OH)D3 sufficient. 20–29 ng/mL considered as 25(OH)D3 insufficient. 19 ng/mL considered as 25(OH) D3 deficient.
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Fig. 2. (2A) TGF b concentration (pg/mL) in maternal and cord blood. (2B) IL-10 concentration (pg/mL) in maternal and cord blood. Different superscripts indicate significant difference at p < 0.05. Values are expressed as mean SE.
Fig. 3. (3A) Treg cell population in maternal and cord blood. Different superscripts indicate significant difference at p < 0.05. Values are expressed as mean SE. (3B) Spearman's rank correlation between maternal 25(OH)D3 and Treg cell population (r = 0.463; p = 0.001). (3C) Spearman's rank correlation between cord blood 25(OH)D3 and Treg cell population (r = 0.512; p = 0.001).
3.4. CD23 and CD21 receptors on B cells in maternal and cord blood and their gene's expressions in placenta tissue The maternal and cord blood B cells with CD23 expression ranged from 0.1 to 0.8% and 0.2 to 0.8%, respectively. In maternal blood, the proportions of B cells with CD23 expression (mean (%) SE) were significantly (p < 0.05) higher in 25(OH)D3 deficient (0.35 0.02) when compared to insufficient (0.22 0.02) and sufficient (0.15 0.02) pregnant women. Similarly, in cord blood the proportion of B cells with CD23 expression (mean (%) SE) was higher (p < 0.05) in 25(OH)D3 deficient (0.41 0.02) when
compared to insufficient (0.32 0.01) and sufficient (0.2 0.01) pregnant women (Fig. 5A). The maternal and cord blood B cells with CD21 expression ranged from 0.1 to 2.9% and 0.2 to 2.1%, respectively. Similar to B cells with CD23 expression, B cells with CD21 expression (mean (%) SE) in maternal blood were significantly higher (p < 0.05) in 25(OH)D3 deficient (1.65 0.04) than in insufficient (0.55 0.05) and sufficient pregnant women (0.21 0.01). In cord blood, the proportion of B cells with CD21 expression (mean (%) SE) was higher (p < 0.05) in 25 (OH)D3 deficient (1.2 0.03) than in insufficient (0.6 0.05) and sufficient pregnant women (0.4 0.02) (Fig. 5B). Maternal 25(OH)
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Fig. 4. mRNA expression (RQ) of CD23, CD21 and FOXP3 genes in placenta tissue of women with 25(OH)D3 deficiency. 25(OH)D3 sufficient group was taken as calibrator and GAPDH was used as internal control. Different superscripts indicate significant difference at p < 0.05. Values are expressed as mean SE.
D3 was inversely correlated with maternal B cells with CD23 (r = 0.389; p = 0.001) and CD21 (r = 0.783; p = 0.001) expression (data not shown); and likewise, maternal blood Treg cell population was inversely correlated with maternal B cells with CD23 (r = 0.298; p = 0.001) and CD21 expression (r = 0.538; p = 0.001) (data not shown). As expected, the placenta tissues CD23 and CD21 mRNA gene expressions were upregulated in 25 (OH)D3 insufficient and deficient women (Fig. 4). 3.5. Vitamin D regulating enzymes and receptor genes Placenta tissue mRNA expressions are expressed in relative quantification (RQ) values and shown in Fig. 6. The VDR was downregulated (p < 0.05) in the placenta tissue of women with 25 (OH)D3 deficiency, but was comparable between 25(OH) D3 sufficient and insufficient pregnant women. The nuclear receptor RXR expression was significantly (p < 0.05) downregulated in 25(OH)D3 deficient and insufficient women. As for the vitamin D activating enzymes, the CYP27B1 was downregulated, while CYP2R1 was upregulated in women with 25(OH) D3 insufficiency and deficiency and were significantly different (p < 0.05) from sufficient women. The vitamin D inactivating
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Fig. 6. mRNA expression (RQ) of VDR, CYP27B1CYP2R1, CYP24A1, VDBP and RXR genes in the placenta tissue of women. Vitamin D sufficient group was taken as calibrator and GAPDH was taken as internal control. Different superscripts indicate significant difference at p < 0.05. Values are expressed as mean SE.
enzyme, CYP24A1 and the VDBP were upregulated in the placenta of women with 25(OH)D3 insufficiency and deficiency (Fig. 6). 4. Discussion The results from the current study show that impaired maternal 25(OH)D3 during pregnancy influences the spectrum of immune cells such as regulatory T cells and B cells with IgE receptors in the maternal and cord blood, which in turn may be important in maintaining optimal fetal health and pregnancy outcomes. Under-nutrition is commonly prevalent in India, where 30% of pregnant women are chronically undernourished (body mass index (BMI), <18.5) and 84.9% are anemic. About 30% of new-borns in India are low birth weight (LBW) babies and 13% of deliveries are preterm [22,23]. The average hemoglobin concentration of pregnant women in India is 10.8 gm/dL, and the mean gestational age, birth weight and hemoglobin concentration observed in this study are close to the national average [23]. The subjects studied here represent the majority of pregnant women population in India and therefore the results can be generalised. As for vitamin D, a few studies have shown that about 84% of pregnant women have inadequate vitamin D concentration [11].
Fig. 5. (5A) B cells with CD23 expression in maternal and cord blood. (5B) B cells with CD21 expression in maternal and cord blood. Different superscripts indicate significant difference at p < 0.05. Values are expressed as mean SE.
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Though India is a tropical country with immense sun shine, only 18 out of 153 women had sufficient 25(OH)D3 concentration in our studies [11,12]. The cultural practice of confining pregnant women to indoors and thus keeping them away from daily activity in agricultural fields or outside homes may be one strong reason for less sunlight exposure and thus vitamin D deficiency. Moreover, significant proportions of pregnant women in our study were Muslims, who are usually covered from head to toe and thus are not exposed to sunshine. So far, human transplacental transport of 1,25(OH)2D3 has not been studied. In rats, however, transport of inactive forms of vitamin D (25(OH)2D3 and 24,25(OH)2D3) but not active form of vitamin D (1,25(OH)2D3) was shown through the placenta [24]. In our study, correlation of the maternal with the cord blood 25(OH) D3 suggests that higher serum concentrations of 25(OH)D3 in maternal blood would facilitate passage of 25(OH)D3 from the mother to the fetus. The hydroxylation of 25(OH)D3 to the active form 1,25 (OH)2D3 is performed by CYP27B1, whereas, CYP24A1 is central for 1,25(OH)2D3 catabolism and degradation [25]. Conversely, CYP2R1 has been suggested to be active in the hydroxylation of both ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3) [25,26]. The CYP27B1 enzyme has been shown to generate a large amount of 1,25(OH)2D3 in the decidua and the placenta. Thus, both CYP27B1 and CYP24A1 play an important role in governing tissue vitamin D concentrations [27–29]. In the current study, increased expression of CYP2R1 on the background of low CYP27B1, and high catabolic CYP24A1 enzyme may be presumed as an attempt to increase the availability of the active form of vitamin D in the vitamin D deficient pregnant women. The observed altered expressions of vitamin D metabolising enzymes suggest altered vitamin D homeostasis in pregnant women with vitamin D deficiency, which is likely to play an important role in the fetal immune development and overall fetal developmentas well. In contrast to our finding, vitamin D deficiency is usually associated with low circulating VDBP, a key carrier binding protein for 25(OH)D3 and 1,25(OH)2D3 [30]. The majority of circulating 25 (OH)D3 is bound to VDBP, and its uptake into cells occurs in both bound and unbound forms [31]. Low VDBP has been suggested to have a protective role against the symptoms associated with lower vitamin D status. Conversely, in the current study we registered an increase in VDBP gene expression, the reason for which is unclear [32]. VDR acts primarily as a heterodimer with the retinoid X receptor (RXR) on vitamin D response elements (VDREs) [33]. VDR is abundantly present in human placenta and plays a critical role in transportation of calcium from mother to fetus. In addition, VDR expression regulates up to 500 vitamin D responsive genes, which influence mineral metabolism, differentiation and proliferation of various cells [34]. Hence, low VDR and RXR expressions in the vitamin D deficient pregnant women as observed in the present study must be viewed critically. Both prenatal and perinatal vitamin D deficiency have been shown to influence early life respiratory morbidity and have been suggested to be vital for lung development and growth. Moreover, low cord blood 25(OH)D3 levels are associated with respiratory syncytial viral bronchiolitis in the first year with a relative risk of 6.2 [35]. Furthermore, some studies have demonstrated prevention of exaggerated autoimmune response in diabetes mellitus upon supplementation of cholecalciferol, which was associated with increased Treg cells percent [36,37]. Vitamin D may play an important role in the maintenance of B cell homeostasis and may be useful in the treatment of B cell-mediated autoimmune disorders [38]. In this study, low Treg cells and increased CD21 and CD23 expression on B cells may have a key role as receptors of IgE antibody and conceivably are linked with increased respiratory morbidity among vitamin D deficient
subjects. From the results of the present study, it may be speculated that vitamin D deficiency may increase early life respiratory morbidity by impairing T cell regulatory function and by increasing B cells with IgE receptors. Treg cell population increases right from the very early stages of pregnancy. Women with impaired Treg cell concentration suffer from spontaneous abortion and infertility and therefore expression of Treg cells has been considered to be essential for successful pregnancy outcome [39]. The migration mechanisms of Treg cells to the pregnant uterus are still unclear, however, human chorionic gonadotropin (hCG), secreted by the trophoblasts after fertilization has been shown to attract Treg cells to the fetal–maternal interface [40]. In pregnant women Treg cell population that comprises less than 1% of peripheral CD4+ cells has the ability to suppress both Th1 and Th2 immunity against paternal/fetal alloantigen, a process, critical for immune regulation and continuation of pregnancy [41]. This is the first study to demonstrate low circulating Treg cells in both maternal and cord blood and impaired FOXP3 gene expression in placenta of vitamin D deficient pregnant women. However, Treg cell response to vitamin D supplementation would have delineated the causal role of vitamin D on Treg cell function. Nevertheless, in view of the proposed link between vitamin D deficiency and adverse events in pregnancy, the results of the current study become more interesting. The study shows that impaired maternal vitamin D during pregnancy may influence a spectrum of immune cells in the mother and placenta, which in turn may be linked to allergy and asthma in neonates. The role of vitamin D in immune function and inflammation in pregnant women must not be underestimated and in light of the existing literature, estimation of serum levels of vitamin D and supplementation in pregnant women must be considered. This is needed especially keeping in view the extent to which low vitamin D status and impaired Treg cell function in utero may lead to the development of chronic diseases in adult life. Conflict of interest The authors report no conflict of interest. Acknowledgments The authors sincerely acknowledge the Golden Triangle Programme (funded by ICMR,CSIR and ICAR) for providing financial support and the Director, National Institute of Nutrition for providing facilities to conduct the study. The authors greatly acknowledge the CSIR-HRDG for the SRF fellowship to Mr. Vijayendra Chary. The authors are thankful to the superintendent of Gandhi Hospital and the nursing staff especially Mrs. Aruna Reddy for their immense support in the recruitment of subjects and collection of samples. References [1] L.M. Bodnar, J.M. Catov, H.N. Simhan, M.F. Holick, R.W. Powers, J.M. Roberts, Maternal vitamin D deficiency increases the risk of preeclampsia, J. Clin. Endocrinol. Metab. 92 (2007) 3517–3522. [2] K.C. Chiu, A. Chu, V.L. Go, Md.F. Saad, Hypovitaminosis D is associated with insulin resistance and beta cell dysfunction, Am. J. Clin. Nutr. 79 (5) (2004) 820–825. [3] A.G. Pittas, J. Lau, F.B. Hu, B. Dawson-Hughes, The role of vitamin D and calcium in type 2 diabetes. A systematic review and meta-analysis, J. Clin. Endocrinol. Metab. 92 (6) (2007) 2017–2029. [4] M. Erkkola, B.I. Nwaru, H.T. Viljakainen, Maternal vitamin D during pregnancy and its relation to immune-mediated diseases in the offspring, Vitam. Horm. 86 (2011) 239–260. [5] S. Dimeloe, A. Nanzer, K. Ryanna, C. Hawrylowicz, Regulatory T cells, inflammation and the allergic response: the role of glucocorticoids and vitamin D, J. Steroid Biochem. Mol. Biol. 120 (2010) 86–95.
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