- Email: [email protected]
CD21 expression with vitamin D in children with asthma
Ann Allergy Asthma Immunol xxx (2016) 1e8
Contents lists available at ScienceDirect
Association of T-regulatory cells and CD23/CD21 expression with vitamin D in children with asthma Anchoju Vijayendra Chary, Mpharm *; Rajkumar Hemalatha, MD *; Machiraju Vasudeva Murali, MD y; Dodle Jayaprakash, PhD z; Bharathraj Dinesh Kumar, PhD x * Department
of Clinical Division, Microbiology and Immunology, National Institute of Nutrition, Hyderabad, Telangana, India Department of Paediatrics, Gandhi hospital, Hyderabad, Telangana, India z Department of Technology, Osmania University, Hyderabad, Telangana, India x Department of Food and Drug Toxicology, National Institute of Nutrition, Hyderabad, Telangana, India y
A R T I C L E
I N F O
Article history: Received for publication November 6, 2015. Received in revised form February 23, 2016. Accepted for publication February 29, 2016.
A B S T R A C T
Background: Children with asthma have low vitamin D levels; however, we do not know whether low vitamin D is associated with impaired T-regulatory (Treg) cell population or high IgE receptors (CD23 and CD21) on B cells. Objectives: To examine Treg cell function, CD23 and CD21 receptors, vitamin D, and vitamin Deregulating enzymes in children with asthma. Methods: Sixty children (2e6 years old) with asthma and 60 age-matched healthy children were selected as study participants. After collecting demographic and clinical data, blood samples were collected. Treg cells and CD23/CD21 expressions were evaluated by flow cytometry, 25-hydroxyvitamin D3 (25[OH]D3) was quantified by high-performance liquid chromatography, and cytokines and total IgE were estimated by enzyme-linked immunosorbent assay. Messenger RNA (mRNA) expressions of FOXP3, CD23, CD21, vitamin D receptors, and vitamin Deregulating enzymes were assessed by reverse transcriptionepolymerase chain reaction. Results: The 25(OH)D3 concentrations and proportion of Treg cells were lower (P < .05) among children with asthma. In contrast, the proportions of B cells with CD23 and CD21 expression were higher (P < .05) in patients with asthma compared with controls. Interleukin 10 and transforming growth factor b were also altered in asthma. The mRNA expression of CD23, CD21, and vitamin D receptors was up-regulated, whereas mRNA expression of vitamin Deregulating enzymes, CYP2R1, CYP27B1, and vitamin D binding protein (except CYP24A1) were up-regulated among children with asthma. Conclusion: The current study found impaired Treg cell population and high numbers of B cells with IgE receptors (CD23 and CD21) and altered regulatory cytokines in children with asthma, suggesting impaired immune regulation. Ó 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Introduction Asthma is a complex and heterogeneous inflammatory disease of the airways.1 The incidence of asthma has increased considerably in the past decade.1 Currently, it is estimated that approximately 300 million individuals with asthma exist worldwide, and this number is expected to increase by another 100 million by 2025, of whom 30 million are from India alone.2 The prevalence of asthma is estimated to range from 3% to 38% in children and from 2% to 12% in adults.3 Reprints: Rajkumar Hemalatha, MD, Department of Clinical Division, Microbiology and Immunology, National Institute of Nutrition, Hyderabad-07, Telangana, India; E-mail: [email protected]. Disclosures: Authors have nothing to disclose. Funding Sources: This study was funded by the National Institute of Nutrition, Hyderabad, Telangana, India.
The various cellular components that drive asthmatic reactions include mast cells, eosinophils, and lymphocytes.1,4 T regulatory (Treg) cells (CD4þ/CD25þ/CD127/FOXP3 Treg cells) are subsets of T lymphocytes described by expression of a specific transcription factor, FOXP3.5 Previous reports have implicated reduction in the numbers and functionality of Treg cells in both human and mouse during allergic conditions.5,6 Treg cells produce regulatory cytokines, such as interleukin 10 (IL-10) and transforming growth factor b (TGF-b).7,8 IL-10 has broad immunosuppressive and anti-inflammatory activity relevant to the inhibition of asthma disease.9 IL-10 acts on antigen-presenting cells and suppresses T-cell activation, including TH2 cells.9 On the other hand, TGF-b induces peripheral expression of the transcription factor FOXP3, which in turn promotes the transformation of Treg cells and inhibits allergic airway disease.10 Interaction between
http://dx.doi.org/10.1016/j.anai.2016.02.018 1081-1206/Ó 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
2
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
IL-10 and TGF-b is likely to be vital in the regulation of IgE and its substrates, which play an essential role in all allergic conditions.10 CD23 (FcεRII) and CD21 are the low-affinity receptors for IgE and are found mainly on the surface of B lymphocytes. CD23 and CD21 interact with each other, and the pairing of these molecules is important in T-cell and B-cell interaction and control of IgE production.11 In an earlier study, we found a strong inverse association between Treg cells and CD23 and CD21 in pregnant women.12 The same study also found impaired Treg cells in pregnant women with vitamin D deficiency.13 Higher vitamin D intake by pregnant mothers reduces asthma risk by as much as 40% in 3- to 5-year-old children.14 Although vitamin D has been suggested to play an important role in the maintenance of B-cell homeostasis, IgE receptor expression on B cells has not been studied in vitamin Dedeficient children with asthma.15 CD23 and CD21 on B cells may have a key role as receptors of IgE antibody and conceivably are linked with increased respiratory morbidity among vitamin Dedeficient individuals.16 It has been speculated that vitamin D deficiency may increase early-life respiratory morbidity by impairing Treg cell population and by increasing B cells with IgE receptors.13 Vitamin D is activated by 2 enzymatic steps. Vitamin D3 (cholecalciferol) is metabolized to 25-hydroxycholecalciferol (calcidiol or 25-hydroxyvitamin D3 [25(OH)D3]) by the hepatic 25hydroxylases (CYP2R1) in the liver. Subsequently, 25(OH)D3 is converted to 1,25-dihydroxycholecaciferol (calcitriol or 1,25 [OH]2D3) by 1a-hydroxylase (CYP27B1) in the kidney.17 The active form of vitamin D3 (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 CYP27B1 enzyme is also produced by several other cell types, including dermal cells, intestinal epithelial cells, lymph nodes, monocytes, and placenta, which enables extra renal activation of 25(OH)D3 to the active hormonal form, 1,25(OH)2D3.19,20 Vitamin D, VDR, RXR and vitamin Deregulating enzymes are known to be responsible for inducing several genes that are important in asthma and allergic conditions.21 In the current study, besides examining the Treg cell population and the IgE receptors on B cells, the serum vitamin D and vitamin Deregulating enzymes (CYP2R1, CYP27B1, and CYP24A1), VDR, and RXR were explored in children with asthma. Methods Ethical Information This study was conducted at the Gandhi Hospital, Hyderabad, according to the Helsinki prescribed guidelines. Ethical approval was obtained to undertake the study, and written informed consent was obtained from parents or guardians before including their children in the study.
classified based on clinical examination and symptoms as described by the Global Initiative for Asthma (GINA) 2008 guidelines.22 Socioeconomic status was calculated with the modified Kuppuswamy socioeconomic scale.23 Blood samples were collected from all the children to assess immune parameters and vitamin D status. Treg cell (CD4þ/CD25þ/CD127/FOXP3þ), CD23, and CD21 expression on B cells was determined by flow cytometry. 25(OH)D3 was quantified with high-performance liquid chromatography. TGF-b, IL-10, and IgE levels were quantified by enzyme-linked immunosorbent assay (ELISA). In a randomly selected subsample of 12 individuals from each group (cases and controls), peripheral blood mononuclear cells (PBMCs) were used for messenger RNA (mRNA) expression of CD23, CD21, FOXP3, CYP2R1, CYP27B1, CYP24A1, vitamin D binding protein (VDBP), VDR, and RXR. The required sample size for mRNA expression of the above mentioned genes was calculated with 90% power after reaching a sample size 12 children in each group. Treg Cell (CD4þ/CD25þ/CD127/FOXP3þ) Population The proportion of Treg cells was estimated in whole blood as described in an earlier study.13 Whole blood (100 mL) was stained with 20 mL of human Treg cell cocktail that contained fluorescein isothiocyanate antihuman CD4 antibody, PE-Cy7 antihuman CD25 antibody, and Alexa Fluor 647 antihuman CD127 antibody (BD Sciences, San Jose, California). The erythrocytes were lysed using red blood cell lysing buffer (BD Sciences). The cells were again washed and were stained with PE mouse antihuman FOXP3 (BD Sciences). The lymphocyte cells were first gated based on forward and side scatter to exclude dead cells and cell debris. Later CD4þ T cells were gated, and CD25þ and CD127 cells were further gated from CD4þ cells. CD4þ/CD25þ/CD127 cells were further gated for FOXP3þ cells (CD4þCD25þ/CD127FOXP3þ or Treg cells). The data were obtained by FACS DIVA software (BD Biosciences, San Jose, California) and was analyzed by FCS Express software (De Novo Software, Glendale, California) (eFig 1). Total IgE and Cytokine Assay (IL-10 and TGF-b) Serum samples were used to estimate total IgE, and cytokines, such as IL-10 and TGF-b, were quantified by sandwich ELISA. Briefly, the microplate was coated with capture antibody. Then, samples that contained an unknown amount of the target analyte of interest were added. After washing, to get rid of the microplate of unbound substances, a horseradish peroxidase conjugate was added, and absorbance was determined according to the manufacturer’s instructions (eBiosciences, San Diego, California). The lower limit of detection for total IgE was 7.8 ng/mL, and the standard curve range given with kit was 7.8 to 1,000 ng/mL. The lower limit of detection for IL-10 was 0.8 ng/mL, and the standard curve range given with kit was 0.8 to 50 ng/mL. The lower limit of detection for TGF-b was 80 pg/mL, and the standard curve range given with kit was 80 to 5000 ng/mL. B Cells With CD23 and CD21 Expression
Enrollment of Study Participants Children aged 2 to 6 years who were visiting the outpatient department of the asthma clinic at Gandhi Hospital were selected as study participants. Children with fever, infections, or skin problems and those taking steroids or antibiotics were excluded from the study. Sixty children with asthma were recruited as cases, and 60 age-matched apparently healthy children without asthma or without any infection were used as controls. After written informed consent was obtained from the parents or guardians of the study participants, a detailed questionnaire was administered on family history of allergy and asthma, history of smoking in the family, demographic profile, and anthropometry data on a pretested semistructured questionnaire. Children with asthma were
To estimate the proportion of B cells with CD23 and CD21 expression, 100 mL of whole blood was stained with 20 mL of Alexa Fluor 700 mouse antihuman CD19 antibody, 20 mL of fluorescein isothiocyanate mouse antihuman CD23 antibody, and 5 mL of Allophycocyanin mouse antihuman CD21 for 20 minutes at room temperature according to the manufacturer’s instructions (BD Sciences). The red blood cells were lysed by BD FACS red blood cell lysing solution. After washing with 2 mL of flow staining buffer, the pellet was suspended in 100 mL of flow staining buffer. A total of 10,000 events were acquired per sample on the FACS ARIA II system. The B cells were gated based on CD19 marker and were further gated for CD19þ/CD23þ and CD19þ/CD21þ cells. The data were obtained by FACS DIVA software (BD Biosciences, San Jose,
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
California) and were analyzed by FCS Express software (De Novo Software, Glendale, California) (eFig 2). Serum Calcium and Hemoglobin Assay Serum calcium was quantified using an ELISA-based kit according to the manufacturer’s instructions (Accucare Labs, Mumbai, India). Hemoglobin was estimated by the Drabkin’s (cyanmethemoglobin) method. 25(OH)D3 Quantification Serum levels of 25(OH)D3 are considered to be the best circulating biomarker of vitamin D metabolic status and reflect contributions from all sources of vitamin D (ie, diet and sun exposure).24 25(OH)D3 levels were categorized as deficient (15 ng/mL) and sufficient (15 ng/mL) on the basis of previous recommendations (Institute of Medicine classification).25 A total of 500 mL of serum was used to quantify 25(OH)D3 by HPLC, using Schimadzu system with Supelcosil column (4.6 mm 25 cm; 5m particle size; Sigma-Aldrich, St Louis, Missouri). The mobile phase used methanol and water (85:15 ratio), with flow rate of 2 mL/min. The injection volume was 25 mL and detection was at 265 nm wavelength. mRNA Expression of FOXP3, CD23, CD21, VDR, CYP2R1, CYP27B1, CYP24A1, VDBP, and RXR in the PBMCs PBMCs were separated from whole blood by density gradient centrifugation using Ficoll reagent. A total of 4 mL of blood was layered on the top of 4 mL of Ficoll Histopaque and was centrifuged for 30 minutes at 100g in 4 C. The whitish buffy coat (PBMCs) formed in the interphase between Histopaque and medium was aspirated. PBMCs were washed 2 times with PBS to remove the Ficoll reagent. Total RNA was extracted from the PBMCs according to the Chomzinsky and Sacchi method.26 Total RNA thus extracted was treated by DNase according to the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, Massachusetts). The quality and the yield of total RNA were verified using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, California) and Nanodrop 1000 (Thermo Fisher Scientific). Electrophoresis was used to check the integrity and purity of the RNA for complementary DNA (cDNA) synthesis by the procedure of Masek et al.27,28
3
Table 1 Primers Used in Reverse TranscriptionePolymerase Chain Reaction Sequence No.
Gene
Primer Sequence
1
GAPDH
2
VDR
3
FOXP3
4
CD23
5
CD21
6
CYP27B1
7
CYP2R1
8
CYP24A1
9
RXR
10
VDBP
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
94 88 105 97 100 100 94 92 105 86
Statistical Analysis For descriptive data, mean values were calculated and differences between groups were assessed with the t test using SPSS software, version 18.0 (SPSS Inc, Chicago, Illinois). Graphical analysis was performed in SigmaPlot 12.3 (Systat Software, Chicago, Illinois). Values are expressed as mean (SE). The mean differences were considered significant when P < .05. Correlations of various parameters were analyzed by the Spearman rank correlation coefficient (r). Results Demographic Information Of the 120 children, 72 were male and the rest were female. The mean (SD) age and weight of the children were 4.3 (1.4) years and 15.38 (3.76) kg, respectively, and the ratios of male vs female were Table 2 Demographic and Clinical Characteristics of the Study Participantsa Parameter
Control Children (n ¼ 60)
Children With Asthma (n ¼ 60)
P Valueb
Male, % Female, % Age, mo Weight, kg Height for age Weight for age BMI for age Weight for height Family history of asthma, % Asthma severity, % Severe persistent Moderate persistent Mild persistent Mild intermittent 25(OH)D3 status, % Sufficient
55 45 46.95 14.36 0.18 1.19 1.92 2.16 .c
65 35 48.87 16.40 0.45 0.02 0.43 0.40 18.3
>.05
cDNA Synthesis and RT-PCR A total of 3 mg of total RNA was transcribed to cDNA using a RevertAid first-strand cDNA synthesis kit (Thermo Fisher Scientific). The cDNA thus obtained was aliquoted and stored at 20 C. For the reverse transcriptionepolymerase chain reaction (RT-PCR) assay, SYBR Green Master Mix (Thermo Fisher Scientific) was used, and primers were designed by Geneious pro software (version 5.4.6; Geneious, Auckland, New Zealand) and synthesized by Imperial Life Sciences (New Delhi, India) (Table 1). GAPDH was used as internal control. To quantify gene expression of target genes such as CD23, CD21, FOXP3, CYP2R1, CYP27B1, CYP24A1, VDBP, VDR, and RXR, the ABI Step One Plus system was used with program of 95 C for 10 minutes followed by 40 cycles of 95 C for 15 seconds and 60 C for 1 minute. The difference in the cycle threshold (CT) value between the control gene (GAPDH) and the target genes was obtained for each sample. The critical 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.
Base Pairs
Deficient
(14.22) (4.54) (1.98) (1.85) (3.07) (2.97)
. . . . 48.33 (19.89 [0.44] ng/mL) 51.66 (13.16 [0.38] ng/mL)
4 26 20 10
(12.96) (2.42) (1.48) (1.11) (1.83) (1.89)
>.05 >.05 >.05 >.05 >.05 >.05
(6.66) (43.33) (33.33) (16.66)
20 (20 [0.67] ng/mL) 80 (11.07 [0.40] ng/mL)
Abbreviations: 25(OH)D3, 25-hydroxyvitamin D3; BMI, body mass index. a Values are expressed as mean (SD) unless otherwise indicated. b P < .05 was considered significant. c Ellipses indicate data not applicable.
>.05 >.05
4
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
1.85:1 and 1.22:1 in asthmatic and control children, respectively. The mean height, weight, and body mass index z scores were comparable between cases and controls (Table 2). Similarly, the mean (SE) hemoglobin levels were comparable between asthmatic (10.3 [1.98] mg/dL) and control children (11.6 [1.87] mg/dL). The serum calcium levels were also comparable between asthmatic (8.3 [0.05] ng/mL) and control children (8.5 [0.08] ng/mL). All the children belonged to middle and low socioeconomic status and as expected had insufficient milk intake. On the basis of the GINA classification, 4 (6.7%) 60 children had severe persistent asthma, 26 (43.3%) had moderate persistent
asthma, 20 (33.3%) had mild persistent asthma, and 10 (16.6%) had mild intermittent asthma. Treg Cell (CD4þ/CD25þ/CD127/FOXP3þ) Population The proportion of the Treg cell population was evaluated within the CD4þ population. The Treg cell population ranged from 0.1% to 0.5% and 0.2% to 0.9% in asthmatic and control children, respectively. Figure 1 shows the mean Treg cell population, which was significantly (P < .05) lower in asthmatic (0.29 [0.01]) compared with control children (0.38 [0.02]).
Figure 1. T-regulatory (Treg) cell population, total IgE levels, and B cells with CD23/CD21 expression in whole blood in asthmatic and control children. Spearman rank correlation of Treg cells with B cells with CD23/CD21. Values are expressed as mean (SE). A, Treg cell population; B, total IgE levels; C, B cells with CD23 expression; D, B cells with CD21 expression; E, correlation of Treg cells and B cells with CD23; and F, correlation of Treg cells and B cells with CD21 in asthma. *P < .05.
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
5
Total IgE Levels
mRNA Expression of Vitamin DeRegulating Enzymes and VDRs
As expected, the mean (SE) serum IgE level was significantly (P < .05) higher in children with asthma (748 [55.8] ng/mL) compared with control children (362.88 [38.53] ng/mL). However, IgE levels were not correlated with either 25(OH)D3 or Treg cell population or B cells with CD23/CD21 expression (Fig 1).
The mRNA expressions of FOXP3, CD23, CD21, IL10, TGF- b, VDR, CYP2R1, CYP27B1, CYP24A1, VDBP, and RXR are presented in RQ values and are compared among children with asthma and the control group. GABDH was used as an internal control (Fig 4). The mRNA expression of the transcription factor of the Treg cell, the FOXP3, was (P < .05) down-regulated in children with asthma. As expected, the mRNA expression of CD23 and CD21 were (P < .05) up-regulated in children with asthma. The mRNA expression of IL-10 was down-regulated, whereas TGF-b gene was up-regulated in children with asthma compared with controls (Fig 4). Both the vitamin Deactivating enzymes, CYP2R1 and CYP27B1, were (P < .05) up-regulated, whereas the vitamin Deinactivating enzyme, CYP24A1, was down-regulated in children with asthma. As for the receptors and binding protein, the VDR, RXR, and VDBP expressions were all up-regulated (P < .05) in children with asthma (Fig 4).
B Cells With CD23/CD21 Expression The mean (SE) percentage of B cells with CD23 expression was higher (P < .05) in children with asthma (1.30% [0.08%]) than in control (0.18% [0.01%]). Similarly, the mean (SE) percentage of B cells with CD21 expression was higher (P < .05) in asthmatic children (1.89% [0.11%]) compared with controls (0.84% [0.03%]) (Fig 1). As expected, the CD23 population was strongly correlated with the CD21 population (n ¼ 60; P < .01; r ¼ 0.729) of B cells (data not shown). However, the Treg cell population was inversely correlated with both CD23 (n ¼ 60; P < .01; r ¼ 0.456) and CD21 (n ¼ 60; P < .01; r ¼ 0.504) population (Fig 1). Regulatory Cytokines (IL-10 and TGF-b) The mean (SE) of IL-10 was lower (P < .05) in asthmatic children (3.4 [0.46] ng/mL) than in controls (5.06 [0.84] ng/mL). In contrast, TGF-b concentration was higher (P < .05) in asthmatic children (4330.46 [162.56] pg/mL) compared with controls (3338.45 [168.17] pg/mL) (Fig 2). Both IL-10 and TGF-b concentrations did not correlate with Treg cell population (data not shown).
25(OH)D3 Levels in Asthmatic and Control Children Twelve children (20%) with asthma had 25(OH)D3 concentrations within the reference range, whereas 29 healthy children (48.3%) had 25(OH)D3 within the reference range (Table 2). However, the mean 25(OH)D3 concentrations were significantly (P < .05) lower in children with asthma (15.54 [0.71] ng/mL) than in controls (18.44 [0.49] ng/mL) (Fig 3). Interestingly, the 25(OH)D3 concentration was directly correlated with Treg cell population (n ¼ 60; P < .01; r ¼ 0.652) in children with asthma (Fig 3). In contrast, the CD23 (n ¼ 60; P < .01; r ¼ 0.670) and CD21 populations (n ¼ 60; P < .01; r ¼ 0.791) were inversely correlated with 25(OH)D3 in children with asthma (Fig 3).
Discussion The current study found impaired Treg cell population and high numbers of B cells with CD23 and CD21 expressions and altered regulatory cytokines in children with asthma. Furthermore, impaired Treg cell population was directly associated with low levels of serum vitamin D. The study also found altered VDR expression and vitamin D regulatory enzymes in children with asthma. The effects of Treg cells on the immune system are important in the intracellular signaling pathways in lymphocytes and antigen-presenting cells.28 That the Treg cells have a central role in suppressing allergic asthma is undisputed. The cytokines most commonly implicated in Treg-mediated control of allergic asthma are TGF-b and IL-1029,30 The regulatory cytokine IL-10 has anti-inflammatory activity and inhibits asthma symptoms.9 On the other hand, TGF-b is known to induce peripheral expression of the Treg cells and its transcription factor FOXP3 and thus inhibits allergic airway disease in asthma.31,32 Although the current study found high TGF-b levels on a low IL-10 background in children with asthma, the source of TGF-b is unclear. A number of host cells, such as Treg cells, effector T cells, or noneT cells, may secrete TGF-b.10 Of the Treg cells, 2 types of FOXP3 Treg cells have been described that produce TGF-b and IL-10, respectively, for their suppressive functions.33 Because there was no correlation between the Treg cell population and TGF-b, we can only speculate that the source of high concentration of TGF-b could not have been Treg
Figure 2. Regulatory cytokines (interleukin-10 [IL-10] and transforming growth factor b [TGF-b]) in the blood. Values are expressed as mean (SE). A, IL-10 levels in asthmatic and control children; B, TGF-b levels in asthmatic and control children. *P < .05.
6
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
Figure 3. 25-hydroxyvitamin D3 (25[OH]D3) concentrations in the blood and Spearman rank correlation of 25(OH)D3 with T-regulatory (Treg) cells and B cells with CD23/CD21 expression in children with asthma. Values are expressed as mean (SE). A, 25(OH)D3 concentrations in asthmatic and control children; B, correlation of 25(OH)D3 and Treg cells; C, correlation of 25(OH)D3 and B cells with CD23 expression; and D, correlation of 25(OH)D3 and B cells with CD21 expression. *P < .05.
cells in children with asthma in the current study. Alternatively, it may be speculated that TGF-b secretion might have been driven by low Treg cell populations as a feedback mechanism to maintain optimum Treg cells and immune regulation.34,35 Although the reason for the low IL-10 concentration is unclear, impaired Treg cell population may be speculated as the mechanism behind low IL-10 levels in asthma.36,37 Interaction between IL-10 and TGF-b is likely to be vital in the regulation of IgE and its substrates, which play an essential role in all allergic conditions.10 CD23 (FcεRII) and CD21 are the low-affinity receptors for IgE and are found mainly on the surface of B lymphocytes. CD23 and CD21 interact with each other, and the pairing of these molecules is important in T-cell and B-cell interaction and control of IgE production.38 In our earlier study, soluble CD23 was increased in children and adults with asthma.39,40 The current study confirmed high expression of CD23 and CD21 on the backdrop of low circulating Treg cells and 25(OH)D3. Even the mRNA expressions of CD23 and CD21 were increased with the concurrent decrease in FOXP3, suggesting altered immune regulation at transcription level in asthma. However, these results should be interpreted with caution because failure to include an IgG isotype control would have influenced the results. Nevertheless, the CD23 or CD21 stained population were clearly separated from unstained population, suggesting little or no background fluorescence. Vitamin D induces Treg cell expression and increases production of IL-10.33 Vitamin D has also been suggested to play an important role in the maintenance of B-cell homeostasis and in the treatment of B-cellemediated autoimmune disorders.15 The vitamin D
regulates IgE levels in the serum via a direct effect on the VDR expression on B cells, However, the role of vitamin D on IgE receptors (CD23/CD21) on B cells is not known, although the present study clearly indicates a strong inverse correlation between 25(OH)D3 and CD23 and CD21 expression on B cells. CD23 and CD21 on B cells may have a key role as receptors of IgE antibody and conceivably are linked with increased respiratory morbidity amongst vitamin Dedeficient individuals.41 Similar to our observation, recent studies have found a high prevalence of vitamin D deficiency in asthmatic patients.42,43 Large cross-sectional studies found an association between low vitamin D levels and reduced lung function in adolescents and adults and an inverse correlation of vitamin D with IgE concentration.44 Although we do not know whether a low vitamin D level in children is a cause or effect of asthma, it may be speculated that low vitamin D level may well be associated with an impaired Treg cell population.45,46 This is the first study, to our knowledge, to report an inverse correlation of vitamin D with CD23 and CD21 expression on B cells in children with asthma. Moreover, the current study describes gene expression of VDBP, VDRs, and vitamin Deregulating enzymes in asthma. VDBP, a key carrier binding protein for 25(OH)D3 and 1,25(OH)2D3, helps in the transportation of these forms of vitamin D into the cells.47 Although the reason for increased expression of VDBP is unclear, a high level of VDBP has been suggested to have a protective role in vitamin D deficiency.48 VDBP has also been implicated in immunomodulatory functions in the lungs and has been described to have a protective role in asthma.49
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
7
Increased expression of VDRs and vitamin Deactivating enzymes and low expression of vitamin Demetabolizing enzymes suggest poor availability of the active form of vitamin D in asthma. More studies are needed to elucidate the role of vitamin D in immune regulation in children with asthma. Acknowledgments We greatly acknowledged the National Institute of Nutrition for providing financial support and facilities to conduct the study. We acknowledge the Council of Scientific & Industrial Research, Human Resource Development Group, for the senior research fellowship to Mr Chary. We are thankful to the Gandhi Hospital superintendent and nursing staff (Aruna Reddy) for their immense support in the recruitment of study participants and collection of samples. Supplementary Data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.anai.2016.02.018. References
Figure 4. Messenger RNA (mRNA) expression of immunologic, vitamin D receptors, and vitamin Deregulating enzymes in peripheral blood mononuclear cells of children with asthma with respect to controls by reverse transcriptionepolymerase chain reaction. The control group was used as the calibrator. GAPDH was used as the internal control. Values are expressed as mean (SE). A, mRNA expression (relative quantification [RQ]) of CD23, CD21, FOXP3, transforming growth factor b (TGF-b), and interleukin 10 (IL-10); B, mRNA expression (RQ) of vitamin D receptor (VDR), CYP27B1, CYP2R1, CYP24A1, vitamin D binding protein (VDBP), and 9-cis-retinoic acid receptor (RXR). *P < .05.
Immune cells, such as T and B lymphocytes, macrophages, and dendritic cells, express VDR and are affected by vitamin D deficiency during their maturation process.50 Several genetic studies have reported VDR polymorphism in asthma.51 VDR acts primarily as a heterodimer with the RXR on vitamin D response elements.52 VDR is abundantly present in various cells in the body and plays a critical role in transportation of calcium.24 In addition, VDR expression regulates up to 500 vitamin D responsive genes, which influence mineral metabolism, differentiation, and proliferation of various cells.53 Hence, high VDR and RXR expressions in children with asthma as observed in the present study must be viewed critically. The active form of vitamin D (1,25(OH)2D3) is converted by CYP27B1 CYP2R1, whereas CYP24A1 is central for 1,25(OH)2D3 catabolism and degradation.54,55 The enzyme CYP2R1, which converts cholecalciferol to 25(OH)D3, is directly inhibited by 25(OH)D3. On the other hand, 1,25(OH)2D3 is metabolized to the inactive 1,24,25(OH)3D3 by CYP24A1. 1,25(OH)2D3 vitamin D in turn inhibits CYP27B1 and stimulates the 24-hydroxylase enzymes, This tightly regulated negative feedback mechanism helps maintain serum concentrations of 25(OH)D3 within a physiologic range (30e100 ng/mL).56 Therefore, it may be speculated that low vitamin D drives up-regulation of CYP2R1, CYP27B1 and down-regulation of CYP24A1 enzymes in children with asthma.
[1] Holgate ST. The sentinel role of the airway epithelium in asthma pathogenesis. Immunol Rev. 2011;242:205e219. [2] Cruz AA, Bousquet J, Khaltaev N. Global Surveillance, Prevention and Control of Chronic Respiratory Diseases: A Comprehensive Approach. Geneva, Switzerland: World Health Organization; 2007. [3] Cavkaytar O, Sekerel BE. Baseline management of asthma control. Allergol Immunopathol (Madr). 2014;42:162e168. [4] Lotvall J, Akdis CA, Bacharier LB, et al. Asthma endotypes: a new approach to classification of disease entities within the asthma syndrome. J Allergy Clin Immunol. 2011;127:355e360. [5] Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP3þ regulatory T cells in the human immune system. Nat Rev Immunol. 2010;10:490e500. [6] Corthay A. How do regulatory T cells work? Scand J Immunol. 2009;70: 326e336. [7] O’Garra A, Barrat FJ, Castro AG, Vicari A, Hawrylowicz C. Strategies for use of IL-10 or its antagonists in human disease. Immunol Rev. 2008;223:114e131. [8] Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Transforming growth factorbeta regulation of immune responses. Annu Rev Immunol. 2006;24:99e146. [9] Martin H, Taube C. Regulatory T cells and regulation of allergic airway disease. Am J Clin Exp Immunol. 2012;1:166. [10] Taylor A, Verhagen J, Blaser K, Akdis M, Akdis CA. Mechanisms of immune suppression by interleukin-10 and transforming growth factor-b: the role of T regulatory cells. Immunology. 2006;117:433e442. [11] Rabatic S, Gagro A, Medar-Lasic M. CD21-CD23 ligand pair expression in children with allergic asthma. Clin Exp Immunol. 1993;94:337e340. [12] Vijayendra Chary A, Hemalatha R, Seshacharyulu M, et al. Vitamin D deficiency in pregnant women impairs regulatory T cell function. J Steroid Biochem Mol Biol. 2015;147:48e55. [13] Litonjua AA, Weiss ST. Is vitamin D deficiency to blame for the asthma epidemic? J Allergy Clin Immunol. 2007;120:1031e1035. [14] Berraies A, Hamzaoui K, Hamzaoui A. Link between vitamin D and airway remodeling. J Asthma Allergy. 2014;7:23e30. [15] Chen S, Sims GP, Chen XX, et al. Modulatory effects of 1,25-dihydroxyvitamin D3 on human B cell differentiation. J Immunol. 2007;179:1634e1647. [16] Hartmann B, Heine G, Babina M, et al. Targeting the vitamin D receptor inhibits the B cell-dependent allergic immune response. Allergy. 2011;66:540e548. [17] Hewison M, Burke F, Evans KN, et al. Extra-renal 25-hydroxyvitamin D31alpha-hydroxylase in human health and disease. J Steroid Biochem Mol Biol. 2007;103:316e321. [18] Foong RE, Zosky GR. Vitamin D deficiency and the lung: disease initiator or disease modifier? Nutrients. 2013;5:2880e2900. [19] Shinkyo R, Sakaki T, Kamakura M, Ohta M, Inouye K. Metabolism of vitamin D by human microsomal CYP2R1. Biochem Biophys Res Commun. 2004;324:451e457. [20] Zehnder D, Evans KN, Kilby MD, et al. The ontogeny of 25-hydroxyvitamin D 3 1a-hydroxylase expression in human placenta and decidua. Am J Pathol. 2002; 161:105e114. [21] Hansdottir S, Monick MM. Vitamin D effects on lung immunity and respiratory diseases. Vitam Horm. 2011;86:217. [22] Bateman E, Hurd S, Barnes P, et al. Global strategy for asthma management and prevention: GINA executive summary. Eur Respir Jl. 2008;31:143e178. [23] Bairwa M, Rajput M, Sachdeva S. Modified Kuppuswamy’s socioeconomic scale: social researcher should include updated income criteria, 2012. Indian J Community Med. 2013;38:185e186. [24] Hollis BW. Circulating 25-hydroxyvitamin D levels indicative of vitamin D sufficiency: implications for establishing a new effective dietary intake recommendation for vitamin D. J Nutr. 2005;135:317e322.
8
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
[25] Ross AC, Manson JE, Abrams SA, et al. The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what clinicians need to know. J Clin Endocrinol Metab. 2011;96:53e58. [26] Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc. 2006;1:581e585. [27] Aranda PS, LaJoie DM, Jorcyk CL. Bleach gel: a simple agarose gel for analyzing RNA quality. Electrophoresis. 2012;33:366e369. [28] Masek T, Vopalensky V, Suchomelova P, Pospisek M. Denaturing RNA electrophoresis in TAE agarose gels. Anal Biochem. 2005;336:46e50. [29] Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4þCD25þ T cell-mediated suppression by dendritic cells. Science. 2003;299:1033e1036. [30] Yalcin AD, Bisgin A, Gorczynski RM. IL-8, IL-10, TGF-, and GCSF levels were increased in severe persistent allergic asthma patients with the anti-IgE treatment. Mediators Inflamm. 2012;2012:720976. [31] Chen W, Jin W, Hardegen N, et al. Conversion of peripheral CD4þCD25- naive T cells to CD4þCD25þ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med. 2003;198:1875e1886. [32] Krogulska A, Polakowska E, Wa˛ sowska-Królikowska K, Małachowska B, Młynarski W, Borowiec M. Decreased FOXP3 mRNA expression in children with atopic asthma and IgE-mediated food allergy. Ann Allergy Asthma Immunol. 2015; 115:415e421. [33] Ito T, Hanabuchi S, Wang YH, et al. Two functional subsets of FOXP3þ regulatory T cells in human thymus and periphery. Immunity. 2008;28:870e880. [34] Urry Z, Xystrakis E, Dimeloe S, Richards D, Brown Z, Hawrylowicz C. 1a, 25-dihydroxyvitamin D3 differentially promotes Foxp3 and IL-10 expression by human CD4þ T cells. Immunology. 2010;131. 184e184. [35] Joetham A, Takeda K, Taube C, et al. Naturally occurring lung CD4(þ)CD25(þ) T cell regulation of airway allergic responses depends on IL-10 induction of TGF-beta. J Immunol. 2007;178:1433e1442. [36] Chatila TA. Role of regulatory T cells in human diseases. J Allergy Clin Immunol. 2005;116:949e959. [37] Halwani R, Al-Muhsen S, Al-Jahdali H, et al. Role of transforming growth factoreb in airway remodeling in asthma. Am J Respir Cell Mol Biol. 2011;44:127e133. [38] Makinde T, Murphy RF, Agrawal DK. The regulatory role of TGF-beta in airway remodeling in asthma. Immunol Cell Biol. 2007;85:348e356. [39] Bonnefoy J, Gauchat J, Life P, Graber P, Mazzei G, Aubry J. Pairs of surface molecules involved in human IgE regulation: CD23-CD21 and CD40-CD40L. Eur Respir J Suppl. 1996;22:63se66s. [40] Vijayendrachary A, Hemalatha R, Narendrababu K, Rameshkumar R, Sivaramaprasad K, Dineshkumar B. CD23, Th1/Th2 cytokines in bronchial asthma, bronchiolitis and bronchial pneumonia in pediatric patients. Asian J Pharm Clin Res. 2012;5:111e114.
[41] Vijayendrachary A, Hemalatha R, Narendrababu K, Rameshkumar R, Chowdary K, Dineshkumar B. CD 23, total IgE and Th1/Th2 cytokines in asthma patients. Int J Pharm Pharm Sci. 2012;4:175e177. [42] Baeke F, Etten EV, Gysemans C, et al. Vitamin D signaling in immunemediated disorders: evolving insights and therapeutic opportunities. Mol Aspects Med. 2008;29:376e387. [43] Sandhu MS, Casale TB. The role of vitamin D in asthma. Ann Allergy Asthma Immunol. 2010;105:191e199. quiz 200-202, 217. [44] Brehm JM, Schuemann B, Fuhlbrigge AL, et al. Serum vitamin D levels and severe asthma exacerbations in the Childhood Asthma Management Program study. J Allergy Clin Immunol. 2010;126:52e58. [45] Provoost S, Maes T, van Durme YM, et al. Decreased FOXP3 protein expression in patients with asthma. Allergy. 2009;64:1539e1546. [46] Von Essen MR, Kongsbak M, Schjerling P, et al. Vitamin D controls T cell antigen receptor signaling and activation of human T cells. Nature immunology. 2010;11:344e349. [47] Ge L, Trujillo G, Miller EJ, Kew RR. Circulating complexes of the vitamin D binding protein with G-actin induce lung inflammation by targeting endothelial cells. Immunobiology. 2014;219:198e207. [48] Gupta A, Dimeloe S, Richards DF, et al. Vitamin D binding protein and asthma severity in children. J Allergy Clin Immunol. 2012;129:1669e1671. [49] Navas-Nazario A, Li FY, Shabanova V, et al. Effect of vitamin Debinding protein genotype on the development of asthma in children. Ann Allergy Asthma Immunol. 2014;112:519e524. [50] Poon AH, Laprise C, Lemire M, et al. Association of vitamin D receptor genetic variants with susceptibility to asthma and atopy. Am J Respir Crit Care Med. 2004;170:967e973. [51] Raby BA, Lazarus R, Silverman EK, et al. Association of vitamin D receptor gene polymorphisms with childhood and adult asthma. Am J Respir Crit Care Med. 2004;170:1057e1065. [52] Carlberg C. Mechanisms of nuclear signalling by vitamin D3. Interplay with retinoid and thyroid hormone signalling. Eur J Biochem. 1995;231: 517e527. [53] Wang TJ, Zhang F, Richards JB, et al. Common genetic determinants of vitamin D insufficiency: a genome-wide association study. Lancet. 2010;376: 180e188. [54] Jones G, Prosser DE, Kaufmann M. 25-Hydroxyvitamin D-24-hydroxylase (CYP24A1): its important role in the degradation of vitamin D. Arch Biochem Biophys. 2012;523:9e18. [55] Jones G, Prosser DE, Kaufmann M. Cytochrome P450-mediated metabolism of vitamin D. J Lipid Res. 2014;55:13e31. [56] Tsiaras WG, Weinstock MA. Factors influencing vitamin d status. Acta Derm Venereol. 2011;91:115e124.
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
8.e1
eFigure 1. Gating strategy of T-regulatory (Treg) cells (CD4þ/CD25þ/CD127/FOXP3þ cells) in whole blood. A, The lymphocyte cells were first gated based on forward scatter (FSC) and side scatter (SSC) to exclude dead cells and cell debris. B, CD4þ T cells were gated based on the expression of CD4 marker. C, The CD4þ T cells were gated for CD25þ and CD127 cells. D, The CD4þ/CD25þ/CD127 cells were further gated for FOXP3þ cells (CD4þCD25þ/CD127FOXP3þ or Treg cells). The data were obtained by FACS DIVA software (BD Biosciences, San Jose, California) and analyzed by FCS Express software (De Novo Software, Glendale, California).
8.e2
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
eFigure 2. Gating strategy of B cells with CD23/CD21 expression in whole blood. A, The lymphocyte cells were first gated based on forward scatter (FSC) and side scatter (SSC) to exclude dead cells and cell debris. B, The B cells were gated based on CD19 marker. C, The CD19 B lymphocytes were further gated for CD19þ/CD23þ. D, The CD19 B lymphocytes were gated CD19þ/CD21þ cells. The data were obtained by FACS DIVA software (BD Biosciences, San Jose, California) and analyzed by FCS Express software (De Novo Software, Glendale, California).
Contents lists available at ScienceDirect
Association of T-regulatory cells and CD23/CD21 expression with vitamin D in children with asthma Anchoju Vijayendra Chary, Mpharm *; Rajkumar Hemalatha, MD *; Machiraju Vasudeva Murali, MD y; Dodle Jayaprakash, PhD z; Bharathraj Dinesh Kumar, PhD x * Department
of Clinical Division, Microbiology and Immunology, National Institute of Nutrition, Hyderabad, Telangana, India Department of Paediatrics, Gandhi hospital, Hyderabad, Telangana, India z Department of Technology, Osmania University, Hyderabad, Telangana, India x Department of Food and Drug Toxicology, National Institute of Nutrition, Hyderabad, Telangana, India y
A R T I C L E
I N F O
Article history: Received for publication November 6, 2015. Received in revised form February 23, 2016. Accepted for publication February 29, 2016.
A B S T R A C T
Background: Children with asthma have low vitamin D levels; however, we do not know whether low vitamin D is associated with impaired T-regulatory (Treg) cell population or high IgE receptors (CD23 and CD21) on B cells. Objectives: To examine Treg cell function, CD23 and CD21 receptors, vitamin D, and vitamin Deregulating enzymes in children with asthma. Methods: Sixty children (2e6 years old) with asthma and 60 age-matched healthy children were selected as study participants. After collecting demographic and clinical data, blood samples were collected. Treg cells and CD23/CD21 expressions were evaluated by flow cytometry, 25-hydroxyvitamin D3 (25[OH]D3) was quantified by high-performance liquid chromatography, and cytokines and total IgE were estimated by enzyme-linked immunosorbent assay. Messenger RNA (mRNA) expressions of FOXP3, CD23, CD21, vitamin D receptors, and vitamin Deregulating enzymes were assessed by reverse transcriptionepolymerase chain reaction. Results: The 25(OH)D3 concentrations and proportion of Treg cells were lower (P < .05) among children with asthma. In contrast, the proportions of B cells with CD23 and CD21 expression were higher (P < .05) in patients with asthma compared with controls. Interleukin 10 and transforming growth factor b were also altered in asthma. The mRNA expression of CD23, CD21, and vitamin D receptors was up-regulated, whereas mRNA expression of vitamin Deregulating enzymes, CYP2R1, CYP27B1, and vitamin D binding protein (except CYP24A1) were up-regulated among children with asthma. Conclusion: The current study found impaired Treg cell population and high numbers of B cells with IgE receptors (CD23 and CD21) and altered regulatory cytokines in children with asthma, suggesting impaired immune regulation. Ó 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Introduction Asthma is a complex and heterogeneous inflammatory disease of the airways.1 The incidence of asthma has increased considerably in the past decade.1 Currently, it is estimated that approximately 300 million individuals with asthma exist worldwide, and this number is expected to increase by another 100 million by 2025, of whom 30 million are from India alone.2 The prevalence of asthma is estimated to range from 3% to 38% in children and from 2% to 12% in adults.3 Reprints: Rajkumar Hemalatha, MD, Department of Clinical Division, Microbiology and Immunology, National Institute of Nutrition, Hyderabad-07, Telangana, India; E-mail: [email protected]. Disclosures: Authors have nothing to disclose. Funding Sources: This study was funded by the National Institute of Nutrition, Hyderabad, Telangana, India.
The various cellular components that drive asthmatic reactions include mast cells, eosinophils, and lymphocytes.1,4 T regulatory (Treg) cells (CD4þ/CD25þ/CD127/FOXP3 Treg cells) are subsets of T lymphocytes described by expression of a specific transcription factor, FOXP3.5 Previous reports have implicated reduction in the numbers and functionality of Treg cells in both human and mouse during allergic conditions.5,6 Treg cells produce regulatory cytokines, such as interleukin 10 (IL-10) and transforming growth factor b (TGF-b).7,8 IL-10 has broad immunosuppressive and anti-inflammatory activity relevant to the inhibition of asthma disease.9 IL-10 acts on antigen-presenting cells and suppresses T-cell activation, including TH2 cells.9 On the other hand, TGF-b induces peripheral expression of the transcription factor FOXP3, which in turn promotes the transformation of Treg cells and inhibits allergic airway disease.10 Interaction between
http://dx.doi.org/10.1016/j.anai.2016.02.018 1081-1206/Ó 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
2
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
IL-10 and TGF-b is likely to be vital in the regulation of IgE and its substrates, which play an essential role in all allergic conditions.10 CD23 (FcεRII) and CD21 are the low-affinity receptors for IgE and are found mainly on the surface of B lymphocytes. CD23 and CD21 interact with each other, and the pairing of these molecules is important in T-cell and B-cell interaction and control of IgE production.11 In an earlier study, we found a strong inverse association between Treg cells and CD23 and CD21 in pregnant women.12 The same study also found impaired Treg cells in pregnant women with vitamin D deficiency.13 Higher vitamin D intake by pregnant mothers reduces asthma risk by as much as 40% in 3- to 5-year-old children.14 Although vitamin D has been suggested to play an important role in the maintenance of B-cell homeostasis, IgE receptor expression on B cells has not been studied in vitamin Dedeficient children with asthma.15 CD23 and CD21 on B cells may have a key role as receptors of IgE antibody and conceivably are linked with increased respiratory morbidity among vitamin Dedeficient individuals.16 It has been speculated that vitamin D deficiency may increase early-life respiratory morbidity by impairing Treg cell population and by increasing B cells with IgE receptors.13 Vitamin D is activated by 2 enzymatic steps. Vitamin D3 (cholecalciferol) is metabolized to 25-hydroxycholecalciferol (calcidiol or 25-hydroxyvitamin D3 [25(OH)D3]) by the hepatic 25hydroxylases (CYP2R1) in the liver. Subsequently, 25(OH)D3 is converted to 1,25-dihydroxycholecaciferol (calcitriol or 1,25 [OH]2D3) by 1a-hydroxylase (CYP27B1) in the kidney.17 The active form of vitamin D3 (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 CYP27B1 enzyme is also produced by several other cell types, including dermal cells, intestinal epithelial cells, lymph nodes, monocytes, and placenta, which enables extra renal activation of 25(OH)D3 to the active hormonal form, 1,25(OH)2D3.19,20 Vitamin D, VDR, RXR and vitamin Deregulating enzymes are known to be responsible for inducing several genes that are important in asthma and allergic conditions.21 In the current study, besides examining the Treg cell population and the IgE receptors on B cells, the serum vitamin D and vitamin Deregulating enzymes (CYP2R1, CYP27B1, and CYP24A1), VDR, and RXR were explored in children with asthma. Methods Ethical Information This study was conducted at the Gandhi Hospital, Hyderabad, according to the Helsinki prescribed guidelines. Ethical approval was obtained to undertake the study, and written informed consent was obtained from parents or guardians before including their children in the study.
classified based on clinical examination and symptoms as described by the Global Initiative for Asthma (GINA) 2008 guidelines.22 Socioeconomic status was calculated with the modified Kuppuswamy socioeconomic scale.23 Blood samples were collected from all the children to assess immune parameters and vitamin D status. Treg cell (CD4þ/CD25þ/CD127/FOXP3þ), CD23, and CD21 expression on B cells was determined by flow cytometry. 25(OH)D3 was quantified with high-performance liquid chromatography. TGF-b, IL-10, and IgE levels were quantified by enzyme-linked immunosorbent assay (ELISA). In a randomly selected subsample of 12 individuals from each group (cases and controls), peripheral blood mononuclear cells (PBMCs) were used for messenger RNA (mRNA) expression of CD23, CD21, FOXP3, CYP2R1, CYP27B1, CYP24A1, vitamin D binding protein (VDBP), VDR, and RXR. The required sample size for mRNA expression of the above mentioned genes was calculated with 90% power after reaching a sample size 12 children in each group. Treg Cell (CD4þ/CD25þ/CD127/FOXP3þ) Population The proportion of Treg cells was estimated in whole blood as described in an earlier study.13 Whole blood (100 mL) was stained with 20 mL of human Treg cell cocktail that contained fluorescein isothiocyanate antihuman CD4 antibody, PE-Cy7 antihuman CD25 antibody, and Alexa Fluor 647 antihuman CD127 antibody (BD Sciences, San Jose, California). The erythrocytes were lysed using red blood cell lysing buffer (BD Sciences). The cells were again washed and were stained with PE mouse antihuman FOXP3 (BD Sciences). The lymphocyte cells were first gated based on forward and side scatter to exclude dead cells and cell debris. Later CD4þ T cells were gated, and CD25þ and CD127 cells were further gated from CD4þ cells. CD4þ/CD25þ/CD127 cells were further gated for FOXP3þ cells (CD4þCD25þ/CD127FOXP3þ or Treg cells). The data were obtained by FACS DIVA software (BD Biosciences, San Jose, California) and was analyzed by FCS Express software (De Novo Software, Glendale, California) (eFig 1). Total IgE and Cytokine Assay (IL-10 and TGF-b) Serum samples were used to estimate total IgE, and cytokines, such as IL-10 and TGF-b, were quantified by sandwich ELISA. Briefly, the microplate was coated with capture antibody. Then, samples that contained an unknown amount of the target analyte of interest were added. After washing, to get rid of the microplate of unbound substances, a horseradish peroxidase conjugate was added, and absorbance was determined according to the manufacturer’s instructions (eBiosciences, San Diego, California). The lower limit of detection for total IgE was 7.8 ng/mL, and the standard curve range given with kit was 7.8 to 1,000 ng/mL. The lower limit of detection for IL-10 was 0.8 ng/mL, and the standard curve range given with kit was 0.8 to 50 ng/mL. The lower limit of detection for TGF-b was 80 pg/mL, and the standard curve range given with kit was 80 to 5000 ng/mL. B Cells With CD23 and CD21 Expression
Enrollment of Study Participants Children aged 2 to 6 years who were visiting the outpatient department of the asthma clinic at Gandhi Hospital were selected as study participants. Children with fever, infections, or skin problems and those taking steroids or antibiotics were excluded from the study. Sixty children with asthma were recruited as cases, and 60 age-matched apparently healthy children without asthma or without any infection were used as controls. After written informed consent was obtained from the parents or guardians of the study participants, a detailed questionnaire was administered on family history of allergy and asthma, history of smoking in the family, demographic profile, and anthropometry data on a pretested semistructured questionnaire. Children with asthma were
To estimate the proportion of B cells with CD23 and CD21 expression, 100 mL of whole blood was stained with 20 mL of Alexa Fluor 700 mouse antihuman CD19 antibody, 20 mL of fluorescein isothiocyanate mouse antihuman CD23 antibody, and 5 mL of Allophycocyanin mouse antihuman CD21 for 20 minutes at room temperature according to the manufacturer’s instructions (BD Sciences). The red blood cells were lysed by BD FACS red blood cell lysing solution. After washing with 2 mL of flow staining buffer, the pellet was suspended in 100 mL of flow staining buffer. A total of 10,000 events were acquired per sample on the FACS ARIA II system. The B cells were gated based on CD19 marker and were further gated for CD19þ/CD23þ and CD19þ/CD21þ cells. The data were obtained by FACS DIVA software (BD Biosciences, San Jose,
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
California) and were analyzed by FCS Express software (De Novo Software, Glendale, California) (eFig 2). Serum Calcium and Hemoglobin Assay Serum calcium was quantified using an ELISA-based kit according to the manufacturer’s instructions (Accucare Labs, Mumbai, India). Hemoglobin was estimated by the Drabkin’s (cyanmethemoglobin) method. 25(OH)D3 Quantification Serum levels of 25(OH)D3 are considered to be the best circulating biomarker of vitamin D metabolic status and reflect contributions from all sources of vitamin D (ie, diet and sun exposure).24 25(OH)D3 levels were categorized as deficient (15 ng/mL) and sufficient (15 ng/mL) on the basis of previous recommendations (Institute of Medicine classification).25 A total of 500 mL of serum was used to quantify 25(OH)D3 by HPLC, using Schimadzu system with Supelcosil column (4.6 mm 25 cm; 5m particle size; Sigma-Aldrich, St Louis, Missouri). The mobile phase used methanol and water (85:15 ratio), with flow rate of 2 mL/min. The injection volume was 25 mL and detection was at 265 nm wavelength. mRNA Expression of FOXP3, CD23, CD21, VDR, CYP2R1, CYP27B1, CYP24A1, VDBP, and RXR in the PBMCs PBMCs were separated from whole blood by density gradient centrifugation using Ficoll reagent. A total of 4 mL of blood was layered on the top of 4 mL of Ficoll Histopaque and was centrifuged for 30 minutes at 100g in 4 C. The whitish buffy coat (PBMCs) formed in the interphase between Histopaque and medium was aspirated. PBMCs were washed 2 times with PBS to remove the Ficoll reagent. Total RNA was extracted from the PBMCs according to the Chomzinsky and Sacchi method.26 Total RNA thus extracted was treated by DNase according to the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, Massachusetts). The quality and the yield of total RNA were verified using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, California) and Nanodrop 1000 (Thermo Fisher Scientific). Electrophoresis was used to check the integrity and purity of the RNA for complementary DNA (cDNA) synthesis by the procedure of Masek et al.27,28
3
Table 1 Primers Used in Reverse TranscriptionePolymerase Chain Reaction Sequence No.
Gene
Primer Sequence
1
GAPDH
2
VDR
3
FOXP3
4
CD23
5
CD21
6
CYP27B1
7
CYP2R1
8
CYP24A1
9
RXR
10
VDBP
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
94 88 105 97 100 100 94 92 105 86
Statistical Analysis For descriptive data, mean values were calculated and differences between groups were assessed with the t test using SPSS software, version 18.0 (SPSS Inc, Chicago, Illinois). Graphical analysis was performed in SigmaPlot 12.3 (Systat Software, Chicago, Illinois). Values are expressed as mean (SE). The mean differences were considered significant when P < .05. Correlations of various parameters were analyzed by the Spearman rank correlation coefficient (r). Results Demographic Information Of the 120 children, 72 were male and the rest were female. The mean (SD) age and weight of the children were 4.3 (1.4) years and 15.38 (3.76) kg, respectively, and the ratios of male vs female were Table 2 Demographic and Clinical Characteristics of the Study Participantsa Parameter
Control Children (n ¼ 60)
Children With Asthma (n ¼ 60)
P Valueb
Male, % Female, % Age, mo Weight, kg Height for age Weight for age BMI for age Weight for height Family history of asthma, % Asthma severity, % Severe persistent Moderate persistent Mild persistent Mild intermittent 25(OH)D3 status, % Sufficient
55 45 46.95 14.36 0.18 1.19 1.92 2.16 .c
65 35 48.87 16.40 0.45 0.02 0.43 0.40 18.3
>.05
cDNA Synthesis and RT-PCR A total of 3 mg of total RNA was transcribed to cDNA using a RevertAid first-strand cDNA synthesis kit (Thermo Fisher Scientific). The cDNA thus obtained was aliquoted and stored at 20 C. For the reverse transcriptionepolymerase chain reaction (RT-PCR) assay, SYBR Green Master Mix (Thermo Fisher Scientific) was used, and primers were designed by Geneious pro software (version 5.4.6; Geneious, Auckland, New Zealand) and synthesized by Imperial Life Sciences (New Delhi, India) (Table 1). GAPDH was used as internal control. To quantify gene expression of target genes such as CD23, CD21, FOXP3, CYP2R1, CYP27B1, CYP24A1, VDBP, VDR, and RXR, the ABI Step One Plus system was used with program of 95 C for 10 minutes followed by 40 cycles of 95 C for 15 seconds and 60 C for 1 minute. The difference in the cycle threshold (CT) value between the control gene (GAPDH) and the target genes was obtained for each sample. The critical 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.
Base Pairs
Deficient
(14.22) (4.54) (1.98) (1.85) (3.07) (2.97)
. . . . 48.33 (19.89 [0.44] ng/mL) 51.66 (13.16 [0.38] ng/mL)
4 26 20 10
(12.96) (2.42) (1.48) (1.11) (1.83) (1.89)
>.05 >.05 >.05 >.05 >.05 >.05
(6.66) (43.33) (33.33) (16.66)
20 (20 [0.67] ng/mL) 80 (11.07 [0.40] ng/mL)
Abbreviations: 25(OH)D3, 25-hydroxyvitamin D3; BMI, body mass index. a Values are expressed as mean (SD) unless otherwise indicated. b P < .05 was considered significant. c Ellipses indicate data not applicable.
>.05 >.05
4
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
1.85:1 and 1.22:1 in asthmatic and control children, respectively. The mean height, weight, and body mass index z scores were comparable between cases and controls (Table 2). Similarly, the mean (SE) hemoglobin levels were comparable between asthmatic (10.3 [1.98] mg/dL) and control children (11.6 [1.87] mg/dL). The serum calcium levels were also comparable between asthmatic (8.3 [0.05] ng/mL) and control children (8.5 [0.08] ng/mL). All the children belonged to middle and low socioeconomic status and as expected had insufficient milk intake. On the basis of the GINA classification, 4 (6.7%) 60 children had severe persistent asthma, 26 (43.3%) had moderate persistent
asthma, 20 (33.3%) had mild persistent asthma, and 10 (16.6%) had mild intermittent asthma. Treg Cell (CD4þ/CD25þ/CD127/FOXP3þ) Population The proportion of the Treg cell population was evaluated within the CD4þ population. The Treg cell population ranged from 0.1% to 0.5% and 0.2% to 0.9% in asthmatic and control children, respectively. Figure 1 shows the mean Treg cell population, which was significantly (P < .05) lower in asthmatic (0.29 [0.01]) compared with control children (0.38 [0.02]).
Figure 1. T-regulatory (Treg) cell population, total IgE levels, and B cells with CD23/CD21 expression in whole blood in asthmatic and control children. Spearman rank correlation of Treg cells with B cells with CD23/CD21. Values are expressed as mean (SE). A, Treg cell population; B, total IgE levels; C, B cells with CD23 expression; D, B cells with CD21 expression; E, correlation of Treg cells and B cells with CD23; and F, correlation of Treg cells and B cells with CD21 in asthma. *P < .05.
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
5
Total IgE Levels
mRNA Expression of Vitamin DeRegulating Enzymes and VDRs
As expected, the mean (SE) serum IgE level was significantly (P < .05) higher in children with asthma (748 [55.8] ng/mL) compared with control children (362.88 [38.53] ng/mL). However, IgE levels were not correlated with either 25(OH)D3 or Treg cell population or B cells with CD23/CD21 expression (Fig 1).
The mRNA expressions of FOXP3, CD23, CD21, IL10, TGF- b, VDR, CYP2R1, CYP27B1, CYP24A1, VDBP, and RXR are presented in RQ values and are compared among children with asthma and the control group. GABDH was used as an internal control (Fig 4). The mRNA expression of the transcription factor of the Treg cell, the FOXP3, was (P < .05) down-regulated in children with asthma. As expected, the mRNA expression of CD23 and CD21 were (P < .05) up-regulated in children with asthma. The mRNA expression of IL-10 was down-regulated, whereas TGF-b gene was up-regulated in children with asthma compared with controls (Fig 4). Both the vitamin Deactivating enzymes, CYP2R1 and CYP27B1, were (P < .05) up-regulated, whereas the vitamin Deinactivating enzyme, CYP24A1, was down-regulated in children with asthma. As for the receptors and binding protein, the VDR, RXR, and VDBP expressions were all up-regulated (P < .05) in children with asthma (Fig 4).
B Cells With CD23/CD21 Expression The mean (SE) percentage of B cells with CD23 expression was higher (P < .05) in children with asthma (1.30% [0.08%]) than in control (0.18% [0.01%]). Similarly, the mean (SE) percentage of B cells with CD21 expression was higher (P < .05) in asthmatic children (1.89% [0.11%]) compared with controls (0.84% [0.03%]) (Fig 1). As expected, the CD23 population was strongly correlated with the CD21 population (n ¼ 60; P < .01; r ¼ 0.729) of B cells (data not shown). However, the Treg cell population was inversely correlated with both CD23 (n ¼ 60; P < .01; r ¼ 0.456) and CD21 (n ¼ 60; P < .01; r ¼ 0.504) population (Fig 1). Regulatory Cytokines (IL-10 and TGF-b) The mean (SE) of IL-10 was lower (P < .05) in asthmatic children (3.4 [0.46] ng/mL) than in controls (5.06 [0.84] ng/mL). In contrast, TGF-b concentration was higher (P < .05) in asthmatic children (4330.46 [162.56] pg/mL) compared with controls (3338.45 [168.17] pg/mL) (Fig 2). Both IL-10 and TGF-b concentrations did not correlate with Treg cell population (data not shown).
25(OH)D3 Levels in Asthmatic and Control Children Twelve children (20%) with asthma had 25(OH)D3 concentrations within the reference range, whereas 29 healthy children (48.3%) had 25(OH)D3 within the reference range (Table 2). However, the mean 25(OH)D3 concentrations were significantly (P < .05) lower in children with asthma (15.54 [0.71] ng/mL) than in controls (18.44 [0.49] ng/mL) (Fig 3). Interestingly, the 25(OH)D3 concentration was directly correlated with Treg cell population (n ¼ 60; P < .01; r ¼ 0.652) in children with asthma (Fig 3). In contrast, the CD23 (n ¼ 60; P < .01; r ¼ 0.670) and CD21 populations (n ¼ 60; P < .01; r ¼ 0.791) were inversely correlated with 25(OH)D3 in children with asthma (Fig 3).
Discussion The current study found impaired Treg cell population and high numbers of B cells with CD23 and CD21 expressions and altered regulatory cytokines in children with asthma. Furthermore, impaired Treg cell population was directly associated with low levels of serum vitamin D. The study also found altered VDR expression and vitamin D regulatory enzymes in children with asthma. The effects of Treg cells on the immune system are important in the intracellular signaling pathways in lymphocytes and antigen-presenting cells.28 That the Treg cells have a central role in suppressing allergic asthma is undisputed. The cytokines most commonly implicated in Treg-mediated control of allergic asthma are TGF-b and IL-1029,30 The regulatory cytokine IL-10 has anti-inflammatory activity and inhibits asthma symptoms.9 On the other hand, TGF-b is known to induce peripheral expression of the Treg cells and its transcription factor FOXP3 and thus inhibits allergic airway disease in asthma.31,32 Although the current study found high TGF-b levels on a low IL-10 background in children with asthma, the source of TGF-b is unclear. A number of host cells, such as Treg cells, effector T cells, or noneT cells, may secrete TGF-b.10 Of the Treg cells, 2 types of FOXP3 Treg cells have been described that produce TGF-b and IL-10, respectively, for their suppressive functions.33 Because there was no correlation between the Treg cell population and TGF-b, we can only speculate that the source of high concentration of TGF-b could not have been Treg
Figure 2. Regulatory cytokines (interleukin-10 [IL-10] and transforming growth factor b [TGF-b]) in the blood. Values are expressed as mean (SE). A, IL-10 levels in asthmatic and control children; B, TGF-b levels in asthmatic and control children. *P < .05.
6
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
Figure 3. 25-hydroxyvitamin D3 (25[OH]D3) concentrations in the blood and Spearman rank correlation of 25(OH)D3 with T-regulatory (Treg) cells and B cells with CD23/CD21 expression in children with asthma. Values are expressed as mean (SE). A, 25(OH)D3 concentrations in asthmatic and control children; B, correlation of 25(OH)D3 and Treg cells; C, correlation of 25(OH)D3 and B cells with CD23 expression; and D, correlation of 25(OH)D3 and B cells with CD21 expression. *P < .05.
cells in children with asthma in the current study. Alternatively, it may be speculated that TGF-b secretion might have been driven by low Treg cell populations as a feedback mechanism to maintain optimum Treg cells and immune regulation.34,35 Although the reason for the low IL-10 concentration is unclear, impaired Treg cell population may be speculated as the mechanism behind low IL-10 levels in asthma.36,37 Interaction between IL-10 and TGF-b is likely to be vital in the regulation of IgE and its substrates, which play an essential role in all allergic conditions.10 CD23 (FcεRII) and CD21 are the low-affinity receptors for IgE and are found mainly on the surface of B lymphocytes. CD23 and CD21 interact with each other, and the pairing of these molecules is important in T-cell and B-cell interaction and control of IgE production.38 In our earlier study, soluble CD23 was increased in children and adults with asthma.39,40 The current study confirmed high expression of CD23 and CD21 on the backdrop of low circulating Treg cells and 25(OH)D3. Even the mRNA expressions of CD23 and CD21 were increased with the concurrent decrease in FOXP3, suggesting altered immune regulation at transcription level in asthma. However, these results should be interpreted with caution because failure to include an IgG isotype control would have influenced the results. Nevertheless, the CD23 or CD21 stained population were clearly separated from unstained population, suggesting little or no background fluorescence. Vitamin D induces Treg cell expression and increases production of IL-10.33 Vitamin D has also been suggested to play an important role in the maintenance of B-cell homeostasis and in the treatment of B-cellemediated autoimmune disorders.15 The vitamin D
regulates IgE levels in the serum via a direct effect on the VDR expression on B cells, However, the role of vitamin D on IgE receptors (CD23/CD21) on B cells is not known, although the present study clearly indicates a strong inverse correlation between 25(OH)D3 and CD23 and CD21 expression on B cells. CD23 and CD21 on B cells may have a key role as receptors of IgE antibody and conceivably are linked with increased respiratory morbidity amongst vitamin Dedeficient individuals.41 Similar to our observation, recent studies have found a high prevalence of vitamin D deficiency in asthmatic patients.42,43 Large cross-sectional studies found an association between low vitamin D levels and reduced lung function in adolescents and adults and an inverse correlation of vitamin D with IgE concentration.44 Although we do not know whether a low vitamin D level in children is a cause or effect of asthma, it may be speculated that low vitamin D level may well be associated with an impaired Treg cell population.45,46 This is the first study, to our knowledge, to report an inverse correlation of vitamin D with CD23 and CD21 expression on B cells in children with asthma. Moreover, the current study describes gene expression of VDBP, VDRs, and vitamin Deregulating enzymes in asthma. VDBP, a key carrier binding protein for 25(OH)D3 and 1,25(OH)2D3, helps in the transportation of these forms of vitamin D into the cells.47 Although the reason for increased expression of VDBP is unclear, a high level of VDBP has been suggested to have a protective role in vitamin D deficiency.48 VDBP has also been implicated in immunomodulatory functions in the lungs and has been described to have a protective role in asthma.49
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
7
Increased expression of VDRs and vitamin Deactivating enzymes and low expression of vitamin Demetabolizing enzymes suggest poor availability of the active form of vitamin D in asthma. More studies are needed to elucidate the role of vitamin D in immune regulation in children with asthma. Acknowledgments We greatly acknowledged the National Institute of Nutrition for providing financial support and facilities to conduct the study. We acknowledge the Council of Scientific & Industrial Research, Human Resource Development Group, for the senior research fellowship to Mr Chary. We are thankful to the Gandhi Hospital superintendent and nursing staff (Aruna Reddy) for their immense support in the recruitment of study participants and collection of samples. Supplementary Data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.anai.2016.02.018. References
Figure 4. Messenger RNA (mRNA) expression of immunologic, vitamin D receptors, and vitamin Deregulating enzymes in peripheral blood mononuclear cells of children with asthma with respect to controls by reverse transcriptionepolymerase chain reaction. The control group was used as the calibrator. GAPDH was used as the internal control. Values are expressed as mean (SE). A, mRNA expression (relative quantification [RQ]) of CD23, CD21, FOXP3, transforming growth factor b (TGF-b), and interleukin 10 (IL-10); B, mRNA expression (RQ) of vitamin D receptor (VDR), CYP27B1, CYP2R1, CYP24A1, vitamin D binding protein (VDBP), and 9-cis-retinoic acid receptor (RXR). *P < .05.
Immune cells, such as T and B lymphocytes, macrophages, and dendritic cells, express VDR and are affected by vitamin D deficiency during their maturation process.50 Several genetic studies have reported VDR polymorphism in asthma.51 VDR acts primarily as a heterodimer with the RXR on vitamin D response elements.52 VDR is abundantly present in various cells in the body and plays a critical role in transportation of calcium.24 In addition, VDR expression regulates up to 500 vitamin D responsive genes, which influence mineral metabolism, differentiation, and proliferation of various cells.53 Hence, high VDR and RXR expressions in children with asthma as observed in the present study must be viewed critically. The active form of vitamin D (1,25(OH)2D3) is converted by CYP27B1 CYP2R1, whereas CYP24A1 is central for 1,25(OH)2D3 catabolism and degradation.54,55 The enzyme CYP2R1, which converts cholecalciferol to 25(OH)D3, is directly inhibited by 25(OH)D3. On the other hand, 1,25(OH)2D3 is metabolized to the inactive 1,24,25(OH)3D3 by CYP24A1. 1,25(OH)2D3 vitamin D in turn inhibits CYP27B1 and stimulates the 24-hydroxylase enzymes, This tightly regulated negative feedback mechanism helps maintain serum concentrations of 25(OH)D3 within a physiologic range (30e100 ng/mL).56 Therefore, it may be speculated that low vitamin D drives up-regulation of CYP2R1, CYP27B1 and down-regulation of CYP24A1 enzymes in children with asthma.
[1] Holgate ST. The sentinel role of the airway epithelium in asthma pathogenesis. Immunol Rev. 2011;242:205e219. [2] Cruz AA, Bousquet J, Khaltaev N. Global Surveillance, Prevention and Control of Chronic Respiratory Diseases: A Comprehensive Approach. Geneva, Switzerland: World Health Organization; 2007. [3] Cavkaytar O, Sekerel BE. Baseline management of asthma control. Allergol Immunopathol (Madr). 2014;42:162e168. [4] Lotvall J, Akdis CA, Bacharier LB, et al. Asthma endotypes: a new approach to classification of disease entities within the asthma syndrome. J Allergy Clin Immunol. 2011;127:355e360. [5] Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP3þ regulatory T cells in the human immune system. Nat Rev Immunol. 2010;10:490e500. [6] Corthay A. How do regulatory T cells work? Scand J Immunol. 2009;70: 326e336. [7] O’Garra A, Barrat FJ, Castro AG, Vicari A, Hawrylowicz C. Strategies for use of IL-10 or its antagonists in human disease. Immunol Rev. 2008;223:114e131. [8] Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Transforming growth factorbeta regulation of immune responses. Annu Rev Immunol. 2006;24:99e146. [9] Martin H, Taube C. Regulatory T cells and regulation of allergic airway disease. Am J Clin Exp Immunol. 2012;1:166. [10] Taylor A, Verhagen J, Blaser K, Akdis M, Akdis CA. Mechanisms of immune suppression by interleukin-10 and transforming growth factor-b: the role of T regulatory cells. Immunology. 2006;117:433e442. [11] Rabatic S, Gagro A, Medar-Lasic M. CD21-CD23 ligand pair expression in children with allergic asthma. Clin Exp Immunol. 1993;94:337e340. [12] Vijayendra Chary A, Hemalatha R, Seshacharyulu M, et al. Vitamin D deficiency in pregnant women impairs regulatory T cell function. J Steroid Biochem Mol Biol. 2015;147:48e55. [13] Litonjua AA, Weiss ST. Is vitamin D deficiency to blame for the asthma epidemic? J Allergy Clin Immunol. 2007;120:1031e1035. [14] Berraies A, Hamzaoui K, Hamzaoui A. Link between vitamin D and airway remodeling. J Asthma Allergy. 2014;7:23e30. [15] Chen S, Sims GP, Chen XX, et al. Modulatory effects of 1,25-dihydroxyvitamin D3 on human B cell differentiation. J Immunol. 2007;179:1634e1647. [16] Hartmann B, Heine G, Babina M, et al. Targeting the vitamin D receptor inhibits the B cell-dependent allergic immune response. Allergy. 2011;66:540e548. [17] Hewison M, Burke F, Evans KN, et al. Extra-renal 25-hydroxyvitamin D31alpha-hydroxylase in human health and disease. J Steroid Biochem Mol Biol. 2007;103:316e321. [18] Foong RE, Zosky GR. Vitamin D deficiency and the lung: disease initiator or disease modifier? Nutrients. 2013;5:2880e2900. [19] Shinkyo R, Sakaki T, Kamakura M, Ohta M, Inouye K. Metabolism of vitamin D by human microsomal CYP2R1. Biochem Biophys Res Commun. 2004;324:451e457. [20] Zehnder D, Evans KN, Kilby MD, et al. The ontogeny of 25-hydroxyvitamin D 3 1a-hydroxylase expression in human placenta and decidua. Am J Pathol. 2002; 161:105e114. [21] Hansdottir S, Monick MM. Vitamin D effects on lung immunity and respiratory diseases. Vitam Horm. 2011;86:217. [22] Bateman E, Hurd S, Barnes P, et al. Global strategy for asthma management and prevention: GINA executive summary. Eur Respir Jl. 2008;31:143e178. [23] Bairwa M, Rajput M, Sachdeva S. Modified Kuppuswamy’s socioeconomic scale: social researcher should include updated income criteria, 2012. Indian J Community Med. 2013;38:185e186. [24] Hollis BW. Circulating 25-hydroxyvitamin D levels indicative of vitamin D sufficiency: implications for establishing a new effective dietary intake recommendation for vitamin D. J Nutr. 2005;135:317e322.
8
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
[25] Ross AC, Manson JE, Abrams SA, et al. The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what clinicians need to know. J Clin Endocrinol Metab. 2011;96:53e58. [26] Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc. 2006;1:581e585. [27] Aranda PS, LaJoie DM, Jorcyk CL. Bleach gel: a simple agarose gel for analyzing RNA quality. Electrophoresis. 2012;33:366e369. [28] Masek T, Vopalensky V, Suchomelova P, Pospisek M. Denaturing RNA electrophoresis in TAE agarose gels. Anal Biochem. 2005;336:46e50. [29] Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4þCD25þ T cell-mediated suppression by dendritic cells. Science. 2003;299:1033e1036. [30] Yalcin AD, Bisgin A, Gorczynski RM. IL-8, IL-10, TGF-, and GCSF levels were increased in severe persistent allergic asthma patients with the anti-IgE treatment. Mediators Inflamm. 2012;2012:720976. [31] Chen W, Jin W, Hardegen N, et al. Conversion of peripheral CD4þCD25- naive T cells to CD4þCD25þ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med. 2003;198:1875e1886. [32] Krogulska A, Polakowska E, Wa˛ sowska-Królikowska K, Małachowska B, Młynarski W, Borowiec M. Decreased FOXP3 mRNA expression in children with atopic asthma and IgE-mediated food allergy. Ann Allergy Asthma Immunol. 2015; 115:415e421. [33] Ito T, Hanabuchi S, Wang YH, et al. Two functional subsets of FOXP3þ regulatory T cells in human thymus and periphery. Immunity. 2008;28:870e880. [34] Urry Z, Xystrakis E, Dimeloe S, Richards D, Brown Z, Hawrylowicz C. 1a, 25-dihydroxyvitamin D3 differentially promotes Foxp3 and IL-10 expression by human CD4þ T cells. Immunology. 2010;131. 184e184. [35] Joetham A, Takeda K, Taube C, et al. Naturally occurring lung CD4(þ)CD25(þ) T cell regulation of airway allergic responses depends on IL-10 induction of TGF-beta. J Immunol. 2007;178:1433e1442. [36] Chatila TA. Role of regulatory T cells in human diseases. J Allergy Clin Immunol. 2005;116:949e959. [37] Halwani R, Al-Muhsen S, Al-Jahdali H, et al. Role of transforming growth factoreb in airway remodeling in asthma. Am J Respir Cell Mol Biol. 2011;44:127e133. [38] Makinde T, Murphy RF, Agrawal DK. The regulatory role of TGF-beta in airway remodeling in asthma. Immunol Cell Biol. 2007;85:348e356. [39] Bonnefoy J, Gauchat J, Life P, Graber P, Mazzei G, Aubry J. Pairs of surface molecules involved in human IgE regulation: CD23-CD21 and CD40-CD40L. Eur Respir J Suppl. 1996;22:63se66s. [40] Vijayendrachary A, Hemalatha R, Narendrababu K, Rameshkumar R, Sivaramaprasad K, Dineshkumar B. CD23, Th1/Th2 cytokines in bronchial asthma, bronchiolitis and bronchial pneumonia in pediatric patients. Asian J Pharm Clin Res. 2012;5:111e114.
[41] Vijayendrachary A, Hemalatha R, Narendrababu K, Rameshkumar R, Chowdary K, Dineshkumar B. CD 23, total IgE and Th1/Th2 cytokines in asthma patients. Int J Pharm Pharm Sci. 2012;4:175e177. [42] Baeke F, Etten EV, Gysemans C, et al. Vitamin D signaling in immunemediated disorders: evolving insights and therapeutic opportunities. Mol Aspects Med. 2008;29:376e387. [43] Sandhu MS, Casale TB. The role of vitamin D in asthma. Ann Allergy Asthma Immunol. 2010;105:191e199. quiz 200-202, 217. [44] Brehm JM, Schuemann B, Fuhlbrigge AL, et al. Serum vitamin D levels and severe asthma exacerbations in the Childhood Asthma Management Program study. J Allergy Clin Immunol. 2010;126:52e58. [45] Provoost S, Maes T, van Durme YM, et al. Decreased FOXP3 protein expression in patients with asthma. Allergy. 2009;64:1539e1546. [46] Von Essen MR, Kongsbak M, Schjerling P, et al. Vitamin D controls T cell antigen receptor signaling and activation of human T cells. Nature immunology. 2010;11:344e349. [47] Ge L, Trujillo G, Miller EJ, Kew RR. Circulating complexes of the vitamin D binding protein with G-actin induce lung inflammation by targeting endothelial cells. Immunobiology. 2014;219:198e207. [48] Gupta A, Dimeloe S, Richards DF, et al. Vitamin D binding protein and asthma severity in children. J Allergy Clin Immunol. 2012;129:1669e1671. [49] Navas-Nazario A, Li FY, Shabanova V, et al. Effect of vitamin Debinding protein genotype on the development of asthma in children. Ann Allergy Asthma Immunol. 2014;112:519e524. [50] Poon AH, Laprise C, Lemire M, et al. Association of vitamin D receptor genetic variants with susceptibility to asthma and atopy. Am J Respir Crit Care Med. 2004;170:967e973. [51] Raby BA, Lazarus R, Silverman EK, et al. Association of vitamin D receptor gene polymorphisms with childhood and adult asthma. Am J Respir Crit Care Med. 2004;170:1057e1065. [52] Carlberg C. Mechanisms of nuclear signalling by vitamin D3. Interplay with retinoid and thyroid hormone signalling. Eur J Biochem. 1995;231: 517e527. [53] Wang TJ, Zhang F, Richards JB, et al. Common genetic determinants of vitamin D insufficiency: a genome-wide association study. Lancet. 2010;376: 180e188. [54] Jones G, Prosser DE, Kaufmann M. 25-Hydroxyvitamin D-24-hydroxylase (CYP24A1): its important role in the degradation of vitamin D. Arch Biochem Biophys. 2012;523:9e18. [55] Jones G, Prosser DE, Kaufmann M. Cytochrome P450-mediated metabolism of vitamin D. J Lipid Res. 2014;55:13e31. [56] Tsiaras WG, Weinstock MA. Factors influencing vitamin d status. Acta Derm Venereol. 2011;91:115e124.
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
8.e1
eFigure 1. Gating strategy of T-regulatory (Treg) cells (CD4þ/CD25þ/CD127/FOXP3þ cells) in whole blood. A, The lymphocyte cells were first gated based on forward scatter (FSC) and side scatter (SSC) to exclude dead cells and cell debris. B, CD4þ T cells were gated based on the expression of CD4 marker. C, The CD4þ T cells were gated for CD25þ and CD127 cells. D, The CD4þ/CD25þ/CD127 cells were further gated for FOXP3þ cells (CD4þCD25þ/CD127FOXP3þ or Treg cells). The data were obtained by FACS DIVA software (BD Biosciences, San Jose, California) and analyzed by FCS Express software (De Novo Software, Glendale, California).
8.e2
A.V. Chary et al. / Ann Allergy Asthma Immunol xxx (2016) 1e8
eFigure 2. Gating strategy of B cells with CD23/CD21 expression in whole blood. A, The lymphocyte cells were first gated based on forward scatter (FSC) and side scatter (SSC) to exclude dead cells and cell debris. B, The B cells were gated based on CD19 marker. C, The CD19 B lymphocytes were further gated for CD19þ/CD23þ. D, The CD19 B lymphocytes were gated CD19þ/CD21þ cells. The data were obtained by FACS DIVA software (BD Biosciences, San Jose, California) and analyzed by FCS Express software (De Novo Software, Glendale, California).