Effect of vitamin D supplementation on cathelicidin, IFN ... - Nature

European Journal of Clinical Nutrition (2014) 68, 338–343 & 2014 Macmillan Publishers Limited All rights reserved 0954-3007/14 www.nature.com/ejcn

ORIGINAL ARTICLE

Effect of vitamin D supplementation on cathelicidin, IFN-g, IL-4 and Th1/Th2 transcription factors in young healthy females M Das1, N Tomar1, V Sreenivas2, N Gupta1 and R Goswami1 OBJECTIVES: We assessed the effect of cholecalciferol and calcium supplementation on mRNA expression of cathelicidin (LL-37), Th1 and Th2 cytokines and their transcription factors in the peripheral blood mononuclear cells (PBMCs) in healthy females with vitamin D deficiency (VDD). SUBJECTS/METHODS: Subjects included 131 females with biochemical VDD randomized to receive (a) oral cholecalciferol (60 000 IU/week for 8 weeks followed by 60 000 IU/fortnight (b) calcium (elemental calcium 500 mg twice/day) (c), dual supplementation and (d) placebo for 6 months. The mRNA expression of cathelicidin, Th1 (IFN-g) and Th2 (IL-4 and its antagonist-IL-4d2) cytokines and their transcription factors (T-bet, STAT4, GATA-3, STAT6) were measured in the PBMC by real-time PCR before and after intervention. RESULTS: Cholecalciferol-supplemented groups showed significant rise of mean serum 25(OH)D (30.6±7.51 and 28.6±8.41 ng/ml). The expression of LL-37, IFN-g, IL-4, IL-4d2 and transcription factors were comparable in the four groups at baseline. Despite significant increase in mean serum 25(OH)D in the cholecalciferol-supplemented groups, their mean mRNA transcripts of LL-37, IFN-g, IL-4, transcription factors and their IFN-g/IL-4 and T-bet/GATA-3 ratios were similar to that of calcium and placebo groups. CONCLUSIONS: Six months of cholecalciferol/calcium supplementation in young females with VDD do not lead to significant alteration in mRNA expression of LL-37, Th1/Th2 cytokines and their transcription factors. European Journal of Clinical Nutrition (2014) 68, 338–343; doi:10.1038/ejcn.2013.268; published online 8 January 2014 Keywords: 25(OH)D deficiency; cholecalciferol supplementation; antimicrobial peptide cathelicidin (LL-37); IFN-g; IL-4; transcription factors

INTRODUCTION Vitamin D deficiency (VDD) and infections such as tuberculosis are common health problems in Asian Indians.1,2 Functional significance of VDD in Asian Indians is reflected in high prevalence of rickets and osteomalacia and low bone mineral density.3,4 In addition to skeletal effects, VDD has been linked with susceptibility to tubercular and bacterial infections of the lungs and autoimmune disorders such as type-1 diabetes and multiple sclerosis.5–9 Bergman et al.10 recently reported a meta-analysis of 11 placebo-controlled randomized clinical trials (RCTs) related to vitamin D supplementation in 5660 patients and observed its protective effect against respiratory tract infection.10 Vitamin D leads to increased expression of antimicrobial peptide cathelicidin (LL-37) in the epithelial and mononuclear cells under in vitro conditions.11–13 Besides, presence of vitamin D receptors in monocytes, dendritic and T cells suggest a role of vitamin D in immune function.14,15 Following wide awareness of skeletal and extraskeletal benefits of vitamin D, physicians are frequently supplementing cholecalciferol and calcium to the asymptomatic general population with VDD. However, there is paucity of RCTs on the effect of vitamin D supplementation on the cathelicidin expression and alteration in Th1 and Th2 cytokines. Most of the earlier studies assessing effect of vitamin D on cathelicidin and change in Th1- and Th2-related cytokines are

under in vitro conditions and report downregulation of Th1 or upregulation of Th2 cytokines following addition of 1,25(OH)2D to the cell culture.16–22 In a RCT involving 9 months of oral cholecalciferol supplementation in 123 patients with congestive heart failure, Schleithoff et al.23 observed significant increase in serum anti-inflammatory interleukin-10 with no significant rise in serum tumor necrosis factor-a unlike placebo group. There are only limited RCTs assessing effect of cholecalciferol supplementation on Th1- and Th2-related cytokines in healthy subjects with biochemical VDD.24–26 Zittermann et al.24 observed significant improvement in cardiovascular disease risk markers including decrease in proinflammatory serum tumor necrosis factor-a after cholecalciferol supplementation in 200 healthy overweight subjects participating in the weight-reduction program.26 To the best of our knowledge, there is no RCT assessing change in cathelicidin expression following cholecalciferol supplementation in asymptomatic healthy volunteers with VDD. However, recently, Lehouck et al.27 observed no significant effect of highdose vitamin D supplementation given for 1 year on the incidence of exacerbations in patients with chronic obstructive pulmonary disease and their plasma cathelicidin levels. Here, we report the effect of cholecalciferol and calcium supplementation on the mRNA expression of (a) cathelicidin and

1 Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi, India and 2Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, India. Correspondence: Professor Dr R Goswami, Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India. E-mail: [email protected] Author contributors: RG has designed and supervised the study. MD and NT have carried out gene expression analysis. VS has analyzed the data. NG has carried out 25 (OH)D and iPTH assays. All authors have contributed equally in the writing of the manuscript. Received 9 September 2013; revised 31 October 2013; accepted 15 November 2013; published online 8 January 2014

Vitamin D supplementation and cathelicidin M Das et al

339 (b) Th1 and Th2 signatory cytokines (IFN-g, IL-4 and IL-4d2 (IL-4 antagonist) and their transcription factors (T-bet, STAT4, GATA-3, STAT6) in the peripheral blood mononuclear cells (PBMCs) in young females with VDD.

MATERIALS AND METHODS Subjects Study subjects were a subset of 173 young healthy female volunteers who had participated in our earlier randomized trial during the year 2010–2011 assessing effect of cholecalciferol supplementation on skeletal muscle strength. The details of the trial including randomization, allocation of intervention and its concealment have been published recently.28 The 131 subjects included for the present report were all those in whom sufficient RNA could be extracted from the PBMC for gene expression analysis at baseline and after 6 months of intervention (Figure 1). Briefly, the interventions groups received (a) cholecalciferol 60 000 IU/week for first 8 weeks followed by 60 000 IU/fortnight for 4 months and placebo tablets, (b) calcium carbonate tablet containing 500 mg of elemental calcium given twice daily for 6 months and placebo sachet, (c) both cholecalciferol and calcium in the above-mentioned doses and (d) placebos of lactose tablets and sachets for 6 months. The active arm and placebo for cholecalciferol and calcium carbonate were manufactured by Cadila Pharmaceuticals Ltd, (Ahmedabad, India) and Elder Pharmaceutical Ltd (Mumbai, India), respectively. The blood for measurement of biochemical parameters and gene expression studies was drawn after written informed consent and ethical approval and trial registration (Clinicaltrials.gov.NCT01190683).

RNA isolation for gene expression Blood for RNA extraction was collected in nuclease-free heparinized tube along with blood drawn for estimation of biochemical parameters, including total serum calcium, inorganic phosphorus, alkaline phosphatase, albumin, 25(OH)D, 1,25(OH)2D, intact parathyroid hormone (iPTH). Though biochemical parameters were assessed at baseline, 2 months and 6 months, blood for RNA isolation was drawn only at baseline and 6 months. Briefly, PBMCs were isolated from 5 ml of blood using Ficoll Paque plus (Amersham Biosciences, Piscataway, NJ, USA) and total RNA was extracted using Tri Reagent (Sigma Aldrich, St Louis, MO, USA). The RNA quality and

quantity was checked by agarose gel electrophoresis and spectrophotometric (GeneQuant-pro, Amersham Biosciences) analysis respectively. First strand of cDNA was synthesized using 2-mg aliquot of total RNA, 500 mM dNTPs, 5 mM random hexamers, 20 U RNase inhibitor and 200 U M-MuLV reverse transcriptase (RevertAid H Minus, MBI, Fermentas, Vilnius, Lithuania) in a 20-ml reaction at 42 1C for 60 min using GenAmp 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). The cDNA was stored at 20 1C till the real-time PCR (RT-PCR) assays.

Quantitative RT-PCR analysis The mRNA expressions of LL-37, Th1 and Th2 cytokines, transcription factors were assessed by RT-PCR. All the RT-PCR assays were put in batches incorporating samples for five genes (cytokines or transcription factors) from six subjects in each PCR plate. A maximum of three RT-PCR assays were run in a day. Gene-specific primers used to amplify glyceraldehyde 3-phosphate dehydrogenase (GAPDH), LL-37, IFN-g, IL-4, IL-4d2, transcription factors and the expected amplicon size are shown in Table 1. The primers for IFN-g were designed using free software Primer3 (http:// frodo.wi.mit.edu/). The primers for GAPDH,29 LL-37,30 IL-4 and IL-4d2,31 and T-bet, GATA-3 and STAT632 and STAT4 were described earlier.33 The RT-PCR (CFX96, Bio-Rad, Hercules, CA, USA) for all the genes assessed was carried out in separate wells using SYBR-I (Sigma Aldrich) fluorescence signal as described earlier.29 The conditions for PCR for all genes were: initial denaturation at 94 1C for 3 min, followed by 40 cycles at 94 1C for 30 s, 60 1C for 30 s, 72 1C for 30 s with final extension at 72 1C for 10 min. The cycle threshold for products was measured at 80 1C for IFN-g, IL-4, IL-4d2 and STAT4 and at 86 1C for LL-37, T-bet, GATA-3 and STAT6 based on melting-curve analysis. Reactions were performed in duplicates, and specificity of the amplified products was checked by post-PCR melting-curve analysis and agarose gel electrophoresis. The mRNA expression of various genes was assessed in relation to GAPDH expression. The inter-assay and intra-assay coefficients of variation for cycle threshold for various genes ranged from 0.37% to 2.35% and 0.29% to 2.18%, respectively (Table 1).

Biochemical estimations The estimations of serum total calcium, inorganic phosphorus, alkaline phosphatase and serum 25(OH)D, 1,25(OH)2D and iPTH were performed by standard methods described earlier.28 Serum 25(OH)D levels were

Randomization after consent (n = 173)

Double placebo (n = 43)

Ca & placebo (n = 43)

Vitamin D & placebo (n = 43)



Ca & Vitamin D (n = 44)

Consent withdrawn (n = 3)

Double placebo (n = 43) RNA available (n) Pre-intervention

(n = 33)

(n = 29)

(n = 34)

Ca & Vitamin D (n = 43) (n = 35)

Withdrawn from study (n = 4) Losses and drop out (13)

Double placebo (n = 37)



Ca & Vitamin D (n = 39)

(n = 32)

(n = 29)

(n = 33)

(n = 36)

RNA available (n) Post- intervention

Figure 1.

Flowchart for the study.

& 2014 Macmillan Publishers Limited

European Journal of Clinical Nutrition (2014) 338 – 343


340 categorized as deficient, insufficient and sufficient based on values o20.0 ng/ml, 20–32 ng/ml, and 432.0 ng/ml respectively.1,34

correlation between various parameters. P-valueso0.05 were considered significant.

Statistical analysis

RESULTS The baseline characteristics of the 131 subjects in the four study groups are shown in Table 2. Daily dietary intake of calories, calcium intake and biochemical parameters including serum 25(OH)D, 1,25(OH)2D and iPTH were comparable among the four groups. The mean 25(OH)D values at baseline in the four study groups were in the deficient range (Table 2), with iPTH in the upper normal range, suggesting a significant

Statistical analysis was performed using the Stata version 12.1. The data were analyzed using per protocol principle and reported as mean±s.d. The mRNA copy numbers were log transformed for analysis. Comparison of the mRNA expression of various genes at baseline and after intervention in the four study groups was assessed by analysis of variance. The significance of change in various indices used for Th1/Th2 ratio was assessed by Kruskal–Wallis test. Baseline-adjusted comparisons were made using analysis of covariance. Pearson correlation was used to assess

Table 1. Gene-specific primers for quantitative real-time PCR to study the expression of cathelicidin (LL-37), cytokines and their transcription factors, amplicon size and assay variations Gene

Forward primer

50 -GCGGTGGTCACTGGTGCTCCTGCTGCT-3 50 -CTCTGCATCGTTTTGGGTTCTCTTGG-30 50 -CGAGTTGACCGTAACAGACAT-30 50 -CAGAGCAGAAGAACACAACTG-30 50 -GTGCCCGAGTACAGCTCCGGA-30 50 -CAAGCAGGGACGGCGGATGT-30 50 -CATCTCAACAATCCGAAGTGATTCA-30 50 -TGGCCGGCTGGATGAAGTCC-30 50 -CCAAGGTCATCCATGACAACTTTGGT-30

LL-37 IFN-g IL-4 IL-4d2 GATA-3 T-bet STAT4 STAT6 GAPDH

Table 2.

Reverse primer

Size (bp)

50 -GAAGAAATCACCCAGCAGGGCAAATC-30 50 -GCGACAGTTCAGCCATCACTTGGAT-30 50 -GTCTTTAGCCTTTCCAAGAAG-30 50 -GTCTTTAGCCTTTCCAAGAAG-30 50 -GAGCCCACAGGCATTGCAGACA-30 50 -TTGGACGCCCCCTTGTTGTTTG-30 50 -GTCAGAGTTTATCCTGTCATTCAGCAG-30 50 -CTCCCGCGCCTGCTTCTCTG-30 50 -TGTTGAAGTCAGAGGAGACCACCTG-30

385 400 279 253 369 276 164 196 381

Assay variation (%) Intra

Inter

1.46 1.08 2.04 1.65 0.29 1.00 0.79 0.56 0.53

2.35 0.73 1.31 1.73 1.58 2.35 0.73 2.35 0.38

Baseline parameters (mean and s.d.) in the four intervention groups

Parameters

Double placebo (n ¼ 33)

Mean age (years) BMI (kg/m2) Serum total Ca (mg/dl) Serum PO4 (mg/dl) SAP (IU/l) Dietary calories (kcal/day) Dietary Ca intake (mg/day) Serum 25(OH)D (ng/ml) Serum 1,25(OH)2D (pg/ml) Serum iPTH (pg/ml)

21.6±3.93 20.4±2.83 9.71±0.50 4.0±0.43 67±21 1552±383 543±306 8.3±3.17 41.5±8.97 62.6±24.72

Calcium alone (n ¼ 29) 22.1±5.02 21.9±4.02 9.9±0.57 4.04±0.33 67±24 1640±423 599±284 10.1±3.60 42.5±12.08 51.5±18.94

mRNA copy number of antimicrobial peptide LL-37 per 103 mRNA copy of GAPDH LL-37 3.6±9.0 (31) 1.4±1.7 (29)

Cholecalciferol alone (n ¼ 34) 21.3±3.10 20.6±2.80 9.9±0.49 3.9±0.57 67.7±22.86 1551±347 589±261 9.05±3.51 40.1±13.67 62.9±22.47

Calcium and cholecalciferol (n ¼ 35) 21.2±2.77 20.3±2.58 9.9±0.65 4.0±0.62 63.75±19.010 1668±394 591±222 9.530±3.68 41.0±11.20 54.1±23.84

P-value 0.77 0.15 0.50 0.67 0.86 0.48 0.83 0.23 0.88 0.09

3.9±9.6 (33)

3.0±4.8 (35)

0.13

46.4±46.3 (32) 3.4±2.8 (33) 0.2±0.3 (30)

30.6±27.0 (30) 4.4±4.6 (35) 0.2±0.2 (24)

0.51 0.95 0.55

23.1±22.8 (35) 520.0±465.0 (34) 27.3±24.2 (34) 860.3±695.5 (33)

0.95 0.74 0.99 0.79

12.9±13.02 (30) 44.3±37.53 (24) 50.9±84.28 (32) 0.74±0.50 (31)

0.33 0.80 0.94 0.50

3

mRNA copy number of Th1 and Th2 cytokines per 10 mRNA copy of GAPDH IFN-g 38.2±31.8 (30) 39.7±28.3 (26) IL-4 4.2±6.1 (32) 3.3±2.9 (28) IL-4d2 0.1±0.2 (27) 0.3±0.5 (24)

mRNA copy number of Th1 and Th2 transcription factors per 103 mRNA copy of GAPDH STAT4 22.8±22.8 (32) 18.5±16.1 (29) 20.0±24.0 (31) T-bet 588.7±497.9 (31) 447.3±355.1 (26) 609.6±513.8 (33) GATA-3 24.4±20.8 (33) 24.6±23.4 (29) 23.2±23.3 (34) STAT6 1036.2±930.7 (33) 1098.7±982.8 (29) 1002.3±750.1 (34) Indices related to Th1/Th2 ratio IFN-g/IL-4 IL-4/IL-4d2 T-bet/GATA-3 T-bet/STAT6

26.3±43.87 (30) 46.7±37.67 (27) 53.8±94.77 (31) 0.79±0.56 (31)

18.3±19.53 (25) 50.9±51.84 (23) 31.2±18.48 (26) 0.63±0.38 (26)

39.2±97.56 (32) 46.8±44.32 (30) 37.4±53.61 (33) 0.61±0.33 (33)

Abbreviations: BMI, body mass index; iPTH, intact parathyroid hormone; SAP, serum alkaline phosphatase. Numbers in the parentheses indicate the subjects in whom the parameters were measured.




341 hypovitaminosis D in them. Post intervention, the groups randomized to cholecalciferol or dual supplementation showed significant rise of serum 25(OH)D levels to 30.6±7.51 and 28.6±8.41 ng/ml, respectively. In comparison, placebo- and calcium-alone-supplemented groups showed no significant change in serum 25(OH)D (Table 3). Figure 2 shows the specific amplification of different genes. The mRNA copy numbers of LL-37, IFN-g, IL-4, IL-4d2, T-bet, STAT4, GATA-3, STAT6 and various indices related to Th1 and Th2 ratios were comparable among the four groups at baseline (Table 2). The pre-intervention mRNA expression levels of the these parameters showed no significant correlation with baseline serum 25(OH)D and 1,25(OH)2D values.

Effect of cholecalciferol and calcium supplementation on LL-37, cytokines, transcription factors mRNA expression Differences in the post-intervention mRNA expression of LL-37, Th1 and Th2 cytokines, and transcriptions factors in the four study groups were analyzed in the unadjusted form and also after adjusting for their expression at baseline. The mean mRNA expression of all the genes was comparable in the four groups in both unadjusted and adjusted analysis after 6 months of intervention (Table 3). Similarly, the delta change (difference between pre and post-intervention) in the expression of the various genes assessed was not significant in both unadjusted and adjusted analysis. Despite significant differences in the serum 25(OH)D in the four groups, the ratios of IFN-g and IL-4 or

Table 3.

Effect of cholecalciferol and calcium supplementation on mRNA expression of cathelicidin (LL-37), cytokines, their transcription factors and their ratios (means and s.d.) Parameters

Double placebo (n ¼ 32)

Calcium alone (n ¼ 29)

BMI (kg/m2) Serum total Ca (mg/dl) Serum PO4 (mg/dl) SAP (IU/l) Serum 25(OH)D (ng/ml) Serum 1,25(OH)2D ( pg/ml) Serum iPTH (pg/ml)

21.2±2.96 9.9±0.58 3.7±0.56 68±19 7.5±3.78 39.7±11.33 72.0±40.71

22.2±4.35 10.1±0.51 3.7±0.53 68±20 7.9±2.49 39.2±13.65 51.0±25.84

Post-intervention mRNA copy number of LL-37 per 103 mRNA copy of GAPDH LL-37 12.1±20.55 (27) 5.5±5.36 (26) Change 9.7±17.9 (27) 4.2±4.4 (26)

P

Pa

0.05 0.11 0.71 0.45 o0.001 0.11 o0.001

— — — — — — —

15.4±18.8 (31) 12.9±18.3 (31)

0.26 0.63

0.36 0.11

77.0±70.7 (28) 46.3±74.0 (28)

0.35 0.43

0.45 0.39

8.0±6.3 (30) 4.6±6.0 (30)

12.4±11.5 (33) 8.3±11.8 (33)

0.27 0.55

0.26 0.66

0.3±0.4 (26) 0.1±0.5 (26)

0.27±0.3 (23) 0.05±0.4 (23)

0.64 0.53

0.69 0.17

7.3±5.7 (35) 15.7±22.0 (35)

0.28 0.42

0.27 0.23

Cholecalciferol alone (n ¼ 33) 20.9±3.03 10.1±0.53 3.6±0.51 64±22 30.6±7.51 45.0±11.90 51.76±17.11

20.2±2.80 10.1±0.50 3.6±0.55 62±18 28.6±8.41 44.8±14.39 42.6±16.69

9.5±12.1 (31) 5.4±15.0 (31)

Post-intervention mRNA copy number of Th1 and Th2 cytokines/103 mRNA copy of GAPDH IFN-g 123.6±97.6 (28) 110.9±85.9 (24) 121.8±104.1 (31) Change 83.9±98.5 (28) 68.2±84.6 (24) 74.7±91.9 (31) IL-4 Change

12.6±13.6 (30) 8.2±14.9 (30)

IL-4d2 Change

0.3±0.4 (23) 0.2±0.5 (23)

10.2±10.9(25) 6.7±10.7 (25) 0.3±0.5 (23) 0.02±0.8 (23)

Calcium and cholecalciferol (n ¼ 36)

Post-intervention mRNA copy number of Th1 and Th2 Transcription factors/mRNA 103 copy of GAPDH STAT4 11.6±16.6 (28) 11.2±8.4 (27) 10.2±8.6 (30) Change 9.3±26.9 (28) 7.7±17.4 (27) 7.5±22.9 (30) T-bet Change

521.8±439.9 (27) 52.9±744.0 (27)

536.2±468.3 (25) 90.1±623.5 (25)

616.2±535.5 (31) 29.0±605.7 (31)

469.8±463.4 (34) 50.2±649.4 (34)

0.53 0.82

0.53 0.54

GATA-3 Change

32.4±20.8 (29) 8.4±28.8 (29)

32.9±27.6 (28) 8.4±39.4 (28)

27.0±10.5 (32) 5.4±23.1 (32)

31.7±25.3 (33) 4.0±34.5 (33)

0.93 0.93

0.92 0.98

STAT6 Change

1309±837 (27) 263±1380 (27)

1015±759 (27) 124±1428 (27)

1154±640 (32) 207±860 (32)

882±612 (33) 22±959 (33)

0.12 0.58

0.10 0.73

13.0±11.03 (32) 13.6±44.39 (28)

20.9±21.02 (27) 1.1±26.49 (22)

27.0±34.02 (31) 13.0±82.51 (29)

13.9±20.76 (34) 2.2±25.21 (28)

0.04 0.57

0.25 0.52

52.6±43.14 (29) 4.1±46.39 (23)

46.6±33.04 (29) 8.9±68.98 (21)

49.5±35.63 (29) 0.5±67.29 (25)

59.9±63.22 (33) 1.8±39.93 (23)

0.79 0.90

0.80 0.92

T-bet/GATA-3 Change

15.7±11.54 (30) 42.4±102.92 (27)

16.0±12.50 (29) 14.9±22.42 (25)

22.0±15.73 (33) 16.5±53.11 (31)

15.8±16.98 (35) 34.3±86.03 (31)

0.04 0.15

0.09 0.18

T-bet/STAT6 Change

0.39±0.23 (28) 0.37±0.51 (25)

0.50±0.30 (28) 0.17±0.37 (24)

0.54±0.31 (32) 0.08±0.33 (30)

0.50±0.49 (36) 0.21±0.69 (31)

0.17 0.14

0.20 0.32

Indices of Th1/Th2 ratio IFN-g/IL-4 Change IL-4/IL-4d2 Change

Abbreviations: BMI, body mass index; iPTH, intact parathyroid hormone; SAP, serum alkaline phosphatase. Numbers in the parentheses indicate the subjects in whom the parameters were measured. aAdjusted for respective baseline values.




GA PD GA H TA -3 Tbe t ST AT 4 ST AT 6 La dd er

GA PD H IF N LL -3 7 IL -4 IL -4 IF 2 N La dd er

342

Figure 2. A 1.5% agarose gel showing the PCR products of (a) cytokines: GAPDH (lane 1), IFN-g (lanes 2 and 6), LL-37 (lane 3), IL-4 (lane 4), IL-4d2 (lane 5) and a 100-bp DNA ladder (lane 7). (b) Transcription factors: GAPDH (lane 1), GATA-3 (lane 2), T-bet (lane 3), STAT4 (lane 4), STAT6 (lane 5) and a 100-bp DNA ladder (lane 6).

transcription factors showed no significant change in Th1 and Th2 predominance with cholecalciferol or calcium supplementation. There was no major change in the mean mRNA expression of the IL-4d2, T-bet, GATA-3 and STAT6. However, the mean mRNA expression of IFN-g and IL-4 increased by two to three folds in all four groups at 6 months in comparison with their baseline values. DISCUSSION There has been an increasing interest in the extraskeletal effect of VDD. Intervention studies are being carried out to explore its beneficial in infections such as tuberculosis.7,8,35 Although the mechanisms linking VDD with disorders of bone mineral homeostasis are well investigated,36 similar information on nonskeletal disorders is limited. Results of the previous in vitro studies have revealed that vitamin D through its nuclear receptor can enhance expression of human-specific antimicrobial peptide LL-37 in immune cells and other tissues.12,13 Vitamin D can inhibit Th1 response through vitamin D response elements on the IFN-g gene.37 It can also indirectly inhibit IFN-g response through inhibition of differentiation of monocytes to antigen-presenting dendritic cells and reduced IL-12 secretion.38,39 There are only limited RCTs assessing effect of cholecalciferol supplementation on Th1- and Th2-related cytokines in healthy subjects with biochemical VDD.24–26 Following cholecalciferol supplementation, Schleithoff et al.23 observed significant increase in serum antiinflammatory interleukin-10 in patients with congestive heart failure, and Zittermann et al.,24 observed significant decrease in proinflammatory serum tumor necrosis factor-a after cholecalciferol supplementation in 200 healthy overweight subjects participating in weight-reduction program.26 On the other hand, vitamin D can also upregulate Th2-specific transcription factor GATA-3.40 The effect of above-described changes on susceptibility to tubercular infections is not clear. Although increased expression of cathelicidin may be helpful in infections such as tuberculosis, exaggeration of Th2 response would have an opposite effect. The present study was carried out to explore the extraskeletal effects of vitamin D and calcium on transcription of antimicrobial peptide ‘cathelicidin’ and IFN-g, IL-4 and Th1- and Th2-related transcription factor genes using doses of cholecalciferol generally used by the physicians to improve vitamin D status. The absence of significant effect of cholecalciferol, calcium or dual supplementation on LL-37 mRNA expression in the PBMC under in vivo conditions is unlike the observations by Bhan et al.,11 reporting significant correlation between plasma LL-37 and serum 25(OH)D. The differences in the results could be because of the differences in the study design and sample size or post-translational effect of vitamin D. The study also revealed no significant alteration in the mRNA transcripts of IFN-g, IL-4 and IL-4d2 gene with vitamin D, calcium or European Journal of Clinical Nutrition (2014) 338 – 343

their dual supplementation. Though the interventions led to major differences in 25(OH)D status, observing no significant differences in their transcriptions factors also supports no alteration in Th1 and Th2 cytokine balance. Further, the observation of no significant correlation between the pre-intervention levels of these cytokines and vitamin D levels also points out to the above view. There are no similar RCTs or controlled studies assessing the effect of cholecalciferol supplementation on Th1- and Th2-related signatory cytokines and their transcription factors on mRNA expression in apparently healthy normal-weight subjects. However, there are two RCTs with findings similar to that of the present study.24,25 Jorde et al.25 observed no significant differences in the serum IFN-g and IL-4 values in overweight and obese subjects following 1 year of cholecalciferol supplementation (20 000–40 000 IU/week).24 Similarly, Yusupov et al.26 observed no significant differences in the serum IFN-g and IL-4 values in the placebo- and cholecalciferol-supplemented (2000 IU/day for 3 months) groups.25 However, in both these trials, the baseline 25(OH)D values were not in the deficient range with mean values 56 nmol/l (22.4 ng/ml) and 64.3 nmol/l (25.8 ng/ml), respectively.24,25 This was unlike the present study, where basal serum 25(OH)D of study groups was in the vitamin D-deficient range. The contrast in pre- and post-supplementation serum 25(OH)D levels observed in the present study would provide effective interpretation in this study. Interestingly, mRNA expression of IFN-g and IL-4 increased in all four groups at repeat sampling after 6 months in March to April. These variations in expression was unrelated to technical or inter-assay variations because the cycle threshold of housekeeping gene GAPDH showed no significant change before and after intervention. These results are also unlikely to represent seasonal bias in the recruitment of the placebo and active groups because study subjects were recruited within a short span of 2 months and secondly block randomization followed in the study would have ensured seasonal synchronization of the study participants. Moreover, mRNA copy numbers of transcription factors showed no such seasonal change. The trend of seasonal variation in the serum IFN-g observed in the present study is similar to that reported by other investigators in healthy individuals.41–43 The results of the present study have some limitations. The cholecalciferol and calcium intervention was carried out in young females for 6 months and therefore cannot be generalized for other age groups, males and for longer duration of supplementation. Further, as the sample size was fixed for the primary objective of assessing the increase in hand-grip strength with vitamin D supplementation, the power of the study to detect significant change in LL-37, IFN-g, IL-4 and IFN-g/IL-4 was only 16%, 9%, 8% and 11%, respectively, with the available number of subjects. With the effect size observed for the above four parameters, a sample size of 500, 436, 622 and 331, respectively, was needed in each group to achieve 80% power. Alternatively, to have 80% power, we need to have 2–2.5 times higher mRNA expression of each parameter after dual supplementation group than the placebo group with the available sample size. Notwithstanding the above limitations, the present study assessing mRNA expression of the cathelcidin, IFN-g and IL-4 genes coupled with the observations of other investigators on serum levels of IFN-g and IL-424,25 allows us to conclude that 6 months of oral cholecalciferol and calcium supplementation in apparently healthy young females with VDD does not result in significant alteration in the expression of these genes and related transcription factors among health individuals. However, there is a need to extend these studies in subjects with compromised nutrition other than vitamin D, with latent chronic infections and susceptible to autoimmune disorders to have better understanding of the effects of vitamin D supplementation. & 2014 Macmillan Publishers Limited


343 CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS The financial support received from Indian Council of Medical Research to carry out this work is acknowledged.

REFERENCES 1 Holick MF. Vitamin D deficiency. N Engl J Med 2007; 357: 266–281. 2 Mithal A, Wahl DA, Bonjour JP, Burckhardt P, Dawson-Hughes B, Eisman JA et al. IOF Committee of Scientific Advisors (CSA) Nutrition Working Group. Global vitamin D status and determinants of hypovitaminosis D. Osteoporos Int 2009; 20: 1807–1820. 3 Ray D, Goswami R, Gupta N, Tomar N, Singh N, Sreenivas V. Predisposition to vitamin D deficiency osteomalacia and rickets in females is linked to their 25(OH)D and calcium intake rather than vitamin D receptor gene polymorphism. Clin Endocrinol 2008; 71: 334–340. 4 Vupputri MR, Goswami R, Gupta N, Ray D, Tandon N, Kumar N. Prevalence and functional significance of 25-hydroxyvitamin D deficiency and vitamin D receptor gene polymorphisms in Asian Indians. Am J Clin Nutr 2006; 83: 1411–1419. 5 Arnson Y, Amital H, Shoenfeld Y. Vitamin D and autoimmunity: new aetiological and therapeutic considerations. Ann Rheum Dis 2007; 66: 1137–1142. 6 Cantorna MT, Mahon BD. Mounting evidence for vitamin D as an environmental factor affecting autoimmune disease prevalence. Exp Biol Med 2004; 229: 1136–1142. 7 Zipitis CS, Akobeng AK. Vitamin D supplementation in early childhood and risk of type 1 diabetes: a systematic review and meta-analysis. Arch Dis Child 2008; 93: 512–517. 8 Munger KL, Levin LI, Hollis BW, Howard NS, Ascherio A. Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis. JAMA 2006; 296: 2832–2838. 9 Goswami R, Marwaha RK, Gupta N, Tandon N, Sreenivas V, Tomar N et al. Prevalence of vitamin D deficiency and its relationship with thyroid autoimmunity in Asian Indians: a community-based survey. Br J Nutr 2009; 102: 382–386. 10 Bergman P, Lindh AU, Bjo¨rkhem-Bergman L, Lindh JD. Vitamin D and respiratory tract infections: a systematic review and meta-analysis of randomized controlled trials. PLoS One 2013; 8: e65835. 11 Bhan I, Camargo Jr CA, Wenger J, Ricciardi C, Ye J, Borregaard N et al. Circulating levels of 25-hydroxyvitamin D and human cathelicidin in healthy adults. J Allergy Clin Immunol 2011; 127: 1302–1304. 12 Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 2006; 311: 1770–1773. 13 Hancock RE, Sahl HG. Antimicrobial and host-defense peptides as new antiinfective therapeutic strategies. Nat Biotechnol 2006; 12: 1551–1557. 14 Brennan A, Katz DR, Nunn JD, Barker S, Hewison M, Fraher LJ et al. Dendritic cells from human tissues express receptors for the immunoregulatory vitamin D3 metabolite, dihydroxycholecalciferol. Immunology 1987; 61: 457–461. 15 Provvedini DM, Tsoukas CD, Deftos LJ, Manolagas SC. 1,25-dihydroxyvitamin D3 receptors in human leukocytes. Science 1983; 221: 1181–1183. 16 Reichel H, Koeffler HP, Tobler A, Norman AW. 1 alpha,25-Dihydroxyvitamin D3 inhibits gamma-interferon synthesis by normal human peripheral blood lymphocytes. Proc Natl Acad Sci USA 1987; 84: 385–3389. 17 Jirapongsananuruk O, Melamed I, Leung DY. Additive immunosuppressive effects of 1,25-dihydroxyvitamin D3 and corticosteroids on TH1, but not TH2, responses. J Allergy Clin Immunol 2000; 106: 981–985. 18 Rausch-Fan X, Leutmezer F, Willheim M, Spittler A, Bohle B, Ebner C et al. Regulation of cytokine production in human peripheral blood mononuclear cells and allergen-specific th cell clones by 1alpha,25-dihydroxyvitamin D3. Int Arch Allergy Immunol 2002; 128: 33–41. 19 Staeva-Vieira TP, Freedman LP. 1,25-dihydroxyvitamin D3 inhibits IFN-gamma and IL-4 levels during in vitro polarization of primary murine CD4 þ T cells. J Immunol 2002; 168: 1181–1189. 20 Boonstra A, Barrat FJ, Crain C, Heath VL, Savelkoul HF, O’Garra A. 1alpha, 25-Dihydroxyvitamin d3 has a direct effect on naive CD4( þ ) T cells to enhance the development of Th2 cells. J Immunol 2001; 167: 4974–4980. 21 Cantorna MT, Woodward WD, Hayes CE, DeLuca HF. 1,25-dihydroxyvitamin D3 is a positive regulator for the two anti-encephalitogenic cytokines TGF-beta 1 and IL-4. J Immunol 1998; 160: 5314–5319.


22 XP Qi, Li P, Li G, Sun Z, Li JS. 1,25-dihydroxyvitamin D(3) regulates LPS-induced cytokine production and reduces mortality in rats. World J Gastroenterol 2008; 14: 3897–3902. 23 Schleithoff SS, Zittermann A, Tenderich G, Berthold HK, Stehle P, Koerfer R. Vitamin D supplementation improves cytokine profiles in patients with congestive heart failure: a double-blind, randomized, placebo-controlled trial. Am J Clin Nutr 2006; 83: 754–759. 24 Zittermann A, Frisch S, Berthold HK, Go¨tting C, Kuhn J, Kleesiek K et al. Vitamin D supplementation enhances the beneficial effects of weight loss on cardiovascular disease risk markers. Am J Clin Nutr 2009; 89: 1321–1327. 25 Jorde R, Sneve M, Torjesen PA, Figenschau Y, Gøransson LG, Omdal R. No effect of supplementation with cholecalciferol on cytokines and markers of inflammation in overweight and obese subjects. Cytokine 2010; 50: 175–180. 26 Yusupov E, Li-Ng M, Pollack S, Yeh JK, Mikhail M, Aloia JF. Vitamin D and serum cytokines in a randomized clinical trial. Int J Endocrinol 2010; 2010: pii: 305054. 27 Lehouck A, Mathieu C, Carremans C, Baeke F, Verhaegen J, Van Eldere J et al. High doses of vitamin D to reduce exacerbations in chronic obstructive pulmonary disease: a randomized trial. Ann Intern Med 2012; 156: 105–114. 28 Goswami R, Vatsa M, Sreenivas V, Singh U, Gupta N, Lakshmy R et al. Skeletal muscle strength in young Asian Indian females after vitamin D and calcium supplementation: a double-blind randomized controlled clinical trial. J Clin Endocrinol Metab 2012; 97: 4709–4716. 29 Goswami R, Mondal AM, Tomar N, Ray D, Chattopadhyay P, Gupta N. Presence of 25(OH)D deficiency and its effect on vitamin D receptor mRNA expression. Eur J Clin Nutr 2009; 63: 446–449. 30 McDonald V, Pollock RC, Dhaliwal W, Naik S, Farthing MJ, Bajaj-Elliott M. A potential role for interleukin-18 in inhibition of the development of Cryptosporidium parvum. Clin Exp Immunol 2006; 145: 555–562. 31 Fletcher HA, Owiafe P, Jeffries D, Hill P, Rook GA, Zumla A et al. Increased expression of mRNA encoding interleukin (IL)-4 and its splice variant IL-4delta2 in cells from contacts of Mycobacterium tuberculosis, in the absence of in vitro stimulation. Immunology 2004; 112: 669–673. 32 De Fanis U, Mori F, Kurnat RJ, Lee WK, Bova M, Adkinson NF et al. GATA3 up-regulation associated with surface expression of CD294/CRTH2: a unique feature of human Th cells. Blood 2007; 109: 4343–4350. 33 Abelson AK, Delgado-Vega AM, Kozyrev SV, Sańchez E, Vela´zquez-Cruz R, Eriksson N et al. STAT4 associates with systemic lupus erythematosus through two independent effects that correlate with gene expression and act additively with IRF5 to increase risk. Ann Rheum Dis 2009; 68: 1746–1753. 34 Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP et al. Guidelines for preventing and treating vitamin D deficiency and insufficiency revisited. J Clin Endocrinol Metab 2012; 97: 1153–1158. 35 Martineau AR, Timms PM, Bothamley GH, Hanifa Y, Islam K, Claxton AP et al. High-dose vitamin D(3) during intensive-phase antimicrobial treatment of pulmonary tuberculosis: a double-blind randomised controlled trial. Lancet 2011; 377: 242–250. 36 Ramasamy I. Recent advances in physiological calcium homeostasis. Clin Chem Lab Med 2006; 44: 237–273. 37 Cippitelli M, Santoni A. Vitamin D3: a transcriptional modulator of the interferongamma gene. Eur J Immunol 1998; 28: 3017–3030. 38 Penna G, Adorini L. 1 Alpha, 25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J Immunol 2000; 164: 2405–2411. 39 D’Ambrosio D, Cippitelli M, Cocciolo MG, Mazzeo D, Di Lucia P, Lang R et al. Inhibition of IL-12 production by 1,25-dihydroxyvitamin D3. Involvement of NF-kappaB downregulation in transcriptional repression of the p40 gene. J Clin Invest 1998; 101: 252–262. 40 Zhu J, Yamane H, Cote-Sierra J, Guo L, Paul WE. GATA-3 promotes Th2 responses through three different mechanisms: induction of Th2 cytokine production, selective growth of Th2 cells and inhibition of Th1 cell-specific factors. Cell Res 2006; 16: 3–10. 41 Stewart N, Taylor B, Ponsonby AL, Pittas F, van der Mei I, Woods G et al. The effect of season on cytokine expression in multiple sclerosis and healthy subjects. J Neuroimmunol 2007; 188: 181–186. 42 Khoo AL, Chai LY, Koenen HJ, Sweep FC, Joosten I, Netea MG et al. Regulation of cytokine responses by seasonality of vitamin D status in healthy individuals. Clin Exp Immunol 2011; 164: 72–79. 43 Khoo AL, Koenen HJ, Chai LY, Sweep FC, Netea MG, van der Ven AJ et al. Seasonal variation in vitamin D levels is paralleled by changes in the peripheral blood human T cell compartment. PLoS One 2012; 7: e29250.
