|Year : 2013 | Volume
| Issue : 1 | Page : 1-5
Vitamin D receptor and cathelicidin expressions in children with atopic dermatitis
Hanan M. Darwish1, Noha M. Ghoneim1, Doaa A. Hassan1, Asmaa S. Farag1, Laila Rashed2
1 Department of Dermatology and Venereology, Faculty of Medicine for Girls, Al-Zahraa Univerisity Hospital, Al-Azhar University, Cairo, Egypt
2 Department of Clinical Biochemistry, Cairo University, Cairo, Egypt
|Date of Submission||23-Mar-2013|
|Date of Acceptance||10-Apr-2013|
|Date of Web Publication||23-Jun-2014|
Hanan M. Darwish
MD, Department of Dermatology and Venereology, Faculty of Medicine for Girls, Al-Zahraa Univerisity Hospital, Al-Azhar University, Abbasia, Cairo
Source of Support: None, Conflict of Interest: None
Atopic dermatitis (AD) is a common chronic inflammatory type of eczema. Vitamin D may enhance the innate immune response by induction of cathelicidin (LL-37), an endogenous antimicrobial peptide. Several recent reports suggest a connection between vitamin D3 and the expression of antimicrobial peptides in keratinocytes. 1,25-dihydroxyvitamin D (1,25(OH)2D3) is the active form of vitamin D and exerts its actions through a specific intracellular vitamin D receptor (VDR).
To evaluate the expression of VDR and cathelicidin in children with AD in Egypt.
The study was carried out on 15 patients with AD and 15 healthy controls. Patients were divided into mild, moderate, and severe groups according to the SCORAD score. All individuals were subjected to skin biopsy. VDR and cathelicidin expressions were estimated using real-time PCR in patients and controls.
A highly significant difference was detected between patients and controls in VDR and cathelicidin expression (P=0.001). A statistically significant correlation was observed between VDR and cathelicidin expressions.
Influencing cathelicidin expression by targeting the vitamin D3 pathway might present a novel therapeutic approach for the treatment of AD.
Keywords: atopic dermatitis, cathelicidin, vitamin D receptor
|How to cite this article:|
Darwish HM, Ghoneim NM, Hassan DA, Farag AS, Rashed L. Vitamin D receptor and cathelicidin expressions in children with atopic dermatitis. Egypt J Dermatol Venerol 2013;33:1-5
|How to cite this URL:|
Darwish HM, Ghoneim NM, Hassan DA, Farag AS, Rashed L. Vitamin D receptor and cathelicidin expressions in children with atopic dermatitis. Egypt J Dermatol Venerol [serial online] 2013 [cited 2022 Dec 10];33:1-5. Available from: http://www.ejdv.eg.net/text.asp?2013/33/1/1/135105
| Introduction|| |
Atopic dermatitis (AD) is a chronic skin condition that affects 10–20% of children and 1–3% of adults 1. Patients with AD show an increased susceptibility to cutaneous infections, especially to pathological colonization with superantigen-secreting Staphylococcus aureus. Recent attention has been focused on antimicrobial peptides (AMP), especially on cathelicidin and human β-defensin 2, which are underexpressed in atopic skin 2.
Vitamin D is a prohormone important for serum calcium and phosphorus homeostasis for proper neuromuscular function and optimal skeletal health. Vitamin D can be obtained from the diet or synthesized in the skin after exposure to ultraviolet B radiation from the sun. Vitamin D is then converted into its major circulating form, 25-hydroxyvitamin D (25(OH)D), by the liver and into its hormonally active form, 1,25-dihydroxyvitamin D (1,25(OH)2D), by the kidney to increase the efficiency of intestinal absorption of calcium as its classic function. 1,25(OH)2D3 is the active form of vitamin D and exerts its actions through a specific intracellular vitamin D receptor (VDR) 3.
Almost all cells of the adaptive immune system express the VDR, a nuclear receptor that renders them responsive to vitamin D3 4. Vitamin D3 suppresses adaptive immunity by inhibition of antigen-presenting cells and by the generation of a T helper type 2 (Th2) micromilieu 5. Upon vitamin D3 treatment of T cells, Th2 cytokines such as interleukin-4 (IL-4), IL-5, and IL-10 were increased whereas IL-2 and IFNγ typical Th1 cytokines required for T-cell proliferation and activation were decreased 6.
The body controls the activity of the VDR through regulation of the vitamin D metabolites. 25(OH)D inactivates the receptor whereas 1,25(OH)2D activates it 2.
Cathelicidin expression is upregulated in infection and injury of the skin 7 and cathelicidin promotes neovascularization, wound healing, and also chemokine expression and migration of immune cells (neutrophils, mast cells, monocytes, and T cells) 8. Although LL-37 is induced in these conditions, the regulation of cathelicidin gene expression was long unclear as mediators of infection did not influence gene expression 9.
1,25(OH)2D3 is a major regulator of the expression of the AMP cathelicidin not only in monocytes but also in epidermal keratinocytes 10. However, certain feedback mechanisms are also in place, which allow the body to limit the production of 1,25(OH)2D to just that amount required for proper transcriptional activation of the VDR.
- When the VDR is activated, it transcribes the gene for the enzyme CYP24A1, which increases conversion of 1,25(OH)2D into inactive metabolites.
- An activated VDR also controls the 1,25(OH)2D concentration by limiting transcription of the gene CYP27B1, which converts 25(OH)D into 1,25(OH)2D 11.
| Patients and methods|| |
Fifteen AD patients ranging in age from 2 to 16 years and 15 healthy controls were included in this study. Patients were recruited from the outpatients’ clinic of dermatology of Al-Zahraa University Hospital.
Each patient was subjected to full assessment of history and clinical examination. A 4-mm punch biopsy specimen was obtained from the AD lesion of each patient. In addition, the intact skin of the 15 healthy individuals was biopsied. Written informed consent was obtained from the parents of all patients and the controls before the biopsy.
The severity of AD was determined by the SCORAD score, which is considered one of the best validated systems suitable for clinical trials. It provides information on the extent, intensity, and subjective symptoms. Therefore, patients were classified into three groups: mild (n=7), moderate (n=6), and severe (n=2).
Severity of atopic dermatitis (SCORAD score) 12
To determine extent, the sites affected by eczema are shaded on a drawing of a body. The rule of 9 is used to calculate the affected area (A) as a percentage of the entire body.
- Head and neck 9%.
- Upper limbs 9% each.
- Lower limbs 18% each.
- Anterior trunk 18%.
- Back 18%.
- 1% each for genitals, each palm, and the back of each hand.
The score for each area is added up. The total area is A, which has a possible maximum of 100%.
A representative area of eczema is selected. In this area, the intensity of each of the following signs is assessed as none (0), mild (1), moderate (2), or severe (3).
- Scratch marks.
- Skin thickening (lichenification).
- Dryness (this is assessed in an area where there is no inflammation).
- The intensity scores are added together to yield B (maximum 18).
Subjective symptoms, that is, itch and sleeplessness, are each scored by the patient or a relative using a visual analogue scale, where 0 is no itch (or no sleeplessness) and 10 is the worst imaginable itch (or sleeplessness). These scores are added to yield C (maximum 20).
SCORAD calculation: A/5+7B/2+C
The severity of AD can be classified into mild (<15), moderate (15–40), and sever (>40).
Reverse transcription and real-time polymerase chain reaction
Total RNA was obtained from skin tissue using the SV RNA isolation kit (Promega, Madison, Wisconsin, USA) according to the manufacturer’s instructions. The RNA concentration was determined spectrophotometrically (Beckman, Texas, USA).
The reverse transcriptase and real-time PCR method was used for the estimation of the concentration of the mRNA of VDR and cathelicidin. Complementary DNA synthesis was carried out using a 36-μl assay mix containing 3 μg total RNA, 10 U RNAse inhibitor, 2 μl random hexa primers, 2 μl dNTP (10 nmol), 10 U M-MLV reverse transcriptase, and 4 μl 10× reaction buffer (100 mmol/l Tris-HCl, 500 mmol/l KCl, and 150 mmol/l MgCl2 in nuclease-free water, Invitrogen, Carlsbad, California, USA).
In a final volume of 25 μl, 5 μl complementary DNA was mixed with 2×SYBR Green PCR master mix (Applied Biosystems, UK) and primers. The primer sequences are presented in [Table 1]: the PCR cycling condition was 95°C for 15 min, followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 20 s. Quantitative real-time PCR was carried out using step 1 Real-Time PCR Systems (Applied Biosystems). mRNA levels were determined by a cycle threshold (C t).
For each sample, a &Dgr;C t value was obtained by subtracting GAPDH values from those of the gene of interest. The average &Dgr;C t value of the control group was then subtracted from the sample to obtain a &Dgr;&Dgr;C t value. The expression of each gene was then evaluated by 2−(&Dgr;&Dgr;C t).
All statistical calculations were carried out using computer programs Microsoft excel version 10 and statistical package for the social science version 20.00 (lowa State Univiversity Press, Ames., lowwa, USA) statistical program at 0.05, 0.01, and 0.001 level of probability. Data were presented as percentage, mean, and SD. Comparison of percentage was carried out using the t-test.
| Results|| |
This study was carried out on 15 patients with AD, nine males (60%) and six females (40%), ranging in age from 4 to 16 years (mean±SD=10.93±2.71). They were classified according to the SCORAD score into 7 mild (47%), 6 moderate (40%), and 2 severe (13%). Patients were compared with 15 healthy age-matched apparently healthy individuals as controls, between 6 and 16 years of age (mean±SD=10.33±1.99).
We found a statistically significant difference in the mean VDR expression in lesions of patients with AD (mean±SD=1.38±0.36) compared with the controls (mean±SD=2.04±0.50) (P<0.001) [Table 2].
|Table 2: Comparison of vitamin D receptor and cathelicidin expression between patients and controls|
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Also, there was a statistically significant difference in the mean VDR expression in patients with severe AD compared with patients with moderate and mild AD (mean±SD=0.16±0.03, 0.18±0.03 and 0.24±0.09, respectively) compared with the controls (mean±SD=1.38±0.36) (P<0.001) [Table 3], [Graph 1].
|Table 3: Comparison of vitamin D receptor and cathelicidin expression in AD patients with different degrees of severity|
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The mean result of cathelicidin expression among patients with AD (mean±SD=2.04±0.50) showed a statistically significant difference compared with cathelicidin expression in the controls (mean±SD=1.10±0.24) (P<0.001) [Table 2].
Compared with the mean expression of cathelicidin in the controls (mean±SD=2.04±0.50), there was a statistically significant difference between the severe, moderate, and mild types of AD (mean±SD=0.96±0.02, 1.06±0.29, and 1.16±0.22, respectively) (P<0.001) [Table 3], [Graph 1].
A statistically significant correlation was observed between VDR and cathelicidin expressions in patients with AD (r=0.439) [Graph 2].
| Discussion|| |
A number of studies have suggested that patients with chronic inflammatory diseases have deficiency in 25(OH)D and that consuming greater quantities of vitamin D, which elevates 25(OH)D levels, alleviates symptoms of the disease. Some years ago, molecular biology identified 25(OH)D as a secosteroid. Secosteroids would typically be expected to depress inflammation, which is in line with the reports of symptomatic improvement. The simplistic first-order mass-action model used to guide the early vitamin studies has given way to a more complex description of action. When active, the VDR affects the transcription of at least 913 genes and impacts processes ranging from calcium metabolism to the expression of key, AMP 13.
Several reports suggest a connection between vitamin D3 and AMP expression in keratinocytes. Vitamin D3 has been considered to play a pivotal role in the antimicrobial immunity of the skin when a vitamin D3 response element was identified in the promoter region of the cathelicidin gene 14. Weber et al. 15 confirmed that cathelicidin is a direct target of vitamin D3 in keratinocytes. Furthermore, conservation of cathelicidin gene regulation by the vitamin D3 pathway in humans and nonhuman primates during evolution has been reported 16.
In the current study, there was a highly significant difference in the percentage of VDR and cathelicidin expressions in patients when compared with controls. Our result showed that a higher mean value of VDR and cathelicidin expression was observed in the mild group of AD compared with moderate and severe groups.
Our study found a significant decrease in VDR and cathelicidin expressions in AD lesions when compared with healthy skin of the control group, suggesting that the increased propensity of patients toward skin infection is because of the lack of AMP expression.
Studies have indicated that the dysregulation of VDR may lead to exaggerated inflammatory responses, raising the possibility that defects in vitamin D and VDR signaling transduction may be linked to bacterial infection and chronic inflammation. Further characterization of vitamin D/VDR will help elucidate the pathogenesis of various human diseases and in the design of new approaches for prevention and treatment 17.
Many studies have evaluated the serum level of vitamin D in AD and reported that there are several biologically plausible explanations for the inverse association between serum vitamin D levels and the presence of AD and particularly with the severity of the disease. Human keratinocytes have the enzymatic apparatus to produce calcitriol, the active compound of vitamin D, from the precursor 7-dehydrocholesterol under the influence of UVB radiation 18. In in-vitro studies, vitamin D3 has been shown to induce cathelicidin expression in keratinocytes, which enhances antimicrobial activity against S. aureus 19 and selectively reduces cutaneous lymphocyte-associated antigen expression. It has been shown that cathelicidin does not influence lymphocyte migration patterns to other tissues, thus specifically decreasing T-lymphocyte homing into the skin 20.
Peroni et al. 21 reported that vitamin D deficiency may be related to the severity of AD and advocate the need for studies evaluating the use of vitamin D as a potential treatment in patients with this disease.
Our results showed that cathelicidin expression is higher in mild AD than moderate and severe AD. A deficient innate antimicrobial barrier in AD patients was first proposed when an impaired expression of AMPs such as cathelicidin and defensins was detected in atopic skin 22. In particular, induction of cathelicidin mRNA transcription in response to wounding is suppressed in AD lesions as compared with healthy skin 23. This could be explained by the altered tissue microenvironment in AD skin: Th2 cytokines such as IL-4and IL-5, which are highly elevated in AD skin, suppress cathelicidin induction in keratinocytes 24. As itching is a hallmark of AD and scratching results in skin wounding, failure to upregulate cathelicidin in response to injury could decrease the cutaneous antimicrobial activity in AD skin 23.
Hata et al. 24 and Howell et al. 25 identified subgroups of AD patients with severe infectious complications and a history of dermatitis herpeticatum in the past, which showed defective upregulation of AMPs.
Kanada et al. 26 reported that systemic vitamin D3 levels are reduced in patients with AD, which may contribute toward the decreased systemic LL-37 levels. LL-37 may systemically potentiate the oncostatin M and IL-31 production in normal donors and patients with AD, whereas vitamin D3 may do so only in normal donors.
The results of this study provide an important background on the relation between VDR and AMP expression and the severity of inflammatory disorders.
| Conclusion|| |
Influencing cathelicidin expression by targeting the vitamin D3 pathway might present a novel therapeutic approach for the treatment of AD.
| References|| |
|1.||Schultz-Larsen F, Hanifin JM.Epidemiology of atopic dermatitis.Immunol Allergy Clin North Am 2002;22:1–24. |
|2.|| Schauber J, Gallo RL.Antimicrobial peptides and the skin immune defense system.J Allergy Clin Immunol 2008;122:261–266. |
|3.|| Bremmer F, Thelen P, Pottek T, Behnes CL, Radzun HJ, Schweyer S.Expression and function of the vitamin D receptor in malignant germ cell tumour of the testis.Anticancer Res 2012;32:341–349. |
|4.|| Van Etten E, Stoffels K, Gysemans C, Mathieu C, Overbergh L.Regulation of vitamin D homeostasis: implications for the immune system.Nutr Rev 2008;66 Suppl 2 S125–S134. |
|5.|| Rigby WF, Stacy T, Fanger MW.Inhibition of T lymphocyte mitogenesis by 1,25-dihydroxyvitamin D3 (calcitriol).J Clin Invest 1984;74:1451–1455. |
|6.|| Boonstra A, Barrat FJ, Crain C, Heath VL, Savelkoul HF, O'Garra A.1alpha,25-dihydroxyvitamin d3 has a direct effect on naive CD4(+) T cells to enhance the development of Th2 cells.J Immunol 2001;167:4974–4980. |
|7.|| Frohm M, Agerberth B, Ahangari G, Stâhle-Bäckdahl M, Lidén S, Wigzell H, et al..The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders.J Biol Chem 1997;272:15258–15263. |
|8.|| Heilborn JD, Nilsson MF, Kratz G, Weber G, Sørensen O, Borregaard N, et al..The cathelicidin anti-microbial peptide LL-37 is involved in re-epithelialization of human skin wounds and is lacking in chronic ulcer epithelium.J Invest Dermatol 2003;120:379–389. |
|9.|| Schauber J, Dorschner RA, Yamasaki K, Brouha B, Gallo RL.Control of the innate epithelial antimicrobial response is cell-type specific and dependent on relevant microenvironmental stimuli.Immunology 2006;118:509–519. |
|10.|| Pasupuleti M, Schmidtchen A, Malmsten M.Antimicrobial peptides: key components of the innate immune system.Crit Rev Biotechnol 2012;32:143–171. |
|11.|| Kato S, Fujiki R, Kim MS, Kitagawa H.Ligand-induced transrepressive function of VDR requires a chromatin remodeling complex, WINAC.J Steroid Biochem Mol Biol 2007;103:372–380. |
|12.|| Severity scoring of atopic dermatitis: the SCORAD index. Consensus Report of the European Task Force on Atopic Dermatitis.Dermatology 1993;186:23–31. |
|13.|| Albert PJ, Proal AD, Marshall TG.Vitamin D: the alternative hypothesis.Autoimmun Rev 2009;8:639–644. |
|14.|| Wang TT, Nestel FP, Bourdeau V, Nagai Y, Wang Q, Liao J, et al..Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression.J Immunol 2004;173:2909–2912. |
|15.|| Weber G, Heilborn JD, Chamorro Jimenez CI, Hammarsjo A, Törmä H, Stahle M.Vitamin D induces the antimicrobial protein hCAP18 in human skin.J Invest Dermatol 2005;124:1080–1082. |
|16.|| Gombart AF, Saito T, Koeffler HP.Exaptation of an ancient Alu short interspersed element provides a highly conserved vitamin D-mediated innate immune response in humans and primates.BMC Genomics 2009;10:321. |
|17.|| Sun J.Vitamin D and mucosal immune function.Curr Opin Gastroenterol 2010;26:591–595. |
|18.|| Schuessler M, Astecker N, Herzig G, Vorisek G, Schuster I.Skin is an autonomous organ in synthesis, two-step activation and degradation of vitamin D(3): CYP27 in epidermis completes the set of essential vitamin D(3)-hydroxylases.Steroids 2001;66:399–408. |
|19.|| Schauber J, Oda Y, Büchau AS, Yun QC, Steinmeyer A, Zügel U, et al..Histone acetylation in keratinocytes enables control of the expression of cathelicidin and CD14 by 1,25-dihydroxyvitamin D3.J Invest Dermatol 2008;128:816–824. |
|20.|| Yamanaka K, Dimitroff CJ, Fuhlbrigge RC, Kakeda M, Kurokawa I, Mizutani H, et al..Vitamins A and D are potent inhibitors of cutaneous lymphocyte-associated antigen expression.J Allergy Clin Immunol 2008;121:148–157. |
|21.|| Peroni DG, Piacentini GL, Cametti E, Chinellato I, Boner AL.Correlation between serum 25-hydroxyvitamin D levels and severity of atopic dermatitis in children.Br J Dermatol 2011;164:1078–1082. |
|22.|| Ong PY, Ohtake T, Brandt C, Strickland I, Boguniewicz M, Ganz T, et al..Endogenous antimicrobial peptides and skin infections in atopic dermatitis.N Engl J Med 2002;347:1151–1160. |
|23.|| Mallbris L, Carlén L, Wei T, Heilborn J, Nilsson MF, Granath F, et al..Injury downregulates the expression of the human cathelicidin protein hCAP18/LL-37 in atopic dermatitis.Exp Dermatol 2010;19:442–449. |
|24.|| Hata TR, Kotol P, Boguniewicz M, Taylor P, Paik A, Jackson M, et al..History of eczema herpeticum is associated with the inability to induce human β-defensin (HBD)-2, HBD-3 and cathelicidin in the skin of patients with atopic dermatitis.Br J Dermatol 2010;163:659–661. |
|25.|| Howell MD, Gallo RL, Boguniewicz M, Jones JF, Wong C, Streib JE, et al..Cytokine milieu of atopic dermatitis skin subverts the innate immune response to vaccinia virus.Immunity 2006;24:341–348. |
|26.|| Kanda N, Hau CS, Tada Y, Sato S, Watanabe S.Decreased serum LL-37 and vitamin D3 levels in atopic dermatitis: relationship between IL-31 and oncostatin M.Allergy 2012;67:804–812. |
[Table 1], [Table 2], [Table 3]