Alopecia areata (AA) is an organ-specific autoimmune disease mediated by cytokines produced by T cells infiltrating peribulbar areas of anagen hair follicles. It varies in clinical presentation from patchy areas of hair loss to alopecia totalis to total body hair loss (alopecia universalis) 1–3. Although both CD4+ and CD8+ T cells have been detected in the peribulbar infiltrate 4, it has been suggested, yet not proven, that CD8+ T lymphocytes are primarily responsible for the follicular damage, whereas CD4+ T cells provide help in the CD8+ response 5.
The interleukin 17 (IL-17) family is an important subclass of cytokines 6. To date, there are six ligands [IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F] and five receptors (IL-17RA, IL-17RB/IL-25R, IL-17RC, IL-17RD, and IL-17RE) belonging to the IL-17 family 7. IL-17A (hereafter referred to as IL-17) is the most intensively studied 8. Originally, IL-17 was thought to be produced exclusively by T cells 9; however, it is now known to be secreted by a variety of innate cells, including macrophages, dendritic cells, and natural killer cells 10. A major development in this field occurred with the recognition that IL-17-producing T cells arise as a specific population of CD4+ T cells, called T helper 17 (Th17) cells, which are distinct from the classic Th1 and Th2 cells 11,12.
IL-17 and other Th17 cytokines are linked to the pathogenesis of diverse autoimmune and inflammatory diseases 8, including multiple sclerosis 13, rheumatoid arthritis 14, and psoriasis 15. IL-17-producing T cells have also been identified in the synovial fluid of patients with Lyme arthritis 16, suggesting the involvement of IL-17 in infection-induced immunopathology 17.
Only few studies have investigated the possible role of IL-17 in the pathogenesis of AA. A study in the Korean population revealed single-nucleotide polymorphisms in the IL-17RA gene to be significantly associated with AA 18. Another study on a mouse model 5 has reported that pathologic T cells primarily express interferon-γ and IL-17 early in the disease. Two recent studies discussed the number of IL-17-producing cells in relation to disease severity and progression 19,20. The aim of this study was to provide more insight into the role of IL-17 in the pathogenesis of AA by studying the level of IL-17 and its gene expression in lesional scalp skin biopsies taken from AA patients.
Patients and methods
This case–control pilot study was approved by the Research Ethical Committee of the Dermatology Department of the Faculty of Medicine, Cairo University (Derma REC). Informed consent forms were signed by adult participants or guardians of non adults.
The study was carried out on 18 patients with different clinical types of AA attending the Outpatient Dermatology Clinic of Kasr Al-Ainy University Hospital during the period from December 2012 to May 2013. Twenty healthy, age-matched and sex-matched volunteers with no systemic or dermatological diseases served as controls.
Patients with other dermatological diseases, those under systemic treatment for AA, those under 16 years, those with heart, liver, or renal failure, immunocompromised patients, and those with other diseases such as Henoch–Schonlein purpura, autoimmune thyroiditis, arrhythmia, Parkinsonism, and optic neuropathy were excluded.
A 4 mm punch skin biopsy was obtained from AA lesional skin of every patient, and a 3 mm punch skin biopsy was obtained from the scalp of each control participant. Biopsies were stored at −20°C.
Each skin biopsy was divided into two parts: one for quantitative assessment of the level of IL-17 in the tissue by enzyme-linked immunosorbent assay (ELISA) and the other for RNA extraction and reverse transcriptase-PCR analysis for the measurement of the expression of IL-17 mRNA. The skin biopsy was weighed, homogenized, and centrifuged at 10 000g. The supernatant was separated and stored at −70°C until processed.
The IL-17 ELISA kit used was supplied by BioSource Europe SA (Nivelles, Belgium). It uses an antibody specific to human IL-17, which is precoated onto the wells. Standards and samples were pipetted into the wells and IL-17 present in the sample bound to the immobilized antibodies in the wells. After washing, biotinylated anti-human IL-17 antibodies were added to the wells. HRP-conjugated streptavidin was then pipetted into the wells. The wells were washed again, and a TMB substrate solution was added to the wells. A color developed in proportion to the amount of bound IL-17. The intensity of the color was measured at 450 nm. The minimum detectable dose of IL-17 is typically less than 80 pg/ml.
Total RNA was isolated using the RNeasy Mini kit (Qiagen, Hamburg, Germany). The concentration of RNA was determined by measuring the absorbance at 260 nm using a Nanodrop ND-1000 instrument (Thermo Fisher Scientific, Wilmington, North Carolina, USA). An aliquot containing 0.2 μg of total RNA was used for the reverse transcription reaction, which was performed on the superscript first-strand cDNA synthesis system (Fermentas, Helsinki, Finland). The IL-17 primers used included the following: forward primer: 5′-AATCTCCACCGCAATGAGGA-3′; reverse primer: 5′-ACGTTCCCATCAGCGTTGA-3′; and probe: FAM-CGGCACTTTGCCTCCCAGATCACA-TAMRA. IL-17 was quantified by real-time PCR (Qiaplex Panel, Qiagen, Hamburg, Germany). The reaction mixture included 3 μl of synthesized cDNA solution, 12.5 μl of 2× Probe PCR Master Mix (Qiagen, Hamburg, Germany), 500 nmol/l of each primer, and 250 nmol/l of the TaqMan probe. The amplification program included a prewarming step (10 min at 94°C), a denaturation step (94°C for 15 s), and an annealing/extension step (60°C for 60 s). β-actin was quantified as a reference gene to normalize the mRNA expression levels of other genes. The relative quantification of gene expression in each sample was analyzed by the 2ΔΔCt method and expressed as the ratio of the related gene to β-actin mRNA.
Data were statistically described in terms of mean and SD, median and range, or frequencies (number of cases) and percentages when appropriate. Numerical variables were compared between the study groups using Student’s t-test for independent samples when comparing two groups and the one-way analysis of variance test with post-hoc multiple two-group comparisons when comparing more than two groups. For comparing categorical data, the χ2-test was used. The exact test was used when the expected frequency was less than 5. The various variables were correlated using Pearson’s moment correlation equation for linear relation in normally distributed variables and Spearman’s rank correlation equation for non-normally distributed variables. P-values less than 0.05 were considered statistically significant. All statistical analyses were carried out using the computer program SPSS (version 15; SPSS Inc., Chicago, Illinois, USA) for Microsoft Windows.
In the patients' group, six were females (33.3%) and 12 were males (66.7%); their ages ranged from 16 to 51 years, with a mean age of 31.23±10.08 years. Family history was positive in one patient (5.6%) and negative in 17 patients (94.4%). Thirteen patients (72.2%) had patchy AA, two patients (11.1%) had alopecia totalis, and three patients (16.7%) had alopecia universalis. Duration of the disease ranged from 0.16 to 21 years, with a mean of 3.6±5.79 years. Ten patients (55.6%) had no previous attacks, three patients (16.7%) had one previous attack, and five patients (27.8%) had two previous attacks.
In the control group, seven were females (35%) and 13 were males (65%); their ages ranged from 19 to 51 years with a mean of 32±8.87 years. No statistically significant difference was detected between patients and controls in either the age (P=0.804) or the sex distribution (P=0.914).
Results of estimation of interleukin-17 and its mRNA expression
Both IL-17 and its mRNA expression were significantly higher in AA lesions compared with controls' skin (P<0.0001; Table 1). Moreover, both parameters were significantly higher in scalp skin biopsies from patients with alopecia totalis compared with alopecia universalis and patchy AA (P<0.0001; Table 2), and a strong significant positive correlation existed between both IL-17 and its mRNA expression (r=0.910 and P<0.0001; Fig. 1).
Both parameters were not significantly affected by the age of the patients (P=0.192 and 0.641, respectively), their sex (P=0.445 and 0.655, respectively), the number of previous attacks (P=0.656 and 0.516, respectively), or by disease duration (P=0.649 and 0.772, respectively).
To the best of our knowledge, this is the first case–control study to report significantly elevated levels of IL-17 and its mRNA in lesional skin of AA patients. This further substantiates preliminary observations pointing to its possible role in this disease 5,18,19.
IL-17 is the key product of Th17 cells; thus, our findings confirm an upregulated Th17 profile in AA patients. Th17 cells are considered to be pathogenic forms of CD4+ T cells driven by IL-23. In addition to IL-17, Th17 cells produce IL-6 and tumor necrosis factor upon antigen-specific stimulation. These cytokines are essential for the establishment of organ-specific inflammation associated with autoimmunity 17, mainly through the pleiotropic activity of IL-17 on fibroblasts, keratinocytes, endothelial cells, neutrophils, and memory T cells 21, which is mediated by the release of various cytokines and chemokines 8.
Interestingly, Th17 cells were also found to produce IL-21, which mediates inflammatory response through IL-21R that is expressed on CD8+ T cells 22, thus linking Th17 with CD8+-mediated cytotoxic effects. It is known that in AA, CD8+ T lymphocytes are primarily responsible for the follicular damage as they infiltrate hair follicles and induce satellite cell necrosis of keratinocytes 19, whereas CD4+ T cells provide help in the CD8+ response 5. The high levels of IL-17 and its mRNA may provide an explanation for the help provided by CD4+ T cells (through its specific Th17 subset) in the CD8+ response in AA.
In our study, we found significantly higher levels of IL-17 in the skin biopsies of patients with alopecia totalis compared with patchy AA and alopecia universalis. It appears that the level of IL-17 in the skin plays important role in the progression of the disease from the patchy to the totalis type. The higher levels of IL-17 and its mRNA in patients with alopecia totalis compared with alopecia universalis in our study may suggest that progression to the universalis form may involve other factors such as increased serum levels of IL-17, however this needs further verification.
Studies on the number of IL-17-producing cells in different severity types of AA are controversial. Whereas Kubo et al.19 reported a positive correlation between the severity and progression of alopecia and the increase in the number of interferon-γ-producing Th1 cells or IL-17-producing Th17 cells, a recently published study by Tojo et al.20 reported that the ratio of IL-17-producing cells in acute, diffuse, and total alopecia was significantly lower than that in multiple patchy AA; however, both researchers did not evaluate the actual levels of IL-17 or its gene expression.
IL-17 appears to be involved in the autoimmune inflammation in AA and may provide CD4+-derived help to the CD8+ cytotoxic cells primarily involved in this disease. Further studies are needed to explore the exact mechanism of action of IL-17 in the pathogenesis of AA and its exact relation with the progression of the disease. Biopsies from apparently normal (nonlesional) skin of patients with patchy AA are also recommended. Therapeutic targeting of IL-17 may provide a potential therapeutic option in this chronic autoimmune disease.
Conflicts of interest
There are no conflicts of interest.
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