Gene Expression of CD70 and CD27 Is Increased in Alopecia Areata Lesions and Associated with Disease Severity and Activity

Background Alopecia areata (AA) is an acquired hair loss disorder induced by a cell-mediated autoimmune attack against anagen hair follicles. CD27-CD70 is a receptor-ligand complex which enhances T helper and cytotoxic T cell activation, survival, and proliferation. The overstimulation of this complex can lead to a lack of tolerance and the development of autoimmunity. Objectives This study aimed to assess the gene expression of CD27 and CD70 in patients with AA. Methods CD70 and CD27 mRNA expressions were evaluated by a quantitative real-time polymerase chain reaction in scalp biopsies from 40 AA patients (both AA lesions and non-lesional areas) and 40 healthy controls (HCs). The Severity of Alopecia Tool (SALT) score was used to assess AA severity. Patients were evaluated for signs of AA activity, including a positive hair pull test and dermoscopic features of black dots, broken hairs, and tapering hairs. Results The gene expression of CD70 and CD27 was significantly higher in AA lesions than in non-lesional areas (p < 0.001 for both) and HCs (p=0.004, p=0.014, respectively). There were significant positive correlations between AA severity and gene expression of CD70 (p < 0.001) and CD27 (p=0.030) in AA lesions. Significant associations were detected between signs of AA activity and lesional gene expression of CD70 and CD27. Additionally, CD70 and CD27 gene expression was significantly lower in non-lesional biopsies compared to HCs (p < 0.001). Conclusion Gene expression of CD70 and CD27 was increased in AA lesions and was associated with disease severity and activity. Thus, both molecules can be a predictor of AA severity and activity. Furthermore, the expression was reduced in non-lesional scalp areas. Thus, a lack of CD27 and CD70 expression may initially predispose to immunological dysregulation and the development of AA.


Introduction
Alopecia areata (AA) is an acquired non-cicatricial hair loss disorder affecting 0.1%-0.2% of individuals worldwide [1]. A collapse of the hair follicle (HF) immune privilege causing a CD8+ cytotoxic T (cT) cell assault against anagen HFs is implicated in AA development [2,3]. However, the precise molecular mechanisms are still poorly established.
Cluster differentiation (CD) 27 is a member of the tumor necrosis factor receptor family. CD27 is expressed on natural killer cells, T helper ( ) cells, cT cells, and hematopoietic stem cells [4]. CD70, the uniquely identified ligand of CD27, belongs to the tumor necrosis factor family and is expressed exclusively on activated T cells, B cells, natural killer cells, and dendritic cells. e expression of CD70 is triggered by T cell activation upon antigen receptor attachment, toll-like receptor, or CD40 signaling. e interaction between CD70 on stimulated antigen presenting cells (APCs) and CD27 on T cells generates costimulatory signals that enhance and cT cell activation, survival, proliferation, chemotaxis, and cytokine production such as interleukin 2 and interferon-c [5]. Moreover, CD27-CD70 interaction induces the production of CXCL10 chemokine by activated cT cells, which enhances the chemotaxis of additional activated cT cells [6]. CD70 was also found to stimulate 1 cell differentiation independent of interleukin 12 [7]. Soluble CD27 can be shed from the activated T cells' surface via cleavage by matrix metalloproteinases to be released in the circulation. It was reported that serum soluble CD27 could enhance T cells and antigen-primed B cells' activation and increase immunoglobulin G production [4,5]. Interestingly, it was proposed that CD70 signaling generated by unaroused, premature APCs can inevitably lead to a lack of tolerance and autoimmunity development [8]. Significant evidence indicates that the dysregulation of CD27-CD70 complex signaling is involved in the pathogenesis of several immune-related disorders [5], but the pathway has been poorly investigated in AA. CD70 is merely expressed upon T cell activation, and thus blocking the C27-CD70 complex has been proposed as an appealing treatment target for autoimmune disorders [9].
is study aimed to assess the expression of CD27 and CD70 genes in patients with AA and to evaluate the association between this expression and AA severity and activity.

Participants.
A total of 40 patients with AA were enrolled in this descriptive analytical case-control study. Patients were recruited from the Dermatology Outpatient Clinic, Suez Canal University Hospital, Ismailia, Egypt. e biochemical analyses were conducted at the molecular laboratory of the Oncology Diagnostic Unit, Faculty of Medicine, Suez Canal University, Egypt. e study was performed between February and September 2021, in line with the guidelines of the Helsinki Declaration and the items of the STROBE statement. Approval was granted by the Institutional Review Board and the Research Ethics Committee, Faculty of Medicine, Suez Canal University, on 25 January 2021, with the approval code: 4454. Age and sex-matched HCs with no concurrent infections, history of AA, autoimmune disorders, atopy, or cancer were included in the study. All participants signed written informed consent form.
Alopecia areata was diagnosed clinically through detecting patches of total hair loss with normal scalp skin, and the diagnosis was confirmed by dermoscopy examination. Exclusion criteria were patients who had any systemic treatment for three months or topical applications for 2 weeks prior to the study or had regrowth of hair, concurrent infection, or a history of other autoimmune diseases or atopy or cancer.
Patients' complete history was recorded including demographic data (age and sex) and significant clinical data (duration of the present AA lesions, disease course, age of disease onset, AA in other body sites, prior attacks of AA, family history of AA, atopy, or other immune-mediated disorders), and dermatological examination was performed to detect site of patches, their number and pattern, and presence of nail abnormalities. AA clinical pattern was categorized into patchy (single patch and multiple patches), ophiasis, alopecia totalis, and alopecia universalis. e "Severity of Alopecia Tool" (SALT) score [10] was used to evaluate AA severity. AA activity was assessed via the subjective history of progression, an objective examination of the hair pull test at the edges of each patch, and the presence of black dots, broken hairs, and tapering hair on dermoscopy examination (DermLite, 3 Gen LLC, San Juan Capistrano, CA, USA) (magnification × 10).

Assessment of the Expression of CD70 and CD27 Genes.
Punch scalp biopsies (4 mm) were taken from each patient from AA lesions and non-lesional areas. One scalp biopsy was taken from the patient with alopecia universalis and HCs. Biopsies were soon submerged in the RNA stabilizing solution (Qiagen, USA) and moved to −80 C for storage until handling. According to the manufacturer's instructions, we isolated the total RNA using RNeasy Mini Kit (QIAGEN, USA). We also assessed RNA purity by NanoDrop ND1000 spectrophotometer at the absorbance ratio of 260/280 nm (NanoDrop Tech., Inc., Wilmington, DE, USA). To assess the integrity of the RNA, we ran it at 1% agarose gel electrophoresis.
Quantitative real-time polymerase chain reactions (PCR) for CD27 and CD70 genes were performed on step one real-time PCR instrument (Applied Biosystems, UK) using COSMO cDNA synthesis kits (WF-1020500X, Willowfort, UK), HERA plus SYBR Green-qPCR Kit (WF1030800X, Willowfort, UK), and the specific primers for the target genes (Table 1). e amplification program included two stages, an initial denaturation stage at 95 C for 3 min, followed by 40 cycles of 95 C denaturation for 15 s and annealing at 57 C for GAPDH and 60 C for (CD27 or CD70) for 60 s. After amplification, a melting curve analysis was performed to confirm PCR amplicons by collecting the fluorescence data. GAPDH was used as an internal control. e relative amounts of the target genes were calculated using the delta CT method.

Statistical Analysis.
Statistical analysis was done via the Social Sciences Statistical Package. Categorical values were represented by using numbers and percentages. e normality of distribution was tested by the Kolmogorov-Smirnov test. Numerical values were represented using the range, median, mean, and standard deviation. Chisquare test, Monte Carlo correction, Mann-Whitney test, Kruskal-Wallis test, Wilcoxon signed rank test, and Spearman coefficient were used to measure the significance. e results were considered significant at a p value less than 0.05 (confidence level of 5%).

Clinical and Demographic Data of AA Patients.
In patients with AA, the age ranged from 15 to 62 years (mean age � 2 8.93 ± 12.21 years). Twenty (50%) patients were males, and 20 (50%) were females. In HCs, the age ranged from 18 to 59 (mean age � 29.80 ± 10.16 years). Eighteen (45%) HC individuals were males, and 22 (55%) were females. ere was no statistically significant difference in age or gender between patients with AA and HCs (Supplementary Table 1). Clinical data of AA patients are shown in Table 2.

Expression of CD70 and CD27 Genes.
e mean mRNA expression of the CD70 gene in AA lesions and non-lesional scalp skin was 2.41 fold and 0.16 fold, respectively, relative to the expression in HCs. e relative expression of CD70 gene was significantly higher in AA lesions compared to nonlesional areas (p < 0.001) and HCs (p � 0.004). In addition, the gene expression was significantly lower in non-lesional areas than in HCs (p < 0.001) (Figure 1). e mean mRNA expression of the CD27 gene in AA lesions and non-lesional scalp skin was 3.19 fold and 0.25 fold, respectively, relative to the expression in HCs. e relative expression of the CD27 gene was significantly higher in AA lesions compared to non-lesional areas (p < 0.001) and HCs (p � 0.014). e CD27 gene expression was significantly lower in non-lesional biopsies compared to HCs (p < 0.001) (Figure 2). e study revealed significant positive correlations between AA severity (SALT) and the relative mRNA expressions of CD70 (p < 0.001) (Figure 3(a)) and CD27 (p � 0.030) (Figure 3(b)) in AA lesions.
Furthermore, there were significant associations between signs of AA activity (positive hair pull test, and the presence of black dots, broken hairs, and tapering hair on dermoscopy examination) and the relative gene expressions of CD70 and CD27 (Table 3). Apart from that, there was no significant relation between CD70 or CD27 gene expression and other data of AA patients (age, sex, disease course, age of onset, duration of AA lesions, clinical pattern, nail abnormalities or family history of AA, atopy, or immune-mediated disorders) (Supplementary Tables 2-5).

Discussion
In this study, the relative gene expression of CD70 and CD27 in AA lesions was significantly higher than that in nonlesional areas and HCs, correlated with AA severity, and was associated with signs of AA activity, including positive hair pull test at patch margins and dermoscopic features such as black dots, broken hairs, and tapering hair. AA is generally believed to be an autoimmune assault upon anagen HF, orchestrated by 1 and cT lymphocytes. Inflammatory infiltrate of , cT cells, and APCs was identified in the peribulbar region of anagen HFs in active AA lesions and was found to be correlated with AA severity. ese kinds of infiltrate trigger apoptosis in anagen HF keratinocytes,    Figure 2: e relative expression of CD27 gene in patients with AA (n � 40) compared to HCs (n � 40). e relative CD27 gene expression in AA lesions ranged from 0.09 to 11.96 (mean 3.19 ± 3.65, median 1.58), while in non-lesional areas, it ranged from 0.0 to 1.68 (mean 0.25 ± 0.38, median 0.14). Wilcoxon signed rank test (p1) was used to compare the expression in AA lesions and non-lesional areas. Mann-Whitney test (p2) was used to compare the expression in AA lesions and HCs. Mann-Whitney test (p3) was used to compare the expression in non-lesional areas and HCs. * Significant at p < 0.05. AA: alopecia areata; HCs: healthy controls; CD: cluster differentiation.   Dermatology Research and Practice causing their arrest and regression into the telogen or dystrophic anagen states [2]. Clinically, this is manifested by a sudden stoppage of hair shaft growth, resulting in tapered and broken hair shafts with a positive hair pull test at the active patch margin [11]. e CD27-CD70 complex is mostly expressed on activated T cells, B cells, natural killer cells, and dendritic cells, and its interaction signaling facilitates 1 and cTcell activation, survival, proliferation, and chemotaxis [5]. us, the higher gene expression of CD70 and CD27 in active AA lesions can be explained by the increased infiltration of immune cells expressing these molecules.
is infiltration was present mostly in active AA lesions and was associated with AA severity; this fact may clarify the association between CD27 and CD70 gene expression and AA severity and signs of activity. As a result, CD27 and CD70 gene expression can be used to predict the severity and activity of AA. To date, no previous studies have evaluated the gene expression of CD27-CD70 complex in AA.
is expression correlated with the hypo or demethylation of the CD70 gene promoter area, which causes a failure to repress CD70 expression when it is triggered by T cell activation [15,16]. Regarding CD27, its soluble serum level was elevated in patients with active vitiligo and was suggested as a marker of disease progression [17,18]. Serum soluble CD27 was downregulated upon treatment of psoriasis [19]. CD27 expression was significantly elevated in the lesional skin and serum of patients with systemic sclerosis, with a significant association with disease severity [20]. e expression of CD27+ B cells and serum soluble CD27 was increased in SLE patients and correlated with disease activity [21]. e pathogenesis of most of these diseases entails cellmediated autoimmune inflammatory pathways, and their association with AA is well established [2]. In the same manner, the expression of CD70 was increased in human contact dermatitis, which is a 1-mediated inflammation [22].
Additionally, the present study revealed that CD27 and CD70 gene expression was significantly lower in the nonlesional areas compared to HCs. Interestingly, Abolhassani [23] reported two family members with genetic abnormalities in the CD70-CD27 signaling cascade associated with clinical features of AA, Behcet's disease, recurrent viral pneumonia, central nervous system infection, and Hodgkin lymphoma induced by Epstein-Barr virus. e patients' clinical and immunologic data revealed an abnormality in B-cell differentiation, impaired functional activity of effector T cells, and decreased antibody production, which increased vulnerability to recurrent viral illness. e authors proposed an association between CD70 deficiency and an increased risk of alopecia areata due to either recurrent uncontrolled viral infections or decreased proliferation and activity of T-regulatory cells. is study's findings are consistent with our results of decreased CD70 and CD27 gene expression in the non-lesional scalp areas of AA patients, suggesting that a deficiency of CD70 and CD27 expression may predispose to immunological dysregulation and the development of AA. After that, the recruitment of autoreactive T cells against anagen hair follicles in active AA lesions may cause local overexpression of CD70 and CD27 expressed on the infiltrating immune cells.
Notably, several in vivo studies have suggested that monoclonal antibodies targeting CD27-CD70 complex could be a potential therapeutic modality in autoimmune diseases [5]. Anti-CD70 antibodies lowered the antibody titer and decreased joint disease's severity in murine collagen-induced arthritis [9]. In addition, anti-CD70 antibodies repressed immunoglobulin secretion by B cells triggered by T cells isolated from SLE patients [24]. Colitis was prevented along with a decrease in colitis-associated 1 cytokines in a mouse model using anti-CD70 antibodies [25]. Accordingly, our study findings may shed new light on targeting CD27-CD70 complex for medical treatment of AA, especially severe cases resistant to traditional medical treatment.
Indeed, the conclusions of this study should be considered against its limitations, which include small sample size and the lack of evaluation of CD27 and CD70 tissue expression in AA scalp lesions compared to non-lesional areas and HCs. Moreover, additional research investigating the molecular functions of CD27 and CD70 in AA and comparing the expression of both molecules during AA activity and after recovery and hair regrowth is needed.

Conclusion
e expression of CD27 and CD70 genes was increased in AA scalp lesions and was associated with AA severity and activity. CD27-CD70 interaction can therefore be a predictor of AA severity and activity. Furthermore, the expression of both molecules was lower in non-lesional scalp areas. us, a lack of CD27 and CD70 expression may predispose to immunological dysregulation and the development of AA.

Data Availability
e data and materials related to the present work are included within this article. 6 Dermatology Research and Practice