Differential proteomics of lesional vs. non-lesional biopsies revealed non-immune mechanisms of alopecia areata

Alopecia areata (AA) is one of the common hair disorders for which treatment is frequently ineffective and associated with relapsing episodes. Better understanding of disease mechanisms and novel therapeutic targets are thus required. From 10 AA patients, quantitative proteomics using LTQ-Orbitrap-XL mass spectrometer revealed 104 down-regulated, 4 absent, 3 up-regulated and 11 newly present proteins in lesional vs. non-lesional biopsies. Among these, the decreased levels of α-tubulin, vimentin, heat shock protein 70 (HSP70), HSP90, annexin A2 and α-enolase were successfully confirmed by Western blotting. Protein-protein interactions network analysis using STRING tool revealed that the most frequent biological processes/networks of the down-regulated proteins included tissue development, cell differentiation, response to wounding and catabolic process, whereas those for the up-regulated proteins included biological process, metabolic process, cellular transport, cellular component organization and response to stimulus. Interestingly, only 5 increased/newly present proteins were associated with the regulation of immune system, which may not be the predominant pathway in AA pathogenic mechanisms as previously assumed. In summary, we report herein the first proteome dataset of AA demonstrating a number of novel pathways, which can be linked to the disease mechanisms and may lead to discovery of new therapeutic targets for AA.

Confirmation of the proteomic data by Western blotting. Because proteins whose levels were significantly decreased in the lesional biopsies were predominate in the list, we confirmed the decreased levels of α-tubulin, vimentin, HSP70, HSP90, annexin A2 and α-enolase determined by quantitative proteomics by another method, i.e., Western blotting. Because quantitative proteomics revealed significant changes of proteins that are commonly used as loading controls, i.e., actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) -see Table 2, whereas there were no significant changes in level of HSP60 observed (as such, this protein was not included in the list of significantly altered proteins shown in Table 2), HSP60 was selected to serve as the loading control to normalize levels of all the aforementioned proteins in our present study. The data showed that levels of α-tubulin, vimentin, HSP70, HSP90, annexin A2 and α-enolase normalized with HSP60 were significantly decreased in all 10 individual AA patients (Fig. 3), consistent with the quantitative proteomics data.
Functional classification and global protein network analysis. The differentially expressed proteins were classified by STRING tool. From a total of 108 decreased/absent proteins in lesional biopsies, the top-three most frequent biological processes/networks included tissue development (60 proteins), cell differentiation (37 proteins), response to wounding (25 proteins), and catabolic process (25 proteins) ( Fig. 4A and B). For the 14 increased/newly present proteins in lesional biopsies, the top-three most frequent biological processes/networks included biological process (10 proteins), metabolic process (9 proteins), cellular transport (7 proteins), cellular component organization (7 proteins), and response to stimulus (7 proteins) ( Fig. 5A and B). Interestingly, only 5 increased/newly present proteins were associated with the regulation of immune system ( Fig. 5A and B), which may not be the predominant pathway in AA pathogenic mechanisms as previously assumed.

Discussion
AA, one of the common hair disorders, is characterized by oval or round, well-circumscribed balding patch(es). Some patients with limited area of AA may have spontaneous recovery and experience only a single AA episode in their lifetime. However, a much larger proportion of the patients have persistent AA that is resistant to medical therapy or have chronic relapsing episodes of the disease. Previous knowledge had suggested that the pathogenesis of AA is related to destruction of the hair follicles by immune process, particularly via cooperative roles of both CD8 + and CD4 + T lymphocytes 8,9 . In fact, the pathogenesis of AA and mechanisms of failure in hair follicle formation remain unclear and largely unknown. This study thus aimed to address such mechanisms and to explore previously unknown or hidden mechanisms associated with defective hair follicles and development of AA using a recent advanced technology based on quantitative proteomics followed by protein-protein interactions network analysis. Quantitative proteomics revealed 122 differentially expressed proteins in lesional vs. non-lesional biopsies. From these, we performed Western blotting to confirm the differential expression data obtained from a quantitative proteomics approach. Vimentin and tubulin were selected because of their significant roles in hair follicular development. Vimentin is an intermediate filament cytoskeleton, which is also known to serve as a marker for mesenchymal feature that can be found among mesenchymal cell populations required for the development of hair follicles 10,11 . Tubulin is a main component of microtubule, a structure that also involves in cellular development and function, i.e., mitosis, vesicular trafficking, cell motility, and wound healing 12 . It is also plays a role in pigment transport 13,14 . In addition, we also performed Western blotting to confirm changes in levels of several other proteins, including HSP70, HSP90, annexin A2 and α-enolase. Using HSP60 as the loading control, the Western blot data nicely confirmed significant changes in levels of these proteins (Fig. 3), consistent with the data obtained from quantitative proteomics approach (Table 2).
Global protein network analysis was then performed to obtain functional insights of the identified proteins that were differentially expressed. From 14 increased/newly presented proteins, only five proteins were classified to get involved in immune-mediated mechanisms ( Fig. 5A and B). Among these, fibrinogen alpha and gamma chains were found to be related to various biological functions including immune mechanism (Fig. 5B). Although previous studies have reported the potential roles of fibrinogen in wound healing, angiogenesis and inflammatory response in epithelial cells via both innate and T lymphocyte-mediated pathways 15,16 , the evidence of its association with AA and hair loss had never been reported. Interestingly, a recent study has revealed that fibrinogen could induce activation and recruitment of myelin-specific Th1 cells and peripheral macrophages into the central nervous system causing demyelination and autoimmune encephalomyelitis 17 . However, a precise role of fibrinogen as a driver of immune privilege breakdown of hair follicles observed in AA remains unclear and deserves further investigations.
Hemopexin (HPX) was found as one of the newly present proteins in lesional biopsies. HPX is a heme-binding glycoprotein that also serves as an antioxidant or oxidative stress scavenger. Previous studies have reported decreased plasma concentration of HPX in severe intravascular hemolysis, chronic hemolytic anemia, chronic liver disease, and acute porphyria attack 18 . In the context of autoimmune disease, HPX plays an important role in mercury-induced autoimmunity 19,20 . The data have shown that HPX-null mice had less number of B and activated T cells as well as lower autoantibody production. T cells isolated from mercury-treated HPX-null mice also had a reduction of IFN-γ production. These results suggest that HPX may involve in autoimmune diseases via regulating heme-iron homeostasis and IFN-γ response 19,20 . Additionally, there is a report of the increased HPX in vernal keratoconjuctivitis, a chronic allergic inflammatory disease 21 . However, the role of HPX in hair disorder had not been studied previously. The presence of HPX only in lesional area of AA might suggest its role in inflammatory process leading to chronicity in AA. Nevertheless, the precise role of HPX in AA needs further elucidations.
Another protein that was exclusively expressed in lesional biopsies was α1-acid glycoprotein 2 (also known as orosomucoid 2). This protein has immunomodulatory effect and can inhibit mitogenic response of lymphocytes, in particular, T cell population 22 . On the other hand, orosomucoid 2 can stimulate T cell proliferation at low concentration and induce mononuclear cells to produce several cytokines involving inflammatory response 23,24 . Additionally, it is known that glycosylation patterns of orosomucoid 2 are distinct among inflammatory and autoimmune diseases (e.g., rheumatoid arthritis, SLE, autoimmune thyroiditis, etc.), thereby, affecting their physical properties and function [25][26][27] . Our findings may suggest the role of this protein in stimulation of proliferating T cells and regulation of local inflammation in lesional area. Knockdown of orosomucoid 2 or investigations on its glycan moieties during the disease onset may be useful to gain mechanistic insights of its increase in AA.
In addition to fibrinogen, HPX and orosomucoid 2, x-ray repair complementing defective repair in chinese hamster cells 6 (XRCC6) was also listed as the differentially expressed proteins involving in immune-mediated mechanism. XRCC6 (also known as Ku70) is a single-strand DNA-dependent/ATP-dependent helicase that plays role in chromosome translocation and double-strand break DNA repair. Interestingly, it has been reported that individuals with SLE produced reactive autoantibody to XRCC6 28 . Additionally, polymorphisms of XRCC6 are associated with the risk of SLE susceptibility 29 . In the context of AA, the decrease in XRCC4 mRNA has been previously reported although its precise mechanism remains unknown 30 .

Accession no.
Protein name

MS/MS identification score %Cov MW (kDa) pI
Abundance level (×10 9 arbitrary unit) (Mean ± SEM)  Table 2. Summary of differentially expressed proteins in lesional vs. non-lesional biopsies of AA patients (in alphabetical order). %Cov = %Sequence coverage [(number of the matched residues/total number of residues in the entire sequence) × 100%]. # DIV/0! = Divided by zero (not present in non-lesional area, but newly present in lesional area). Surprisingly, functional classification and protein network prediction did not show immune-mediated mechanism as the predominant pathway involved in AA as we initially anticipated. This surprising result indicated that there should be several other non-immune mechanisms that are involved in the pathogenic mechanisms of AA that might be previously unknown, unexplored and/or hidden by limited knowledge in the past. Using recent advanced proteomic technology helped us to explore previously unknown, unexplored and/or hidden mechanisms/pathways in an unbiased manner as in the case of many other diseases 6,7,[31][32][33][34][35] . Interestingly, a much larger number of the differentially expressed proteins (approximately 89%) had decreased levels or were absent in the AA lesional biopsies. The most frequent biological processes/networks of these down-regulated proteins included tissue development, cell differentiation, response to wounding and catabolic process (Fig. 4).

P-value
In concordance to the previous transcriptomics studies of AA 30,36,37 , we found the decreased expression of both Type I and Type II keratin in the lesional biopsies (including 20 keratin species as follows: K2, K5-K8, K14-17, K71, K75-77, K79, K83, and K85-86). These proteins are essential in hair and nail formation. Among these, K75 and K86 have been previously reported to be increased in response to corticosteroid treatment in AA patients, suggesting that its increase may be used as a biomarker for monitoring response to the steroid therapy 38 . In addition, several genes in S100 family were decreased in AA patients as compared to healthy controls 38 . We also found the decreased levels of S100A7, S100A8, and S100A9 in lesional biopsies. S100A8, S100A9 and S100A8/A9 heterodimers have been reported to play roles in neutrophil chemotaxis and adhesion to inflammatory sites 39,40 . They are also known as myeloid-related proteins that are highly expressed in neutrophils, monocytes, differentiated macrophages and keratinocytes 41 . It is thus plausible that the decreased levels of S100A8 and S100A9 found in AA tissues might reflect restriction of the immune response in the affected area as well as dysregulation of keratinocyte proliferation and differentiation 42 .
In addition, we compared our data to the previously reported differential proteomes of hair follicles at different phases in mice 43 . In concordance to the previous findings, we observed the decreased expression of annexin A1, heat shock protein (HSP)-β1 and vimentin, which were also decreased in telogen-hair follicle, but increased in anagen and catagen phases 43 . These provide further support to the observation that hair follicles in patients with AA rapidly progress from anagen to telogen phase, re-enter to anagen phase, and then strictly reside in anagen III/IV phase. Taken together, the decreased levels of these proteins support the miniature of hair follicles found in AA patients. Moreover, we also compared our proteomic data to the genomic data obtained from genome-wide association study (GWAS) and linkage analysis in AA patients 30,[44][45][46][47][48] . In these studies, several immune-related genes have been identified so far, while only a few of non-immune genes have been reported 49,50 . These genes include ERBB3, VDR, STX17, PRDX5, KIAA0350/CLEC16A and SPATA5. Among these, ERBB3, VDR, STX17 and PRDX5 are skin/hair-related genes 51,52 . Herein, we provide additional dataset of non-immune proteins mainly involved in tissue development and differentiation. Although our data is different from the previous GWAS reports, these discrepancies could be explained by differences in technical approach. Another important factor is the difference in populations of AA patients included among these studies. Since several lines of evidence have suggested that AA is strongly related to genetic basis; therefore, different ethnic populations should be taken into account for such differences.
Besides the genetics, it should be noted that tissue biopsies with different onsets and severities among the studies could also affect the results at transcriptome and proteome levels. Moreover, our present study showed decreased levels of several cytoskeletal/structural proteins in the lesional areas. It was thus possible that their decreases might be a result from the reduction of hair follicles in the lesional areas. Alternatively, these altered proteome may serve as the potentially novel mechanisms leading to AA. Nevertheless, further functional investigations on these candidate biological functions/pathways should be done to strengthen our hypothesis.
In summary, we report herein the first proteome dataset of AA, which implicates that a number of potentially novel mechanisms or biological pathways may be involved in pathogenic mechanisms of AA. Our data offer opportunities to explore previously unknown, unexplored or hidden mechanisms of AA and to define novel biomarkers for diagnostics/prognostics and new therapeutic targets for better clinical outcome for AA.

Materials and Methods
Patients and lesional/non-lesional biopsies. This study was approved by the institutional ethical committee (Siriraj Institutional Review Board) (approval no. Si259/2015). All the experiments involved human subjects and clinical samples were conducted according to the international guidelines, i.e. the Declaration of Helsinki, the Belmont Report, and ICH Good Clinical Practice, and informed consents were obtained from all subjects. Newly diagnosed patchy AA patients were recruited during June -October 2015 and subjected to skin biopsy of both lesional and non-lesional areas. Patients with any of the following exclusion criteria, including psychiatric disorder (e.g., trichotillomania), active scalp infection, systemic conditions affecting the scalp and hairs (e.g., anemia), recurrent AA, history of vitamin and/or mineral supplement, history of coagulopathy, ingestion of anti-platelets and/or anticoagulants, pregnancy and lactation, were excluded. Clinical data from each patient were recorded, including age, gender, duration from disease onset to the visit, family history of AA, location, underlying disease, and Severity of Alopecia Tool Score (SALT score). Finally, ten enrolled patients were included in this study. Thereafter, four punch biopsies were obtained: the first two were from lesional area for histopathological examination to confirm the diagnosis of AA; the third was from the lesional area for proteome analysis; and the fourth was from non-lesional area (defined as the scalp region that was away from the margin of the lesional area more than a diameter of the lesion) for proteome analysis.
Protein extraction. Biopsies were taken from both non-lesional and lesional areas of each patient and proteins were extracted from each biopsy separately. Briefly, the biopsied tissue was chopped into small pieces and washed with pre-chilled phosphate buffered saline (PBS). The sample was snap frozen by liquid nitrogen, ground into powder, extracted by SDT lysis buffer (containing 4% SDS, 100 mM DTT, and 100 mM Tris-HCl; pH 7.6) and incubated on ice for 30 min. The supernatant was collected after centrifugation at 10,000 × g and 4 °C for 30 min and protein concentration was measured by Bio-Rad Protein Assay (Bio-Rad Laboratories; Hercules, CA) based on the Bradford's method.
In-solution tryptic digestion by filter-aided sample preparation (FASP) method. Equal amount of total protein derived from each sample was pooled and digested by trypsin according to FASP protocol 53 . nanoLC-ESI-LTQ-Orbitrap MS/MS. Each sample was run in technical triplicates. Separation of the digested peptides was performed using EASY-nLC II (Thermo Scientific; Waltham, MA). Briefly, peptides were loaded from a cooled (7 °C) autosampler into an in-house, 3-cm-long pre-column containing 5-µm C18 resin (Dr.Maisch GmbH; Ammerbuch, Germany) and then to an in-house, 10-cm-long analytical column packed with