Alopecia areata (AA) is a chronic inflammatory disease of hair follicles manifesting as patchy areas of hair loss on the scalp and body. Development of AA is associated with pen- and intra-follicular inflammation of anagen stage hair follicles, primarily by CD4+ and CD8+ cells. We hypothesized that if cell-mediated cytotoxicy against hair follicles is to be a component of the hair loss disease mechanism, increased expression of genes and products typical of cytotoxic cells, as well as increased apoptosis activity within affected hair follicles, would be expected to occur in the lesional skin compared to the normal skin. Furthermore, we studied gene expression levels of multiple cytokines and characteristic chemokines, using the C3FI/HeJ mouse model of AA. mRNA expression levels of granzyme A, granzyme B, perform Fas, Fas ligand, TNF-cL, TNF-aRl and R2, TRAIL, TRAILR, TRAMP, Thi-, Th2-, and Th17-associated cytokines, as well as multiple chemokines were compared between the skin, draining lymph nodes, thymus and spleens of normal and AA-affected mice using quantitative reverse transcriptase PCR. FasL, granzyme A, granzyme B, pro- and anti-inflammatory cytokines were all highly up-regulated in the skin of AA-affected mice. Immunohistochemical studies of the skin revealed that, although greater numbers of granzyme B and FasL expressing cells were present in AA affected skin, the cells were morphologically diffusely distributed and not exclusively located within the focal pen- and intrafollicular infiltrate. The majority of these cells were further characterized as mast cells, which were also found in substantially greater numbers in the skin of mice with AA compared to their normal haired controls. Almost no perform expressing cells were identified in AA affected mouse skin and TUNEL staining suggested relatively limited apoptosis activity in hair follicle keratinocytes. In conclusion, while granzymes and FasL may play important roles in disease development, the profiles and patterns of expression are not consistent with direct cell-mediated cytotoxic action against the follicular epithelium in chronic mouse AA. Potentially, hair growth inhibiting cytokines may play a more dominant role in AA development than previously thought. Furthermore, mast cells, with their increased presence around hair follicles in the AA affected mouse skin and their ability to express granzyme B and FasL, are suggested as potential key players in the pathogenesis of AA. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Understanding the genetic architecture of complex disorders is important for identifying disease mechanisms and potential molecular targets for therapeutic interventions. Genetic diseases are broadly classified as either Mendelian (monogenic) diseases or complex (polygenic) diseases. Common, polygenic disorders result from inheritance of multiple common variants with low penetrance. In contrast, monogenic, Mendelian disorders are caused by rare variants with high penetrance at a single genetic locus. However, an increasing number of studies support a role for rare variants of moderate effect size in complex diseases. As a result, genetic approaches previously utilized for discovering rare variants in Mendelian diseases, such as next generation sequencing, can now effectively be applied to complex diseases to define the contribution of both rare and common variants to the genetic burden of polygenic traits and diseases. Alopecia Areata (AA) is a complex autoimmune disease characterized by non-scarring hair loss that is due to a combination of both enviornmental and genetic factors. Our previous Genome-wide Association Study (GWAS) identified at least 14 genetic regions contributing to AA disease susceptibility. Although useful in identifying disease-associated loci and surrounding linkage disequilibrium (LD) blocks, GWAS is not sufficiently granular to 1) elucidate causal (association-driving) variants; and 2) discover rare risk variants. This level of resolution can only be achieved by deep sequencing followed by functional validation of variants. The goal of this thesis was to address these challenges in AA using two genetic approaches that have not been previously utilized in the context of this disease. In Chapter 2, I performed a hypothesis-driven analysis of common variants in a GWAS-associated locus using targeted genomic sequencing. In Chapter 3, I utilized whole exome sequencing (WES) in an unbiased approach to assess rare variant contribution in AA disease risk. To conduct these analyses simultaneously, we designed a whole exome sequencing (WES) chip that also included custom capture of 24 Mb of genomic regions covering the 14 genetic loci previously identified using GWAS. I applied these two sequencing approaches in a large AA patient cohort and identified potentially causal variants in several genes. To interrogate the consequences of these variants, I performed functional analyses to determine the effects of disease-causing variants on the target organ of AA attack, the hair follicle (HF). In Chapter 2, I report on the use of targeted genomic sequencing to interrogate the coding and non-coding regions surrounding a previously implicated GWAS locus. This approach provided fine mapping of coding and non-coding common variants in regions that may be contributing to disease risk. In this thesis, I focused on the effect of genetic perturbations on the end-organ HF, and consequentially prioritized my functional analysis using two criteria: 1) genes expressed in the (HF) and; 2) GWAS regions that were not previously implicated in other autoimmune diseases. One of the regions that satisfied these criteria harbored the Syntaxin 17 (STX17) gene, which encodes a SNARE protein involved in autophagy and mitochondrial fission. Targeted genomic sequencing of the STX17 region in 849 AA cases identified 35 non-coding and 0 coding variants in high LD with the GWAS SNP. Thirty-three variants were significantly enriched in cases compared to controls, and the remaining two were nominally significant. Thirty-two of the significantly associated AA variants were confirmed to be AA skin eQTLs that downregulated expression of STX17 in affected scalp skin of AA patients. Downstream analyses incorporated in silico and functional cell assays that uncovered a novel autophagy-independent role for STX17 in melanocyte biology. I discovered that a reduction of STX17 expression was associated with an accumulation of a melanocyte-specific antigen and increased immunogenicity, as seen by CD8+ T cell infiltrates in the skin of AA patients with low levels of STX17 expression. I used a targeted sequencing approach to successfully identify candidate causal variants driving the GWAS association at the STX17 locus, and propose a novel mechanism underlying STX17-dependent melanocyte perturbation and AA disease. In the second section of this thesis, we used the WES feature of the chip to assess the genetic contribution of rare variation in AA, in a genome-wide and unbiased manner. WES data and gene-level burden analyses of 18,653 genes in 849 AA patients was compared to 15,640 controls to identify rare variants associated with AA. Unexpectedly, this analysis identified one gene, encoding a hair-specific keratin, Keratin 82 (KRT82) that harbored significantly more rare damaging mutations in AA cases compared to controls (p=2.68E-06). Eleven rare damaging mutations were found in 51 AA patients in the heterozygous state (6.01%) compared to 2.58% controls. These variants resided in evolutionary conserved amino acid residues, and nine out of the eleven mutations were located in established disease-causing domains in keratin proteins. I determined that KRT82 expression was absent or largely reduced in AA hair follicles, including the bulb region, the site of AA immune attack. Moreover, AA patients with damaging variants and reduced KRT82 expression had increased perifollicular CD8+ T cell infiltrates in comparison to control HFs with intact KRT82 expression remaining. I proposed that damaging mutations in the coding regions of KRT82 resulted in loss of functional protein, thereby weakening the protective HF cuticle and predisposing the HF to immune attack. In summary, I used two genetic approaches (targeted genomic sequencing and WES) to identify common (Chapter 2) and rare variants (Chapter 3) with novel contributions to the complex genetic architecture of AA. I focused my functional studies on genes expressed in the target HF, with the goal of defining the role of unidentified, variant-mediated end-organ disruption in the predisposition of AA patient HFs to aberrant autoimmune attack. Up to now, most efforts in AA mechanistic studies have focused on the aberrant immune response. The work in my thesis uncovered novel roles for perturbations in the HF itself as a participating factor in AA disease risk.

Alopecia areata (AA) is a complex autoimmune disease in which autoreactive T cell-mediated attack of the hair follicle (HF) leads to non-scarring hair loss. Although AA is one of the most prevalent autoimmune diseases, the development of novel effective therapeutics has been limited. Standard of care remains observation for mild cases and steroids for moderate-to-severe cases, which have demonstrated only limited efficacy. The skin is a highly heterogeneous tissue at baseline, comprised of a diverse array of immune and non-immune cell types whose coordinated crosstalk is essential for homeostasis. The skin microenvironment becomes markedly altered as a result of disease-associated inflammation in AA. A pathognomonic histopathologic feature of AA is an intense lymphocytic infiltrate surrounding the lower portion of the HF in the growth phase of the hair cycle, known as anagen. We previously established that CD8+ T cells comprise the majority of this infiltrate in AA skin, and that they are necessary and sufficient to drive disease via JAK/STAT activation. While this discovery led to the pioneering use of JAK inhibitors as a novel class of therapeutics in AA, JAK inhibition is not a curative solution, since patients often experience relapse upon discontinuation of treatment. This not only underscores the continued need for translational drug discovery research in AA, but also reflects an incomplete understanding of the mechanisms that govern disease pathophysiology. Recent advances in single-cell RNA sequencing (scRNAseq) present an unprecedented opportunity to dissect the heterogeneity of complex tissues and disorders. Since its emergence, scRNAseq has proven to be a powerful tool for the discovery of rare cell types and novel therapeutic targets in a variety of contexts that range from cancer to autoimmunity. In this thesis, we leveraged scRNAseq to interrogate the cellular and molecular mechanisms underlying disease pathogenesis in AA at single-cell resolution, together with validation and functional experiments, with the goal of uncovering novel cell types and pathways that can guide the development of innovative therapeutic strategies. In Chapter 2, we performed scRNAseq of skin-infiltrating CD45+ immune cells to dissect lymphocyte heterogeneity in both murine and human AA. Our scRNAseq analyses informed a series of antibody-mediated cell depletion experiments that assessed the in vivo function of specific lymphocyte subsets in murine AA. Our results established CD8+ T cells as the predominant disease-driving cell type in AA. We identified shared mechanisms underlying CD8+ T cell heterogeneity in murine and human AA skin, in which CD8+ T cells form an “effectorness gradient” comprised of interrelated transcriptional states that culminate in increased expression of inflammatory cytokines and T cell effector function. We also demonstrated a role for CD4+ T helper cells in disease initiation, and determined that regulatory T cells possess intact immunosuppressive capacity in AA. In Chapter 3, we expanded upon the studies described in Chapter 2 and performed scRNAseq of skin-infiltrating CD45+ cells at various timepoints throughout disease course (from 3 to 24 weeks post-disease induction) in AA to analyze the temporal dynamics of lymphocyte heterogeneity in AA skin and skin-draining lymph nodes. In conjunction with scRNAseq, we also performed single-cell TCR sequencing to assess the dynamics of T cell clonality alongside changes in T cell transcriptional profiles. We observed a striking increase in CD8+ T cell clonal expansion during disease onset, which increased throughout disease progression and subsequently decreased in chronic AA, when the preclinical mouse model exhibits total body hair loss. Our single-cell analyses suggested that CD8+ T cell clonality and pathogenicity are closely linked, which we validated in vivo by demonstrating that a single expanded clonotypic population of CD8+ T cells is sufficient to induce disease in mice. In Chapter 4, we analyzed single-cell transcriptomic profiles obtained from full-thickness skin in mice with chronic AA to investigate the contributions of the HF and other non-T cell populations in disease. In this study, we also used a network biology-based approach to infer single-cell protein activity, which together with single-cell mRNA gene expression profiles uncovered a multitude of novel findings in AA. Our results revealed a role for necroptosis as a potential HF-intrinsic mechanism of pro-inflammatory signaling in AA, and also identified an MHC Class II signature specific to basal keratinocytes in AA skin. Furthermore, we uncovered a novel, rare population of disease-associated Arg1+ macrophages, which prompted us to revisit our immune-specific scRNAseq datasets described in Chapters 2 and 3 and perform an integrative analysis of this novel cell type in AA. Our preliminary in vivo studies suggested that targeting Arg1+ macrophages and/or arginine metabolism may ameliorate disease in AA. Taken together, this thesis presents a comprehensive, systematic interrogation of AA pathogenesis at single-cell resolution. Importantly, the validation and functional studies that were informed by our scRNAseq data demonstrate proof-of-concept of the use of single-cell technology to accelerate the discovery and translation of novel therapeutic targets in complex diseases. While we undertook a hypothesis-driven approach to design our studies, the data presented in this thesis was also profoundly hypothesis-generating, and has informed a number of ongoing projects in the laboratory with the shared goal of advancing our understanding of disease pathology in AA.

京都大学 / 新制・論文博士 / 博士(農学) / 乙第13497号 / 論農博第2901号 / 新制||農||1093(附属図書館) / 学位論文||R4||N5403(農学部図書室) / (主査)教授 谷 史人, 教授 佐々木 努, 教授 保川 清 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No / Although we have made significant progress in understanding the regulation of the UVR-exposed epidermal-melanin unit, we know relatively little about how human hair follicle pigmentation is regulated. Progress has been hampered by gaps in our knowledge of the hair growth cycle’s controls, to which hair pigmentation appears tightly coupled. However, pigment cell researchers may have overly focused on the follicular melanocytes of the nocturnal and UVR-shy mouse as a proxy for human epidermal melanocytes. Here, I emphasize the epidermis-follicular melanocyte pluralism of human skin, as research models for vitiligo, alopecia areata and melanoma, personal care/cosmetics innovation. Further motivation could be in finding answers to why hair follicle and epidermal pigmentary units remain broadly distinct? Why melanomas tend to originate from epidermal rather than follicular melanocytes? Why multiple follicular melanocyte sub-populations exist? Why follicular melanocytes are more sensitive to aging influences? In this perspective, I attempt to raise the status of the human hair follicle melanocyte and highlight some species-specific issues involved which the general reader of the pigmentation literature (with its substantial mouse-based data) may not fully appreciate.

Yes / Alopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60–70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss. / Canadian Institutes of Health Researches (Funding Reference Number: 134214 and 136945).

Alopecia areata (AA) is a putative autoimmune hair loss disorder. It mainly affects the scalp hair but can also involve body hair, and can also affect the nail and the eye. While there are may be several lines of evidence to support the autoimmune basis of AA, there is still very little information on the hair follicle autoantigen(s) involved in its pathogenesis. In this project, serum antibodies (AA=10, control=10) were used to immunoprecipitate AA-relevant target antigens from normal human scalp hair follicle extracts. These immunoprecipitates were analysed by LC-MALDI-TOF/TOF mass spectrometry for target protein identification. This part of the project involved substantial methods development. Trichohyalin was immunoprecipitated by all AA sera, but by only 5 normal sera. Importantly, the mean Mascot scores of the AA group was significantly higher than the normal group (p=0.005). Keratin 16 was also identified from immunoprecipitates as another potential AA-relevant target antigen. Functional studies by ex vivo whole hair follicle organ culture using commercial antibodies to trichohyalin and keratin 16 significantly inhibited hair fibre elongation compared to controls. Indirect immunofluorescence studies revealed that AA sera contained higher immunoreactivity against normal human scalp anagen hair follicles compared to normal sera. Immunoreactivities were mainly in the outer root sheath and inner root sheath, and less so to the medulla and hair bulb matrix. Double immunofluorescence studies of AA and normal serum with anti-trichohyalin antibody (AE15) revealed co-localisation of 9 of the AA sera antibodies with trichohyalin in the inner root sheath (mostly in Henle¿s, less in Huxley¿s/inner root sheath cuticle), but only weakly in 3 normal sera. This study supports the involvement of an antibody response to anagen-specific hair follicles antigens in AA. Moreover, there may be some evidence that these antibodies may have a pathogenic role.

Yes / The development of androgenetic alopecia is associated with a risk of developing cardiovascular diseases, but the association of alopecia areata with cardiovascular diseases in humans is largely unexplored. We measured the plasma level of two common cardiovascular disease markers, cardiac troponin I and Creactive protein, in alopecia areata and androgenetic alopecia-affected subjects. Also, we investigated the possible presence of pro-apoptotic factors in the plasma of hair loss subjects. The mean plasma cardiac troponin I level was highest in alopecia areata subjects, moderately higher in androgenetic alopecia subjects, and lowest in subjects without hair loss (p < 0.05). Alopecia areata subjects not receiving treatments had highest levels of cardiac troponin I (p < 0.05). Alopecia areata plasma samples with high cardiac troponin I levels also induced significantly higher rates of cardiomyocyte apoptosis in cell culture assays. The results suggest the potential for increased heart remodelling. Close monitoring of cardiovascular health in alopecia areata subjects, as well as subsets of androgenetic alopecia patients, may be appropriate. / Canadian Institutes of Health Research (CIHR; MOP-82927). EW is the recipient of a Banting Postdoctoral Fellowship (SAC-92845).

Alopecia areata (AA) is a putative autoimmune hair loss disorder. It mainly affects the scalp hair but can also involve body hair, and can also affect the nail and the eye. While there are may be several lines of evidence to support the autoimmune basis of AA, there is still very little information on the hair follicle autoantigen(s) involved in its pathogenesis. In this project, serum antibodies (AA=10, control=10) were used to immunoprecipitate AA-relevant target antigens from normal human scalp hair follicle extracts. These immunoprecipitates were analysed by LC-MALDI-TOF/TOF mass spectrometry for target protein identification. This part of the project involved substantial methods development. Trichohyalin was immunoprecipitated by all AA sera, but by only 5 normal sera. Importantly, the mean Mascot scores of the AA group was significantly higher than the normal group (p=0.005). Keratin 16 was also identified from immunoprecipitates as another potential AA-relevant target antigen. Functional studies by ex vivo whole hair follicle organ culture using commercial antibodies to trichohyalin and keratin 16 significantly inhibited hair fibre elongation compared to controls. Indirect immunofluorescence studies revealed that AA sera contained higher immunoreactivity against normal human scalp anagen hair follicles compared to normal sera. Immunoreactivities were mainly in the outer root sheath and inner root sheath, and less so to the medulla and hair bulb matrix. Double immunofluorescence studies of AA and normal serum with anti-trichohyalin antibody (AE15) revealed co-localisation of 9 of the AA sera antibodies with trichohyalin in the inner root sheath (mostly in Henle's, less in Huxley's/inner root sheath cuticle), but only weakly in 3 normal sera. This study supports the involvement of an antibody response to anagen-specific hair follicles antigens in AA. Moreover, there may be some evidence that these antibodies may have a pathogenic role.

Primary scalp alopecia is classically divided into cicatricial (scarring) and non-cicatricial (non-scarring). Challenging cases are assessed with a scalp biopsy. The use of both horizontal and vertical sections (HoVert sections) has dramatically improved the accuracy of histopathological diagnosis. In this work, we have used immunostaining to address diagnostic difficulties, which persist despite all currently available tools. We performed an immunostain panel (CD3, CD4, CD8 and CD20) in order to distinguish pattern hair loss from alopecia aerate in cases which do not have the usual peribulbar lymphocytic infiltrate and showed that CD3+ T-lymphocytes within the empty fibrous follicular tracts favor a diagnosis of alopecia areata. We performed CD123 in order to distinguish lichen planopilaris from alopecia lupus erythematosus in cases with only a superficial lymphocytic infiltrate and an uninvolved interfollicular epidermis and showed that clusters of CD123+ plasmacytoid dendritic cells favor a diagnosis of lupus erythematosus. We performed cytokeratin 15 in order to assess whether the loss of the follicular bulge stem cells has diagnostic value in cicatricial alopecia and demonstrated that the loss of cytokeratin 15+ bulge stem cells is identified in lichen planopilaris, frontal fibrosing alopecia, and lupus erythematous, so cytokeratin 15 has no diagnostic value. We have attempted to integrate the new concepts and our findings into the traditional classifications of alopecia and proposed a new diagnostic algorithm. In conclusion, immunostaining combined with HoVert grossing advances the accuracy of histopathological diagnosis of primary scalp alopecia. / L’alopécie primitive du cuir chevelu est habituellement classée en cicatricielle et non-cicatricielle. Dans les cas difficiles, la biopsie du cuir chevelu peut aider au diagnostic. L’utilisation de coupes, à la fois verticales et horizontales sur le même spécimen (technique HoVert), a radicalement amélioré le diagnostic histopathologique. Dans ce travail, nous avons utilisé l’immunohistochimie pour évaluer les difficultés diagnostiques qui persistent malgré tous les outils actuels. Nous avons utilisé les CD3, CD4, CD8 et CD20 pour différencier l’alopécie androgénique de la pelade dépourvue de l’infiltrat lymphocytaire péribulbaire habituel et nous avons démontré que la présence de lymphocytes CD3+ dans les travées folliculaires fibreuses est en faveur de la pelade. Nous avons utilisé le CD123 pour différencier le lichen plan pilaire du lupus érythémateux alopécie avec infiltrat lymphocytaire superficiel et sans atteinte de l’épiderme interfolliculaire et nous avons démontré que la présence d’amas de cellules dendritiques plasmacytoïdes CD123+ est en faveur du lupus érythémateux. Nous avons utilisé la cytokératine 15 pour évaluer si la perte des cellules souches du bulge a une valeur diagnostique dans l’alopécie cicatricielle et nous avons démontré que cette perte s’observait de manière identique dans le lichen plan pilaire, l’alopécie frontale fibrosante comme dans le lupus érythémateux et n’avait donc aucune valeur diagnostique. Nous avons tenté d’intégrer les nouveaux concepts et nos données dans les classifications traditionnelles des alopécies et nous avons élaboré un nouvel algorithme diagnostique. L’association des immunomarquages avec la technique HoVert ouvre de nouvelles perspectives dans le diagnostic histopathologique des alopécies primaires du cuir chevelu. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished