Human Alopecia Areata Research Articles

Interleukin-15 is a hair follicle immune privilege guardian

The autoimmunity-promoting cytokine, Interleukin-15 (IL-15), is often claimed to be a key pathogenic cytokine in alopecia areata (AA). Yet, rhIL-15 promotes human hair follicle (HF) growth ex vivo. We have asked whether the expression of IL-15 and its receptor (IL-15R) isoforms is altered in human AA and how IL-15 impacts on human HF immune privilege (HF-IP) in the presence/absence of interferon-γ (IFNγ), the well-documented key AA-pathogenic cytokine, as well as on hair regrowth after experimental AA induction in vivo. Quantitative immunohistomorphometry showed the number of perifollicular IL-15+ T cells in AA skin biopsies to be significantly increased compared to healthy control skin, while IL-15, IL-15Rα, and IL-15Rγ protein expression within the hair bulb were significantly down-regulated in AA HFs. In organ-cultured human scalp HFs, rhIL-15 significantly reduced hair bulb expression of MICA, the key “danger” signal in AA pathogenesis, and increased production of the HF-IP guardian, α-MSH. Crucially, ex vivo, rhIL-15 prevented IFNγ-induced HF-IP collapse, restored a collapsed HF-IP by IL-15Rα-dependent signaling (as documented by IL-15Rα−silencing), and protected AA-preventive immunoinhibitory iNKT10 cells from IFNγ-induced apoptosis. rhIL-15 even promoted hair regrowth after experimental AA induction in human scalp skin xenotransplants on SCID/beige mice in vivo. Our data introduce IL-15 as a novel, functionally important HF-IP guardian whose signaling is constitutively defective in scalp HFs of AA patients. Our data suggest that selective stimulation of intrafollicular IL-15Rα signaling could become a novel therapeutic approach in AA management, while blocking it pharmacologically may hinder both HF-IP restoration and hair re-growth and may thus make HFs more vulnerable to AA relapse.

Physiological Importance of the Vitaminoid Thiocyanate and Its Influence on Hair Growth

Thiocyanate is a physiologically and biochemically active metabolite that is not sufficiently formed by endogenous synthesis in the case of diseases, stress, malnutrition, infection, toxic influences or loss through washing out during hair washing. In these siuations, alimentary supplementation or local application become necessary depending on the objective. An important effect of thiocyanate is the stimulation of new cell formation, initially proven in cell culture and in plants by the increase in yield, later also in the animal species mink (improved coat quality), sheep (increased wool yield) and guinea pigs (stimulation of the anagen phase of hair formation). Therapeutic efficacy was even demonstrated in human alopecia areata, with 51% showing complete to good remission 12 months after completion of treatment. The growth-promoting effect was confirmed in the trichogram. The biological activity of the vitaminoid is not based on a uniform mechanism of action but is to be understood as the sum of various partial effects

Functional interrogation of lymphocyte subsets in alopecia areata using single-cell RNA sequencing

Alopecia areata (AA) is among the most prevalent autoimmune diseases, but the development of innovative therapeutic strategies has lagged due to an incomplete understanding of the immunological underpinnings of disease. Here, we performed single-cell RNA sequencing (scRNAseq) of skin-infiltrating immune cells from the graft-induced C3H/HeJ mouse model of AA, coupled with antibody-based depletion to interrogate the functional role of specific cell types in AA in vivo. Since AA is predominantly T cell-mediated, we focused on dissecting lymphocyte function in AA. Both our scRNAseq and functional studies established CD8+ T cells as the primary disease-driving cell type in AA. Only the depletion of CD8+ T cells, but not CD4+ T cells, NK, B, or γδ T cells, was sufficient to prevent and reverse AA. Selective depletion of regulatory T cells (Treg) showed that Treg are protective against AA in C3H/HeJ mice, suggesting that failure of Treg-mediated immunosuppression is not a major disease mechanism in AA. Focused analyses of CD8+ T cells revealed five subsets, whose heterogeneity is defined by an "effectorness gradient" of interrelated transcriptional states that culminate in increased effector function and tissue residency. scRNAseq of human AA skin showed that CD8+ T cells in human AA follow a similar trajectory, underscoring that shared mechanisms drive disease in both murine and human AA. Our study represents a comprehensive, systematic interrogation of lymphocyte heterogeneity in AA and uncovers a novel framework for AA-associated CD8+ T cells with implications for the design of future therapeutics.

Identification of BST2 as a biomarker for alopecia areata in both mice and humans

Abstract Alopecia areata is an autoimmune disease characterized by aberrant non-scarring hair loss. 147 million people worldwide are currently diagnosed with alopecia areata with a lifetime incidence of 2.1%. The hair follicle is a site of immune privilege however in alopecia areata, CD4+ helper T cells and CD8+ cytotoxic T cells infiltrate and attack the hair follicle. To better understand the initiating mechanisms surrounding alopecia areata, we examined the function of epidermal γδ T cells and dermal macrophages in the initiation of or protection from the disease. We show that epidermal γδ T cells and keratinocytes are elevated in the skin of C3H/HeJ mice with alopecia areata. Analysis of publicly available single cell RNA sequencing data delineated the top differentially expressed genes of epidermal γδ T cells and dermal macrophages. Bone marrow stromal antigen 2 (BST2) is significantly upregulated among both cell types and is a novel gene associated with alopecia areata. Using immunofluorescence microscopy we determined that a small population of hair follicle-associated epidermal γδ T cells express BST2 in C3H/HeJ mice and C57Bl/6J mice without alopecia areata. We have also identified a significant increase in BST2 expression in skin sections from mice affected by alopecia areata. BST2+CD11b+ macrophages upregulate genes associated with antiviral activity indicating an association between the pathogenesis of alopecia areata and infectious viral particles. BST2+ epidermal γδ T cells upregulate genes associated with FAK signaling in alopecia areata, which indicates a role for FAK in the increased number of epidermal T cells. These studies identify a role for BST2 in alopecia areata that is mediated by epidermal γδ T cells and macrophages.

004 Cell-based therapy may be effective in alopecia areata: Preclinical evidence that autologous, peripheral regulatory γdT cells are preventive in human ex vivo and therapeutic in human in vivo models

Regulatory T cells (Tregs) have long been thought to exert protective, immunoinhibitory functions in the C3H/HeJ mouse model of alopecia areata (AA), while their role in human AA remains obscure. However, FOXP3+/γdT cell receptor+ Tregs (γdTregs) may be the predominant and more potently immunoinhibitory regulatory T cells. Given that we have recently identified γdT cells as important novel players in human AA pathobiology, we have therefore asked whether γdTregs play a role in human AA and might be recruited for future AA treatment.

014 Tyrosine Kinase 2 Inhibition Ameliorates the Phenotype of Lesional Alopecia Areata Scalp Skin Ex Vivo, and Reverses the Induction of Human Alopecia Areata in a Humanized Mouse Model

Alopecia areata (AA) is an immune-mediated hair follicle (HF) disorder induced by elevated IFNγ levels and a Tc1-driven inflammatory response. We have previously shown that treatment with IL-12 and IL-18 induces IFNγ-dependent, immune privilege collapse of the hair bulb, which can be inhibited by blocking IL-12 receptor signaling with the TYK2 inhibitor, BMS-986202 (BMS), in human microdissected HFs. Here, we further investigate the role of TYK2 and its inhibition in AA pathogenesis. We cultured human full thickness non-lesional and lesional scalp skin biopsies from acute or chronic AA patients ex vivo with BMS.

AIRE Deficiency Leads to the Development of Alopecia Areata‒Like Lesions in Mice

Alopecia areata (AA) is an autoimmune hair loss disorder with no cure. Patients with sequence variation in AIRE are 15 times more likely to develop AA than the general population, yet the roles of AIRE in AA pathogenesis are unknown. In this study, we report that 62% of C57BL/6J female Aire‒/‒ mice spontaneously developed persistent AA-like lesions that displayed several hallmarks of human AA. Lesional Aire‒/‒ skin exhibited hair follicle (HF) dystrophy as determined by a reduced number of anagen HFs, decreased anagen HF proliferation, hair pigmentary changes, and decreased hair width and length. Inflammatory infiltrate comprising CD8+ T cells, CD4+ T cells, CD68+ macrophages, and mast cells was prominent in lesional Aire‒/‒ HFs. From gene expression analyses, we found lesional Aire‒/‒ skin to have significantly increased expression of human AA signature genes, including H2-Ab1, Ifnγ, IFN-γ‒induced chemokines (Ccl5, Cxcl9‒11), γc family cytokine receptor Il2RA, and JAK‒signal transducer and activator of transcription (STAT) signaling components (Stat1, Stat2, Stat4). By immunostaining, lesional Aire‒/‒ HFs also show upregulated major histocompatibility complex class I and downregulated α-melanocyte-stimulating hormone, signifying immune privilege collapse, and increased STAT1 activation in HF keratinocytes. Our study highlights a role for AIRE in HF biology and shows that Aire‒/‒ mice may serve as a valuable model system to study AA pathogenesis.

056 IL-15 prolongs hair growth and operates as a guardian of human hair follicle immune privilege

Effective alopecia areata (AA) therapy requires targeting of the underlying key pathomechanisms, namely hair follicle (HF) immune privilege (IP) collapse and premature catagen. Based on mouse data, the multi-functional, anti-apoptotic, autoimmunity-promoting cytokine, interleukin-15 (IL-15), is often postulated to be a key pathogenic cytokine in human AA, even though it is unclear how IL-15 affects HF-IP and catagen. Here, we show that lesional skin of AA patients shows an increased number of perifollicular IL-15+ T and NK cells.

700 Tyrosine kinase 2 inhibition rescues hair follicles from IL-12–mediated immune privilege collapse and reverses the induction of human alopecia areata in a humanized mouse model

Alopecia areata (AA), an immune-mediated hair follicle (HF) disorder, leads to hair loss due to collapse of the HF’s physiologic immune privilege (IP), induced by elevated IFNγ levels and a Tc1-driven inflammatory response dominated by T and NK cells. Given that IL-12 signaling is crucial for IFNγ production by immune cells, we explored for the first time the role of IL-12 and tyrosine kinase 2 (TYK2)-mediated signaling in the induction and prevention of HF IP collapse via a selective TYK2 inhibitor (BMS-986202).

LB708 ILC1-like innate lymphocytes in human autoimmunity: Lessons from Alopecia Areata

Innate lymphoid cells type 1 (ILC1) express NKG2D and produce large amounts of IFN-γ, i.e. two key elements in the pathogenesis of alopecia areata (AA). In this study, we aimed to explore a possible involvement of ILC1-like cells in human AA by using ex-vivo and in-vivo models for human AA. Triple immunofluorescence staining of AA sections revealed pathological infiltrates of NKG2D+ ILC1-like cells in and around the anagen hair bulb of lesional AA HFs, but also (more discretely) in/around non-lesional AA HFs, together with a dominant infiltrate of CD8+/NKG2D+ cells.

The Alopecia Areata Phenotype Is Induced by the Water Avoidance Stress Test In cchcr1-Deficient Mice

We recently discovered a nonsynonymous variant in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene within the alopecia areata (AA) risk haplotype. We also reported that the engineered mice with this risk allele exhibited. To investigate more about the involvement of the CCHCR1 gene in AA pathogenesis, we developed an AA model using C57BL/6N cchcr1 gene knockout mice. In this study, mice (6–8 weeks) were divided into two groups: cchcr1−/− mice and wild-type (WT) littermates. Both groups were subjected to a water avoidance stress (WAS) test. Eight weeks after the WAS test, 25% of cchcr1−/− mice exhibited non-inflammatory foci of alopecia on the dorsal skin. On the other hand, none of wild-type littermates cause hair loss. The foci resembled human AA in terms of gross morphology, trichoscopic findings and histological findings. Additionally, gene expression microarray analysis of cchcr1−/− mice revealed abnormalities of hair related genes compared to the control. Our results strongly suggest that CCHCR1 is associated with AA pathogenesis and that cchcr1−/− mice are a good model for investigating AA.

Selective inhibition of JAK3 signaling is sufficient to reverse alopecia areata.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) are key intracellular mediators in the signal transduction of many cytokines and growth factors. Common γ chain cytokines and interferon-γ that use the JAK/STAT pathway to induce biological responses have been implicated in the pathogenesis of alopecia areata (AA), a T cell–mediated autoimmune disease of the hair follicle. We previously showed that therapeutic targeting of JAK/STAT pathways using the first-generation JAK1/2 inhibitor, ruxolitinib, and the pan-JAK inhibitor, tofacitinib, was highly effective in the treatment of human AA, as well as prevention and reversal of AA in the C3H/HeJ mouse model. To better define the role of individual JAKs in the pathogenesis of AA, in this study, we tested and compared the efficacy of several next-generation JAK-selective inhibitors in the C3H/HeJ mouse model of AA, using both systemic and topical delivery. We found that JAK1-selective inhibitors as well as JAK3-selective inhibitors robustly induced hair regrowth and decreased AA-associated inflammation, whereas several JAK2-selective inhibitors failed to restore hair growth in treated C3H/HeJ mice with AA. Unlike JAK1, which is broadly expressed in many tissues, JAK3 expression is largely restricted to hematopoietic cells. Our study demonstrates inhibiting JAK3 signaling is sufficient to prevent and reverse disease in the preclinical model of AA.

Single-Cell mRNA Sequencing of Murine and Human Alopecia Areata Identifies Immune Cell Profiles Predictive of Human Disease State

Abstract Alopecia areata (AA) is one of the most common autoimmune conditions, presenting initially with loss of hair without overt skin changes. The unremarkable appearance of the skin surface contrasts with the complex immune activity occurring at the hair follicle. AA pathogenesis is due to the loss of immune privilege of the hair follicle leading to autoimmune attack. Although the literature has focused on CD8+ T cells, vital roles for CD4+ T cells and antigen-presenting cells have been suggested. Here, we use single-cell mRNA sequencing to reveal distinct expression profiles of immune cells in AA. We found clonal expansions of both CD4+ and CD8+ T cells, with shared clonotypes across varied transcriptional states. Demonstrating distinct gene and clonotypic variations, AA murine data were used to generate highly predictive models of human AA disease. In order to corroborate the results, single-cell sequencing of T cells in human AA recapitulated the clonotypic findings and the gene expression of the predictive models. Taken together, this work demonstrates the unique transcriptomic environment of AA skin, not just limited to CD8+ T cells. This work also represents the first single-cell sequencing for the autoimmune condition AA.

Pro-inflammatory Vδ1+T-cells infiltrates are present in and around the hair bulbs of non-lesional and lesional alopecia areata hair follicles

BackgroundIt is widely accepted that NKG2D+cells are critically involved in alopecia areata (AA) pathogenesis. However, besides being expressed in CD8+T-cells and NK cells, NKG2D is also found in human γδT-cells. AA lesional hair follicles (HFs) overexpress NKG2D and γδTCR activating ligands, e.g. MICA and CD1d, and chemoattractants for γδT-cells, such as CXCL10. ObjectiveTo investigate whether abnormal activities of γδT-cells may be involved in AA pathogenesis. MethodsWe analyzed the number and activation status of γδT-cells in human healthy, lesional and non-lesional AA scalp biopsies by FACS and/or quantitative (immuno-)histomorphometry. ResultsIn healthy human scalp skin, the few skin-resident γδT-cells were found to be mostly Vδ1+, non-activated (CD69−NKG2Ddim) and positive for CXCL10, and CXCL12 receptors. These Vδ1+T-cells predominantly localized in/around the HF infundibulum. In striking contrast, the number of Vδ1+T-cells was significantly higher around and even inside the proximal (suprabulbar and bulbar) epithelium of lesional AA HFs. These cells also showed a pro-inflammatory phenotype, i.e. higher NKG2D, and IFN-γ and lower CD200R expression. Importantly, more pro-inflammatory Vδ1+T-cells were seen also around non-lesional AA HFs. Lesional AA HFs also showed significantly higher expression of CXCL12. ConclusionOur pilot study introduces skin-resident γδT-cells as a previously overlooked, but potentially important, mostly (auto-)antigen-independent, new innate immunity protagonist in AA pathobiology. The HF infiltration of these activated, IFN-γ-releasing cells already around non-lesional AA HFs suggest that Vδ1+T-cells are involved in the early stages of human AA pathobiology, and may thus deserve therapeutic targeting for optimal AA management.

A transcriptomic map of murine and human alopecia areata.

Alopecia areata (AA) is a common autoimmune condition, presenting initially with loss of hair without other overt skin changes. The unremarkable appearance of the skin surface contrasts with the complex immune activity occurring at the hair follicle. AA pathogenesis is due to the loss of immune privilege of the hair follicle, leading to autoimmune attack. Although the literature has focused on CD8+ T cells, vital roles for CD4+ T cells and antigen-presenting cells have been suggested. Here, we use single-cell sequencing to reveal distinct expression profiles of immune cells in murine AA. We found clonal expansions of both CD4+ and CD8+ T cells, with shared clonotypes across varied transcriptional states. The murine AA data were used to generate highly predictive models of human AA disease. Finally, single-cell sequencing of T cells in human AA recapitulated the clonotypic findings and the gene expression of the predictive models.

Bioinformatics analysis of differentially expressed genes on alopecia.

The molecular mechanism of alopecia areata (AA) is still elusive and here we utilized bioinformatics methods to analyze AA-related differentially expressed genes. In this study, GSE45512 and GSE45513 were downloaded from the NCBI sub-database Gene Expression Omnibus (GEO). The gene expressions of AA and normal samples were analyzed using the R package limma, which showed significant differences between AA and normal samples in two species. These genes were subject to functional annotation and protein interaction networks. At the same time, gene set enrichment analysis was conducted for all differentially expressed genes. The study revealed that a total of 225 differentially expressed genes were screened from human AA samples, and a total of 337 differentially expressed genes were screened from spontaneous AA skin samples in C3H/HeJ mice. There are 23 differentially expressed genes in the two species. GO and protein interaction network analysis shown gene enrichment in immune-related functions, and these proteins interact with each other. Gene set enrichment analysis showed that differential genes from both species were significantly enriched to chemokine signaling pathways, cytokine-cytokine receptor interactions, staphylococcus aureus infection, and antigen processing and presentation. Moreover, the human down-regulated differential gene not only maps to the alopecia in human phenotype ontology, but also maps to the pathologically relevant phenotype of the skin appendage. In brief, 23 significant differentially expressed genes were screened out coexisting in AA human and mouse by bioinformatics methods. In addition, the result demonstrated that AA is closely related to the immune process and skin appendage lesions. These results provide new ideas for the diagnosis and treatment of AA.

Phytotherapeutic Interventions using Rodent Models of Hair Loss: Current Research and Future Strategy

Hair follicles are special additions to mammalian skin with intricate differentiation and growth characteristics. They constantly undergo cycles of regression and regeneration throughout the lifespan of animal. Several rodent models have been studied for androgenetic alopecia and alopecia areata in humans with only handful of products approved by United States Food and Drug Administration; topical minoxidil (men and women) and oral finasteride (only men) for hair loss indication. This review summarizes rodent models of human hair loss to emphasize their role in testing traditional medicinal plants as hair growth promoters. Review also discusses the mechanisms of action of various herbs. Currently, both natural and synthetic products are in use to remedy alopecia. Although, use of natural products has shown measurable efficacy, the option is open to test chemically synthesized active fractions of plants as monotherapy or polytherapy to facilitate approval by regulatory agencies. Overall, purpose of this review is to discuss the rodent models of efficacy and how modifications in herbal preparations may bring more alternatives and a better value to the products in future.

IL-12/IL-23 neutralization is ineffective for alopecia areata in mice and humans

Ustekinumab is currently being explored for the treatment of alopecia areata. Here, our work in a murine model and in human patients indicates that neutralization of the IL-12 and IL-23 pathways does not ameliorate disease.

High-throughput T cell receptor sequencing identifies clonally expanded CD8+ T cell populations in alopecia areata

Alopecia areata (AA) is an autoimmune disease in which cytotoxic T cells specifically target growing hair follicles. We used high-throughput TCR sequencing in the C3H/HeJ mouse model of AA and in human AA patients to gain insight into pathogenic T cell populations and their dynamics, which revealed clonal CD8+ T cell expansions in lesional skin. In the C3H/HeJ model, we observed interindividual sharing of TCRβ chain protein sequences, which strongly supports a model of antigenic drive in AA. The overlap between the lesional TCR repertoire and a population of CD8+NKG2D+ T cells in skin-draining lymph nodes identified this subset as pathogenic effectors. In AA patients, treatment with the oral JAK inhibitor tofacitinib resulted in a decrease in clonally expanded CD8+ T cells in the scalp but also revealed that many expanded lesional T cell clones do not completely disappear from either skin or blood during treatment with tofacitinib, which may explain in part the relapse of disease after stopping treatment.

307 Computer-assisted epitope prediction revealed potential autoantigens associated with human alopecia areata

307 Computer-assisted epitope prediction revealed potential autoantigens associated with human alopecia areata