Role of neurogenic inflammation in the pathogenesis of alopecia areata.

Alopecia areata refers to an autoimmune illness indicated by persistent inflammation. The key requirement for alopecia areata occurrence is the disruption of immune-privileged regions within the hair follicles. Recent research has indicated that neuropeptides play a role in the damage to hair follicles by triggering neurogenic inflammation, stimulating mast cells ambient the follicles, and promoting apoptotic processes in keratinocytes. However, the exact pathogenesis of alopecia areata requires further investigation. Recently, there has been an increasing focus on understanding the mechanisms of immune diseases resulting from the interplay between the nervous and the immune system. Neurogenic inflammation due to neuroimmune disorders of the skin system may disrupt the inflammatory microenvironment of the hair follicle, which plays a crucial part in the progression of alopecia areata.

Single-cell transcriptome profiling reveals cell type-specific variation and development in HLA expression of human skin.

Skin, the largest organ of body, is a highly immunogenic tissue with a diverse collection of immune cells. Highly polymorphic human leukocyte antigen (HLA) molecules have a central role in coordinating immune responses as recognition molecules. Nevertheless, HLA gene expression patterns among diverse cell types within a specific organ, like the skin, have yet to be thoroughly investigated, with stromal cells attracting much less attention than immune cells. To illustrate HLA expression profiles across different cell types in the skin, we performed single-cell RNA sequencing (scRNA-seq) analyses on skin datasets, covering adult and fetal skin, and hair follicles as the skin appendages. We revealed the variation in HLA expression between different skin populations by examining normal adult skin datasets. Moreover, we evaluated the potential immunogenicity of multiple skin populations based on the expression of classical HLA class I genes, which were well represented in all cell types. Furthermore, we generated scRNA-seq data of developing skin from fetuses of 15 post conception weeks (PCW), 17 PCW, and 22 PCW, delineating the dynamic expression of HLA genes with cell type-dependent variation among various cell types during development. Notably, the pseudotime trajectory analysis unraveled the significant variance in HLA genes during the evolution of vascular endothelial cells. Moreover, we uncovered the immune-privileged properties of hair follicles at single-cell resolution. Our study presents a comprehensive single-cell transcriptomic landscape of HLA genes in the skin, which provides new insights into variation in HLA molecules and offers a clue for allogeneic skin transplantation.

CD80 on skin stem cells promotes local expansion of regulatory T cells upon injury to orchestrate repair within an inflammatory environment.

Following tissue damage, epithelial stem cells (SCs) are mobilized to enter the wound, where they confront harsh inflammatory environments that can impede their ability to repair the injury. Here, we investigated the mechanisms that protect skin SCs within this inflammatory environment. Characterization of gene expression profiles of hair follicle SCs (HFSCs) that migrated into the wound site revealed activation of an immune-modulatory program, including expression of CD80, major histocompatibility complex class II (MHCII), and CXC motif chemokine ligand 5 (CXCL5). Deletion of CD80 in HFSCs impaired re-epithelialization, reduced accumulation of peripherally generated Treg (pTreg) cells, and increased infiltration of neutrophils in wounded skin. Importantly, similar wound healing defects were also observed in mice lacking pTreg cells. Our findings suggest that upon skin injury, HFSCs establish a temporary protective network by promoting local expansion of Treg cells, thereby enabling re-epithelialization while still kindling inflammation outside this niche until the barrier is restored.

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.

Microbiota and maintenance of skin barrier function.

Human skin forms a protective barrier against the external environment and is our first line of defense against toxic, solar, and pathogenic insults. Our skin also defines our outward appearance, protects our internal tissues and organs, acts as a sensory interface, and prevents dehydration. Crucial to the skin's barrier function is the colonizing microbiota, which provides protection against pathogens, tunes immune responses, and fortifies the epithelium. Here we highlight recent advances in our understanding of how the microbiota mediates multiple facets of skin barrier function. We discuss recent insights into pathological host-microbiota interactions and implications for disorders of the skin and distant organs. Finally, we examine how microbiota-based mechanisms can be targeted to prevent or manage skin disorders and impaired wound healing.

Essential Role of STAT3 Signaling in Hair Follicle Homeostasis.

Dominant-negative mutations associated with signal transducer and activator of transcription 3 (STAT3) signaling, which controls epithelial proliferation in various tissues, lead to atopic dermatitis in hyper IgE syndrome. This dermatitis is thought to be attributed to defects in STAT3 signaling in type 17 helper T cell　specification. However, the role of STAT3 signaling in skin epithelial cells remains unclear. We found that STAT3 signaling in keratinocytes is required to maintain skin homeostasis by negatively controlling the expression of hair follicle-specific keratin genes. These expression patterns correlated with the onset of dermatitis, which was observed in specific pathogen-free conditions but not in germ-free conditions, suggesting the involvement of Toll-like receptor-mediated inflammatory responses. Thus, our study suggests that STAT3-dependent gene expression in keratinocytes plays a critical role in maintaining the homeostasis of skin, which is constantly exposed to microorganisms.

Resident human dermal γδT-cells operate as stress-sentinels: Lessons from the hair follicle.

Murine γδT-cells have stress-surveillance functions and are implicated in autoimmunity. Yet, whether human γδT-cells are also stress sentinels and directly promote autoimmune responses in the skin is unknown. Using a novel (mini-)organ assay, we tested if human dermis resident γδT-cells can recognize stressed human scalp hair follicles (HFs) to promote an alopecia areata (AA)-like autoimmune response. Accordingly, we show that γδT-cells from healthy human scalp skin are activated (CD69+), up-regulate the expression of NKG2D and IFN-γ, and become cytotoxic when co-cultured with autologous stressed HFs ex vivo. These autologous γδT-cells induce HF immune privilege collapse, dystrophy, and premature catagen, i.e. three hallmarks of the human autoimmune HF disorder, AA. This is mediated by CXCL12, MICA, and in part by IFN-γ and CD1d. In conclusion, human dermal γδT-cells exert physiological stress-sentinel functions in human skin, where their excessive activity can promote autoimmunity towards stressed HFs that overexpress CD1d, CXCL12, and/or MICA.

Disruption of the endopeptidase ADAM10-Notch signaling axis leads to skin dysbiosis and innate lymphoid cell-mediated hair follicle destruction.

Hair follicles (HFs) function as hubs for stem cells, immune cells, and commensal microbes, which must be tightly regulated during homeostasis and transient inflammation. Here we found that transmembrane endopeptidase ADAM10 expression in upper HFs was crucial for regulating the skin microbiota and protecting HFs and their stem cell niche from inflammatory destruction. Ablation of the ADAM10-Notch signaling axis impaired the innate epithelial barrier and enabled Corynebacterium species to predominate the microbiome. Dysbiosis triggered group 2 innate lymphoid cell-mediated inflammation in an interleukin-7 (IL-7) receptor-, S1P receptor 1-, and CCR6-dependent manner, leading to pyroptotic cell death of HFs and irreversible alopecia. Double-stranded RNA-induced ablation models indicated that the ADAM10-Notch signaling axis bolsters epithelial innate immunity by promoting ß-defensin-6 expression downstream of type I interferon responses. Thus, ADAM10-Notch signaling axis-mediated regulation of host-microbial symbiosis crucially protects HFs from inflammatory destruction, which has implications for strategies to sustain tissue integrity during chronic inflammation.

Cutaneous Lymphadenoma Is a Distinct Trichoblastoma-like Lymphoepithelial Tumor With Diffuse Androgen Receptor Immunoreactivity, Notch1 Ligand in Reed-Sternberg-like Cells, and Common EGFR Somatic Mutations.

The term "cutaneous lymphadenoma" was coined in this journal for an unusual lymphoepithelial cutaneous adnexal neoplasm, possibly with immature pilosebaceous differentiation. Some authors further proposed that cutaneous lymphadenoma was an adamantinoid trichoblastoma. However, although a hair follicle differentiation is widely accepted, the fact that this is a lymphoepithelial tumor is not appropriately explained by the trichoblastoma hypothesis. Our goal was to further clarify the phenotypic and genotypic features of cutaneous lymphadenoma in a series of 11 cases. Histologically, a lobular architecture surrounded by a dense fibrous stroma was present in all cases. The lobules were composed of epithelial cells admixtured with small lymphocytes and isolated or clustered large Reed-Sternberg-like (RS-L) cells. The epithelial cells were diffusely positive for the hair follicle stem cell markers CK15, PHLDA1, and for androgen receptor. No immunostaining for markers of sebaceous differentiation was found. Intraepithelial lymphocytes were predominantly CD3+, CD4+, FoxP3+ T cells. RS-L cells showed both strong Jagged-1 and Notch1 cytoplasmic immunostaining. Androgen-regulated NKX3.1 nuclear immunostaining was present in a subset of large intralobular cells in all cases. Double immunostaining showed coexpression of NKX3.1 and CD30 in a subset of RS-L cells. No immunostaining for lymphocytic or epithelial markers was present in RS-L cells. EGFR, PIK3CA, and FGFR3 somatic mutations were found by next-generation sequencing in 56% of the cases. We consider that cutaneous lymphadenoma is a distinct benign lymphoepithelial tumor with androgen receptor and hair follicle bulge stem cell marker expression, RS-L cell-derived Notch1 ligand, and common EGFR gene mutations.

T-Cell-Driven Fibroinflammation Inducing Follicular Dedifferentiation in Alopecia Areata and IgG4-Modified Disease.

ABSTRACT: The definition of IgG4-related diseases incorporates a broad range of systemic diseases particularly a subset dominated by fibroinflammation. CD4+cytotoxic T cells have emerged as the major driving force for the fibroinflammation, and the pathogenetic role of IgG4 still remains to be determined. Cutaneous involvement is uncommon and is not well defined as elevated tissue IgG4 plasma cells are not a specific marker and prominent cutaneous fibroinflammation is often absent in cutaneous disease. We report the case of a patient with longstanding alopecia universalis and severe atopic dermatitis who presented with diffuse induration and mottled dyspigmentation of his scalp. Multiple scalp biopsies revealed diffuse interfollicular fibroinflammation and IgG4 plasma cells with induction of distinctive dedifferentiated follicles not seen in alopecia areata. This complex case may provide insight into the role of specific subsets of T cells not only in respect to the fibroinflammation linked to IgG4-related diseases but also the capacity to modify disease, follicular stem cell activation, immune privilege, cytotoxicity in alopecia areata, and the presence of atopy that may have contributed to the pathogenesis of this case.

Argan (Argania Spinosa) press cake extract enhances cell proliferation and prevents oxidative stress and inflammation of human dermal papilla cells.

BACKGROUND: Hair follicle undergoes a growth cycle under the regulation of dermal papilla cells. Due to their enormous roles, these fibroblast cells have been used in various in vitro studies as a screening model to evaluate the effect of hair growth regulating agents. OBJECTIVE: In the current study, we aim to check the hair growth potential effect of Argan press cake (APC) extracted using 50 or 80 % aqueous ethanol on human hair follicle dermal papilla cells (HFDPCs) and to determine the molecular mechanism. METHODS: APC were applied to HFDPCs, then cell proliferation assays, mitochondrial biogenesis assay, and oxidative stress assay were assessed. DNA microarray was performed from the cells treated with our samples and minoxidil. Validation of the results was done using Quantitative Real-Time PCR with primers for hair-growth related genes. GC/MS analysis was used to determine the compounds contained in APC 50 and 80 %. RESULTS: APC enhanced cell proliferation along with the stimulation of the ATP content. Additionally, APC had an anti-oxidant activity against H2O2 mediated oxidative stress preventing dermal papilla cell senescence. Consistent with this, global gene profiling analysis showed an activation of hair growth-related pathway, and a downregulation of inflammation- and oxidative stress-related genes by APC extracts. GC/MS analysis revealed that these extracts contained pure fatty acids, derived sugar chains, and pure compounds including tocopherols, squalene, and spinasterol. CONCLUSION: Taken together, here we showed that APC extracts had an effect on stimulating hair growth while inhibiting the inflammation and the oxidative stress of HFDPCs and thus can potentially contribute to an anti-hair loss drug development.

Induction of alopecia areata in C3H/HeJ mice using cryopreserved lymphocytes.

BACKGROUND: Alopecia areata (AA) is an autoimmune disease resulting in non-scarring hair loss. Animal models are useful means to identify candidates for therapeutic agents. The C3H/HeJ mouse AA model induced via transferring cultured lymphoid cells isolated from AA-affected mice is widely used for AA research. However, this conventional method requires the continuous breeding of AA mice. OBJECTIVE: We aimed to establish a new method to generate AA model using the transfer of cryopreserved cells, which allows the rapid induction of a large number of AA mice when needed. METHODS: We cryopreserved lymph node cells soon after isolation from AA-affected mice and injected thawed-cultured cells into recipient mice. H&E staining, immunohistochemical staining, quantitative real-time PCR and ELISA were conducted to identify pathological characteristics. Flow cytometry was performed to reveal the profile of transferred cells. RESULTS: More than 90 % of recipient mice developed AA-like hair loss and showed inflammatory cell infiltration around anagen hair follicles, markedly increased mRNA expressions of interferon-γ, CXCL11, and granzyme B, and elevated interferon-α protein levels in the skin compared with naïve mice. Higher percentages of effector memory T cells and dendritic cells in transferred cells resulted in a higher incidence of AA. CONCLUSION: This is the first report to establish a method for generating AA mice using cryopreserved lymphocytes. These AA mice have similar pathological characteristics to AA mice generated with the conventional method and AA patients. This convenient and reproducible method is expected to be valuable for AA study.

High-fat diet induces a predisposition to follicular hyperkeratosis and neutrophilic folliculitis in mice.

BACKGROUND: Neutrophilic folliculitis is an inflammatory condition of hair follicles. In some neutrophilic folliculitis, such as in patients with acne and hidradenitis suppurativa, follicular hyperkeratosis is also observed. Neutrophilic folliculitis is often induced and/or exacerbated by a high-fat diet (HFD). However, the molecular mechanisms by which an HFD affects neutrophilic folliculitis are not fully understood. OBJECTIVE: Our aim was to elucidate how an HFD promotes the development of neutrophilic folliculitis. METHODS: Mice were fed an HFD, and their skin was subjected to histologic, RNA sequencing, and imaging mass spectrometry analyses. To examine the effect of an HFD on neutrophil accumulation around the hair follicles, phorbol 12-myristate 13-acetate (PMA) was used as an irritant to the skin. RESULTS: Histologic analysis revealed follicular hyperkeratosis in the skin of HFD-fed mice. RNA sequencing analysis showed that genes related to keratinization, especially in upper hair follicular keratinocytes, were significantly upregulated in HFD-fed mice. Application of PMA to the skin induced neutrophilic folliculitis in HFD-fed mice but not in mice fed a normal diet. Accumulation of neutrophils in the skin and around hair follicles was dependent on CXCR2 signaling, and CXCL1 (a CXCR2 ligand) was produced mainly by hair follicular keratinocytes. Imaging mass spectrometry analysis revealed an increase in fatty acids in the skin of HFD-fed mice. Application of these fatty acids to the skin induced follicular hyperkeratosis and caused PMA-induced neutrophilic folliculitis even in mice fed a normal diet. CONCLUSION: An HFD can facilitate the development of neutrophilic folliculitis with the induction of hyperkeratosis of hair follicles and increased neutrophil infiltration around the hair follicles via CXCR2 signaling.

CCL13 is upregulated in alopecia areata lesions and is correlated with disease severity.

Alopecia areata (AA) is a multi-factors disease characterized by non-scarring hair loss. AA could be classified into three main clinical phenotypes including patchy type AA (AAP), alopecia totalis (AT) and alopecia universalis (AU) based on the severity and areas of hair loss. Recent studies suggested immunological factor was critical in AA, but the precise aetiology and pathogenesis of AA still need exploration. In the work, we screened two gene expression profiles (GSE45512 and GSE68801) from Gene Expression Omnibus (GEO). Based on the two data sets, 10 upregulated genes and 107 downregulated genes in AA skin biopsies were identified. CCL13, as one of the remarkably upregulated genes, was found to have potential biological functions in aberrant immune response of AA according to the GO and KEGG analyses. The PPI network showed CCL13 was associated with multiple immune-related genes. The expression of CCL13 was increased depending on the severity of disease in AA patients. Cytotoxic lymphocytes, T cells and myeloid dendritic cells accumulated remarkably in scalp tissue depending on the severity of AA, and CCL13 was significantly correlated to cytotoxic lymphocytes, T cells and myeloid dendritic cells in AA patients. Our RT-PCR and ELISA results found CCL13 was upregulated in skin biopsy and serum of AA patients, and the immunohistochemistry (IHC) detection showed CCL13 was expressed by both the hair follicle epithelium and infiltrating immune cells. In conclusion, the upregulated of CCL13 and subsequent immune cell infiltration was related to AA, which could be a promising target for diagnosis and therapy in AA patients.

The PD-1/PD-L1 pathway in murine hair cycle transition: a potential anagen phase regulator.

Programmed cell death protein-1 (PD-1) is primarily recognized as an inhibitory receptor involved in the regulation of immunological tolerance. However, recent studies have indicated that PD-1/PD-L1 signaling could also regulate the functions of nonimmune cells and may be involved in regulating hair biology. In this study, we showed in a mouse model of depilation-induced hair cycling that PD-1/PD-L1 are expressed in the murine epidermis and hair follicle (HF) in a hair cycle-dependent manner. During HF morphogenesis, PD-1 expression was strongly decreased during the anagen phase compared with the catagen and telogen phases. PD-L1 expression was enhanced during the catagen phase compared with the anagen and telogen phases. Moreover, direct blockade of PD-L1 not only accelerated hair anagen phase onset but also delayed catagen progression. In conclusion, our findings indicated that PD-1/PD-L1 signaling may act as a negative regulator of hair cycle transition. Anti-PD-1/PD-L1 therapy may thus be a promising strategy for treating anagen-reduced hair loss.

Evaluation of platelet-rich plasma on hair regrowth and lesional T-cell cytokine expression in alopecia areata: A randomized observer-blinded, placebo-controlled, split-head pilot study.

BACKGROUND: Platelet-rich plasma has shown some promise in the treatment of alopecia areata. OBJECTIVE: To evaluate the effect of platelet-rich plasma on hair regrowth and lesional T-cell cytokine expression in alopecia areata. METHODS: This was a randomized, placebo-controlled, split-head study involving 27 patients with alopecia areata (Severity of Alopecia Tool score ≥25%). Alopecia patches on either side of the scalp were randomized to receive 3 intradermal injections of platelet-rich plasma or normal saline at monthly intervals and evaluated 3 months after the last session. Lesional T-cell cytokine messenger RNA expression was compared pre- and posttreatment in the platelet-rich plasma-treated sites. RESULTS: The mean Severity of Alopecia Tool score did not change significantly compared with baseline with either platelet-rich plasma or placebo injections at any visit; however, the mean percentage reduction in the score in the platelet-rich plasma arm was more than in the placebo arm (9.05% ± 36.48% vs 4.99% ± 33.88%; P = .049) at final assessment. The mean interferon gamma (P = .001) and interleukin 17 cytokine (P = .009) messenger RNA expression decreased, whereas the mean interleukin 10 (P = .049) and FOXP3 (P = .011) messenger RNA expression increased significantly after platelet-rich plasma treatment. LIMITATIONS: Small sample size and a relatively short follow-up. CONCLUSION: Platelet-rich plasma was found to have limited efficacy in alopecia areata. However, it may play a role in restoring immune balance in the alopecic patches.

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

BACKGROUND: It 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. OBJECTIVE: To investigate whether abnormal activities of γδT-cells may be involved in AA pathogenesis. METHODS: We 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. RESULTS: In 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. CONCLUSION: Our 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.