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.

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.

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.

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.

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.

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.

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.

Hair follicle immune privilege and its collapse in alopecia areata.

Anagen stage hair follicles (HFs) exhibit "immune privilege (IP)" from the level of the bulge downwards to the bulb. Both passive and active IP mechanisms protect HFs from physiologically undesired immune responses and limit immune surveillance. IP is relative, not absolute, and is primarily based on absent, or greatly reduced, intra-follicular antigen presentation via MHC class I and II molecules, along with prominent expression of "no danger" signals like CD200 and the creation of an immunoinhibitory signalling milieu generated by the secretory activities of HFs. Perifollicular mast cells, Tregs and other immunocytes may also contribute to HF IP maintenance in healthy human skin. Collapse of anagen hair bulb IP is an essential prerequisite for the development of alopecia areata (AA). In AA, lesional HFs are rapidly infiltrated by NKG2D + T cells and natural killer (NK) cells, while perifollicular mast cells acquire a profoundly pro-inflammatory phenotype and interact with autoreactive CD8+ T cells. Using animal models, significant functional evidence has accumulated that demonstrates the dominance of the immune system in AA pathogenesis. Purified CD8+T-cell and NK cell populations alone, which secrete fγ, suffice to induce the AA phenotype, while CD4+T-cells aggravate it, and Tregs and iNKT cells may provide relative protection against AA development. While IP collapse may be induced by exogenous agents, inherent IP deficiencies might confer increased susceptibility to AA for some individuals. Thus, a key goal for effective AA management is the re-establishment of a functional HF IP, which will also provide superior protection from disease relapse.

The Effect of JAK Inhibitor on the Survival, Anagen Re-Entry, and Hair Follicle Immune Privilege Restoration in Human Dermal Papilla Cells.

Topical or systemic administration of JAK inhibitors has been shown to be a new treatment modality for severe alopecia areata (AA). Some patients show a good response to JAK inhibitors, but frequently relapse after cessation of the treatment. There have been no guidelines about the indications and use of JAK inhibitors in treating AA. The basic pathomechanism of AA and the relevant role of JAK inhibitors should support how to efficiently use JAK inhibitors. We sought to investigate the effect of JAK1/2 inhibitor on an in vitro model of AA and to examine the possible mechanisms. We used interferon gamma-pretreated human dermal papilla cells (hDPCs) as an in vitro model of AA. Ruxolitinib was administered to the hDPCs, and cell viability was assessed. The change of expression of the Wnt/ß-catenin pathway, molecules related to the JAK-STAT pathway, and growth factors in ruxolitinib-treated hDPCs was also examined by reverse transcription PCR and Western blot assay. We examined immune-privilege-related molecules by immunohistochemistry in hair-follicle culture models. Ruxolitinib did not affect the cell viability of the hDPCs. Ruxolitinib activated several molecules in the Wnt/ß-catenin signaling pathway, including Lef1 and ß-catenin, and suppressed the transcription of DKK1 in hDPCs, but not its translation. Ruxolitinib reverted IFN-γ-induced expression of caspase-1, IL-1ß, IL-15, and IL-18, and stimulated several growth factors, such as FGF7. Ruxolitinib suppressed the phosphorylation of JAK1, JAK2 and JAK3, and STAT1 and 3 compared to IFN-γ pretreated hDPCs. Ruxolitinib pretreatment showed a protective effect on IFN-γ-induced expression of MHC-class II molecules in cultured hair follicles. In conclusion, ruxolitinib modulated and reverted the interferon-induced inflammatory changes by blocking the JAK-STAT pathway in hDPCs under an AA-like environment. Ruxolitinib directly stimulated anagen-re-entry signals in hDPCs by affecting the Wnt/ß-catenin pathway and promoting growth factors in hDPCs. Ruxolitinib treatment prevented IFN-γ-induced collapse of hair-follicle immune privilege.

Alopecia areata susceptibility variant in MHC region impacts expressions of genes contributing to hair keratinization and is involved in hair loss.

BACKGROUND: Alopecia areata (AA) is considered a highly heritable, T-cell-mediated autoimmune disease of the hair follicle. However, no convincing susceptibility gene has yet been pinpointed in the major histocompatibility complex (MHC), a genome region known to be associated with AA as compared to other regions. METHODS: We engineered mice carrying AA risk allele identified by haplotype sequencing for the MHC region using allele-specific genome editing with the CRISPR/Cas9 system. Finally, we performed functional evaluations in the mice and AA patients with and without the risk allele. FINDINGS: We identified a variant (rs142986308, p.Arg587Trp) in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene as the only non-synonymous variant in the AA risk haplotype. Furthermore, mice engineered to carry the risk allele displayed a hair loss phenotype. Transcriptomics further identified CCHCR1 as a novel component interacting with hair cortex keratin in hair shafts. Both, these alopecic mice and AA patients with the risk allele displayed morphologically impaired hair and comparable differential expression of hair-related genes, including hair keratin and keratin-associated proteins (KRTAPs). INTERPRETATION: Our results implicate CCHCR1 with the risk allele in a previously unidentified subtype of AA based on aberrant keratinization in addition to autoimmune events. FUNDING: This work was supported by JSPS KAKENHI (JP16K10177) and the NIHR UCLH Biomedical Research center (BRC84/CN/SB/5984).