Mouse Model Of Alopecia Areata Research Articles

MiR-199a-3p Regulates the PTPRF/β-Catenin Axis in Hair Follicle Development: Insights into the Pathogenic Mechanism of Alopecia Areata.

Alopecia areata is an autoimmune disease characterized by the immune system attacking self hair follicles, mainly in the scalp. There is no complete cure, and the pathogenesis is still not fully understood. Here, sequencing of skin tissues collected from 1-month-old coarse- and fine-wool lambs identified miR-199a-3p as the only small RNA significantly overexpressed in the fine-wool group, suggesting a role in hair follicle development. MiR-199a-3p expression was concentrated in the dermal papillae cells of sheep hair follicles, along with enhanced β-catenin expression and the inhibition of PTPRF protein expression. We also successfully constructed a mouse model of alopecia areata by intracutaneous injection with an miR-199a-3p antagomir. Injection of the miR-199a-3p agomir resulted in hair growth and earlier anagen entry. Conversely, local injection with the miR-199a-3p antagomir resulted in suppressed hair growth at the injection site, upregulation of immune system-related genes, and downregulation of hair follicle development-related genes. In vivo and in vitro analyses demonstrated that miR-199a-3p regulates hair follicle development through the PTPRF/β-catenin axis. In conclusion, a mouse model of alopecia areata was successfully established by downregulation of a small RNA, suggesting the potential value of miR-199a-3p in the study of alopecia diseases. The regulatory role of miR-199a-3p in the PTPRF/β-catenin axis was confirmed, further demonstrating the link between alopecia areata and the Wnt-signaling pathway.

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.

Generation of autoreactive CD8 T cell in a mouse model of alopecia areata

Abstract Alopecia areata (AA) is the most frequent form of an organ-specific autoimmune disease in humans mediated by NKG2D +CD8 T cells destroying hair follicles. C3H/HeJ mice naturally develop AA similar to humans, and we found it accompanies the expansion of exhausted phenotype (CD101 +Tim3 +) memory CD8 T cells not only in the skin and skin-draining lymph nodes (SDLNs), but also in the liver and lungs. Before the disease induction, we found these CD8 T cells are activated in the lungs and liver, suggesting they are pre-activated in these organs. In the skin, expanded CD8 T cells additionally upregulated CD39, PD-1, and CTLA-4, which produced less IFNg than those in the SDLN, liver and lungs when stimulated. These features suggest that autoreactive CD8 T cells in the skin are exhausted by chronic antigenic stimulation. However, they did not upregulate TOX, a hallmark of T cell exhaustion. Intriguingly, CD44 highNKG2D-expressing CD8 T cells appeared in the thymi of aged C3H/HeJ mice before disease induction. These cells express TBET and EOMES without overt expansion of PLZF-expressing T cells. Collectively these features suggest AA is mediated by autoreactive activation CD8 T cells that are not explained by previously known mechanisms. This research was supported by the National Research Foundation of Korea (NRF-2022R1A2C1007692)

1108 Comparison of ruxolitinib and a collagen-targeted PTH-based hair cycle stimulant on hair follicle counts and hair growth in the C3H/HeJ engrafted mouse model of alopecia areata

1108 Comparison of ruxolitinib and a collagen-targeted PTH-based hair cycle stimulant on hair follicle counts and hair growth in the C3H/HeJ engrafted mouse model of alopecia areata

Selective Janus kinase 1 inhibition resolves inflammation and restores hair growth offering a viable treatment option for alopecia areata.

Janus Kinase (JAK) inhibition has recently demonstrated therapeutic efficacy in both restoring hair growth and resolving inflammation in Alopecia Areata (AA). These effects are dose dependent and mainly efficacious at ranges close to a questionable risk profile. We explored the possibility to separate the beneficial and adverse effects of JAK inhibition by selectively inhibiting JAK1 and thereby avoiding side effects associated with JAK2 blockade. The C3H/HeJ mouse model of AA was used to demonstrate therapeutic efficacy in vivo with different regimens of a selection of JAK inhibitors in regards to systemic versus local drug exposure. Human peripheral blood lymphocytes were stimulated in vitro to demonstrate translation to the human situation. We demonstrate that selective inhibition of JAK1 produces fast resolution of inflammation and complete restoration of hair growth in the C3H/HeJ mouse model of AA. Furthermore, we show that topical treatment does not restore hair growth and that treatment needs to be extended well beyond that of restored hair growth in order to reach treatment-free remission. For translatability to human disease, we show that cytokines involved in AA pathogenesis are similarly inhibited by selective JAK1 and pan-JAK inhibition in stimulated human peripheral lymphocytes and specifically in CD8+ T cells. This study demonstrates that systemic exposure is required for efficacy in AA and we propose that a selective JAK1 inhibitor will offer a treatment option with a superior safety profile to pan-JAK inhibitors for these patients.

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.

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

Alopecia areata (AA) is one of the most prevalent autoimmune diseases, yet the development of innovative therapeutic strategies has been hampered by an incomplete understanding of disease pathogenesis, such as the complex immunological mechanisms that underlie AA pathogenesis. Here, we performed single-cell RNA sequencing (scRNAseq) of skin-infiltrating immune cells from the well-established graft-induced C3H/HeJ mouse model of AA, together with antibody-based depletion experiments to systematically interrogate the role of specific immune cell types in disease pathophysiology in vivo.

829 Cytotoxic T lymphocytes target henle’s layer in alopecia areata

Autoimmune disease occurs when a specific adaptive immune response is mounted against self-antigen(s) and immune-mediated tissue damage is sustained until the self-antigen is eliminated. We recently identified immunogenic cell death via necroptosis in the hair follicle (HF) epithelium as the primary initiating mechanism in alopecia areata (AA). Here, we aimed to identify the major target cells of T cell-mediated cytotoxicity in AA, after the autoimmune response was initiated. Using the C3H/HeJ mouse model of AA, we first established a spatiotemporal map of caspase-8-driven immune-mediated apoptosis in HF epithelial cells during premature HF regression in AA.

139 Pathogenic CD8+ T cell infiltration and immune synapse formation in alopecia areata hair follicles

Alopecia Areata (AA) involves CD8+ NKG2D+ T cell-mediated hair follicle (HF) damage that causes hair loss. Despite the identification of CD8+ T cells as primary pathogenic cells involved in AA, the molecular mechanisms used by these cells to kill their target cells within HF are not known. CD8+ T cells form immune synapses (IS) to contact, recognize and destroy their target cells, therefore we postulated that CD8+ T cells employ a cytolytic IS for target cell killing in AA. Here, we aimed to directly visualize the interactions of CD8+ T cells with HF target cells in a C3H/HeJ mouse model of AA using high-resolution confocal microscopy.

567 Gut dysbiosis is associated with the development of alopecia areata

Alopecia areata (AA) is a highly prevalent autoimmune disease (AD) leading to hair loss in affected individuals, in which both genetic and environmental factors likely play a role. Associations between changes in the microbiome composition and many ADs have been reported, however, a characterization of the role of gut microbiota in the development of AA has not been undertaken. To investigate the functional relevance of gut microbiota to AA development in vivo, we depleted the gut microbiome in the C3H/HeJ mouse model of AA with broad-spectrum antibiotics.

040 The C3H/HeJ mouse model of alopecia areata as a resource for training graduate and undergraduate students in clinical laboratory research techniques

Alopecia areata (AA) is a cell-mediated autoimmune disease that targets actively growing hair follicles in both humans and mice, resulting in hair loss [1]. Alopecia Areata occurs naturally in about 20% of C3H/HeJ mice by 12 months of age [2]. Transplantation of skin or cells from the lymph nodes of affected animals into younger, histocompatible C3H/HeJ mice will reliably produce AA symptoms within 10-20 weeks of transplant [2, 3] providing both a useful tool for investigating the underlying mechanisms of AA and a preclinical model for drug testing.

Increased proliferation of epidermal gamma delta T cells and expression of the transmembrane protein, BST2, in Alopecia areata

Abstract Alopecia areata is an autoimmune disease characterized by patchy non-scarring hair loss in affected individuals. Typically, research has focused on hair follicle infiltration and disruption via CD8+ and CD4+ T cells. Our research attempts to elucidate the role of resident epidermal γδ T cells in disease. Analysis of publicly available bioinformatics data shows a statistically significant downregulation in a number of immune-related genes after patients were treated with a JAK inhibitor, Tofacitinib. We have focused on one of these proteins, bone marrow stromal antigen 2 (BST2), in vitro and in vivo using the C3H mouse model of alopecia areata. We have identified populations of epidermal γδ T cells expressing BST2 in the follicle using immunofluorescence microscopy. In addition, BST2 expression is upregulated in unaffected skin from mice with active alopecia areata suggesting BST2 expression may precede loss of hair follicle immune privilege. Furthermore, epidermal γδ T cell number is increased in skin affected by alopecia areata. Together our findings suggest that BST2 is expressed by epidermal T cells prior to hair loss and is followed by an increase in epidermal γδ T cell number. Our lab is focused on how these changes in epidermal γδ T cell number and BST2 expression impacts disease. Supported by grants from NIH Supported by grants from NSF

Altered T cell subpopulations and serum anti-TYRP2 and tyrosinase antibodies in the acute and chronic phase of alopecia areata in the C3H/HeJ mouse model

BackgroundC3H/HeJ mouse models progress gradually in hair loss from acute to chronic phase and reflect the symptoms of patients with alopecia areata (AA). However, the underlying pathological characteristics alteration associated with disease progression and autoantigens remain unclear. ObjectiveWe aimed at elucidating the pathological differences between acute and chronic-AA in the C3H/HeJ mouse model. MethodsWe analyzed populations of PBMCs, skin-draining lymph node (SDLN) cells, and cutaneous cells of AA mice using flow cytometry. The cytokine and chemokine expressions in the serum and skin were determined using multiplex assay and qPCR. The antibody serum levels were determined using ELISA and the antigen-specific T cells were detected using the MHC class I tetramer. ResultsThe CD8+NKG2D+ T and CD8+ TEM cell percentage in the chronic-AA SDLNs or among the unaffected and acute-AA mice PBMCs increased. The Th1 and CD4+ TEM cell percentage in the SDLNs and among PBMCs increased in the unaffected and AA mice. The percentage of CD8+ TEM/TRM cells and MHC class I expression increased in the lesions of acute-AA or the non-lesions and lesions of chronic-AA. The Th1 cells, dendritic cell-related cytokines, CD11c+ cells and MHC class II expression increased in the skin of AA mice. The antibody levels and TYRP2 and tyrosinase-specific CD8+ T cell percentages were upregulated in AA mice. ConclusionThese results suggest that the CD8+ and CD4+ T cell subpopulations, cytokine and chemokine expressions differ between the disease phases. Moreover, TYRP2 and tyrosinase are potential autoreactive targets in the AA mouse model.

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.

Myeloid Adherent Cells Are Involved in Hair Loss in the Alopecia Areata Mouse Model.

Alopecia areata (AA), which is defined as an autoimmune hair loss disease, has a serious impact on the quality of life for patients with AA worldwide. In this study, to our knowledge, a previously unreported method of AA induction in C3H mice has been established and validated. Using this method, we showed that dermal injection of 1-3 million of a mixture of skin cells freshly isolated from AA-affected skin induces AA in more than 80% of healthy mice. Contrary to the previous protocol, the induction of AA by this approach does not need any surgical AA skin grafting, cell manipulation, or high number of activated T cells. We also showed that dermal injection of adherent myeloid cells (mainly CD11b+) in healthy mice is as potent as a mixture of none adherent CD3+ T cells and CD19+ B cells in the induction of AA. Interestingly, most of the mice (7 out of 8) that received non-adherent cells developed AA universalis, whereas most of the mice (5 out of 7) that received adherent cells developed patchy AA. Finally, we found a high number of stage-specific embryonic antigen-expressing cells whose expression in monocytes in an inflammatory disease causes the release of inflammatory cytokines, TNF-α and IL-1β, from these cells in AA-affected skin.

Increase in IL-15 levels in patients with alopecia areata

Introduction and purpose: Alopecia areata (AA) is a condition that causes non-scarring hair loss (often with acute onset). Alopecia areata in the population occurs in 0.1-0.2% of people, with a similar frequency in men and women. Alopecia areata is an example of an autoimmune disease of the hair follicle. Hair loss in alopecia areata is caused by lymphocytic infiltration around the hair follicle and IFN-γ. IgG antibodies against hair follicle cells are also found in people suffering from alopecia areata. Recent studies have shown a significant increase in IL-15 in AA. The aim of the study was to review the current knowledge on the use of IL-15 in the treatment of alopecia areata.A brief description of the state of knowledge: Interleukin-15 (IL-15) is a pleiotropic cytokine that exhibits multidirectional biological effects on various cell types. It affects the functions of the immune system, both innate and acquired, and therefore plays an important role in inflammation and during the immune response to infections and infestations. In the AA mouse model, antibody-mediated blockade of IFN-γ, interleukin-2 (IL-2), or interleukin-15 receptor β (IL-15Rβ) prevented disease progression by minimizing the accumulation of CD8 (+) NKG2D (+) T cells in the skin and reducing the cutaneous IFN response. The concentration of IL-15 in patients with alopecia areata was significantly higher than in the control group. Moreover, the concentration of IL-15 increased in direct proportion to the area of alopecia, the highest value in patients with total alopecia.For this reason, it is important to search for new medical treatments that will enable patients to stay physically healthy, and what is equally important, to remain mental health. Conclusions: In addition, studies have shown an increase in IL-15 levels in patients with alopecia areata, which correlated with the duration of the disease. However, too few studies conducted so far do not allow conclusions to be drawn regarding the use of IL-15 as a therapeutic point.

Toward Predicting the Spatio-Temporal Dynamics of Alopecia Areata Lesions Using Partial Differential Equation Analysis.

Hair loss in the autoimmune disease, alopecia areata (AA), is characterized by the appearance of circularly spreading alopecic lesions in seemingly healthy skin. The distinct spatial patterns of AA lesions form because the immune system attacks hair follicle cells that are in the process of producing hair shaft, catapults the mini-organs that produce hair from a state of growth (anagen) into an apoptosis-driven regression state (catagen), and causes major hair follicle dystrophy along with rapid hair shaft shedding. In this paper, we develop a model of partial differential equations (PDEs) to describe the spatio-temporal dynamics of immune system components that clinical and experimental studies show are primarily involved in the disease development. Global linear stability analysis reveals there is a most unstable mode giving rise to a pattern. The most unstable mode indicates a spatial scale consistent with results of the humanized AA mouse model of Gilhar et al. (Autoimmun Rev 15(7):726-735, 2016) for experimentally induced AA lesions. Numerical simulations of the PDE system confirm our analytic findings and illustrate the formation of a pattern that is characteristic of the spatio-temporal AA dynamics. We apply marginal linear stability analysis to examine and predict the pattern propagation.

098 Simvastatin decreases AA-associated inflammation through effects on isoprenoid metabolites

Alopecia areata (AA) is a T-cell-mediated disorder in which lymphocytic infiltrate attacks and damages the hair follicle during the anagen phase of growth, leading to varying patterns of hair loss. At present, there is no treatment approved by the FDA for AA, and those treatments currently in use are associated with worrying side effects, prohibitive expense, or low rates of response. Previously, we have demonstrated the effectiveness of the lipid-lowering treatments simvastatin and ezetimibe in restoring T regulatory cell levels and hair growth in a mouse model of AA. Here, we aim to elucidate the mechanisms by which simvastatin exerts its effects on the autoimmune condition. Hair re-growth in the mouse model was associated with reduction of inflammatory markers in the skin, as well as reduced activation of STAT1 and reduced expression of NKG2D in skin-draining lymph nodes. These changes corresponded to a significant decrease in serum cholesterol and triglycerides in mice who responded with complete hair re-growth. Simvastatin was similarly able to decrease in vitro proliferation of both the IL-2-dependent cytotoxic T cell line CTLL-2 and primary mouse CD4+ and CD8+ T lymphocytes. This effect was shown to be at least partially mediated through the statin’s effects on isoprenoid metabolite production, as the addition of exogenous farnesyl pyrophosphate and geranyl geranyl pyrophosphate restored the proliferative ability of statin-treated CTLL-2 cells. Taken together with previous findings, these results indicate an immediate effect of simvastatin on the lymphocytic subpopulations responsible for hair follicle damage in AA through disruption of isoprenylation.

085 Autoantigen screening in C3H/HeJ mouse model of alopecia areata revealed high antigenicity of melanocyte-associated antigen epitopes

085 Autoantigen screening in C3H/HeJ mouse model of alopecia areata revealed high antigenicity of melanocyte-associated antigen epitopes

A randomized placebo-controlled single-center pilot study of the safety and efficacy of apremilast in subjects with moderate-to-severe alopecia areata.

Alopecia areata (AA) is a common autoimmune disease that results in non-scarring hair loss. AA pathogenesis is thought to involve multiple inflammatory cytokines. Apremilast is a phosphodiesterase 4 (PDE4) inhibitor that reduces pro-inflammatory cytokine production. Recent studies demonstrate upregulation of PDE4 in human scalp lesions of AA patients and hair regrowth in a humanized AA mouse model upon apremilast treatment, suggesting a possible potential of apremilast in AA. To assess the efficacy and safety of apremilast in AA, we conducted a double-blind, placebo-controlled single-center pilot study in 30 moderate-to-severe AA patients (≥ 50% scalp involvement) that were randomized 2:1 to receive apremilast (n = 20) or placebo (n = 10) orally for 24weeks. The primary endpoint was the percentage of patients achieving 50% reduction in severity of alopecia tool (SALT) score (SALT50) at 24weeks compared to baseline, and the secondary endpoints included the percent change in SALT score at weeks 24 and 48. Eight patients in the apremilast arm withdrew prior to week 24 along with two patients in the placebo group, mostly due to lack of efficacy and adverse events. At 24weeks, only 1 of 12 apremilast-treated subjects achieved SALT50, and similarly 1 of 8 placebo-treated subjects achieved SALT50. The difference between the mean percent improvement in SALT score at week 24 compared to baseline of the two study arms was not statistically significant (p = 0.38). The lack of treatment response in most of our patients argues against a pathogenic role for PDE4 specifically in moderate-to-severe AA, but targeting this pathway may still be of value in patients with mild AA as there is less of an inflammatory burden in this population. However, future larger studies may be needed to conclude apremilast's lack of efficacy in moderate-to-severe AA.