RESUMO
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.
Assuntos
Alopecia em Áreas , Folículo Piloso , Privilégio Imunológico , Interferon gama , Interleucina-15 , Interleucina-15/metabolismo , Interleucina-15/imunologia , Folículo Piloso/imunologia , Folículo Piloso/metabolismo , Humanos , Animais , Alopecia em Áreas/imunologia , Alopecia em Áreas/metabolismo , Camundongos , Interferon gama/metabolismo , Feminino , Receptores de Interleucina-15/metabolismo , Receptores de Interleucina-15/imunologia , Masculino , Adulto , Pessoa de Meia-Idade , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Subunidade alfa de Receptor de Interleucina-15/imunologia , Pele/imunologia , Pele/metabolismo , Pele/patologia , Modelos Animais de DoençasRESUMO
OBJECTIVE: Skin adhesives offer many advantages over traditional wound-closure devices. Recently, the current research group reported on tissue adhesives composed of natural polymers (gelatin and alginate), which are biocompatible with mechanical properties suitable for tissue adhesion. The objective of the present study was to conduct clinical and histologic assessment of this hemostatic bioadhesive in the healing of long skin incisions (≥4 cm) in comparison with traditional and commercially available methods. METHODS: Researchers created 24 long incisions on the ventral side of two domestic pigs to compare four different treatment modalities: two topical bioadhesives based on gelatin and alginate combined with the hemostatic agent kaolin, nylon sutures, and commercial tissue adhesive N-butyl-2-cyanoacrylate. The bioadhesive compounds were spread on the incision surface and then mixed either manually or with a double-headed syringe. After 14 days, clinical and histologic measurements were performed to evaluate the healing phase of the wounds. RESULTS: The bioadhesive formulation that contained a relatively low crosslinker concentration demonstrated superior results to the formulation that contained a standard crosslinker concentration. However, no significant statistical differences were observed compared with the control incisions (sutures and commercial adhesive N-butyl-2-cyanoacrylate). This was verified by immunohistochemical analysis for epithelial integrity and scar formation as well as by clinical assessment. CONCLUSIONS: This newly developed bioadhesive demonstrated suitable properties for the closure of long incisions in a porcine skin model.
Assuntos
Embucrilato , Hemostáticos , Ferida Cirúrgica , Adesivos Teciduais , Suínos , Animais , Hemostáticos/farmacologia , Hemostáticos/uso terapêutico , Adesivos Teciduais/farmacologia , Adesivos Teciduais/uso terapêutico , Gelatina , AlginatosRESUMO
Here, we have explored the involvement of innate lymphoid cells-type 1 (ILC1) in the pathogenesis of alopecia areata (AA), because we found them to be significantly increased around lesional and non-lesional HFs of AA patients. To further explore these unexpected findings, we first co-cultured autologous circulating ILC1-like cells (ILC1lc) with healthy, but stressed, organ-cultured human scalp hair follicles (HFs). ILClc induced all hallmarks of AA ex vivo: they significantly promoted premature, apoptosis-driven HF regression (catagen), HF cytotoxicity/dystrophy, and most important for AA pathogenesis, the collapse of the HFs physiological immune privilege. NKG2D-blocking or IFNγ-neutralizing antibodies antagonized this. In vivo, intradermal injection of autologous activated, NKG2D+/IFNγ-secreting ILC1lc into healthy human scalp skin xenotransplanted onto SCID/beige mice sufficed to rapidly induce characteristic AA lesions. This provides the first evidence that ILC1lc, which are positive for the ILC1 phenotype and negative for the classical NK markers, suffice to induce AA in previously healthy human HFs ex vivo and in vivo, and further questions the conventional wisdom that AA is always an autoantigen-dependent, CD8 +T cell-driven autoimmune disease.
Assuntos
Alopecia em Áreas , Camundongos , Animais , Humanos , Alopecia em Áreas/patologia , Autoimunidade , Imunidade Inata , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Linfócitos/patologia , Camundongos SCID , Folículo PilosoRESUMO
BACKGROUND: The key signals that suffice to induce atopic dermatitis (AD) in human skin remain incompletely understood. Also, current mouse models reflect human AD only unsatisfactorily. Therefore, we have asked whether a humanized AD mouse model can be developed that reflects human AD more faithfully and permit to identify key signals that suffice to induce AD lesions in previously healthy human skin in vivo. METHODS: Healthy human skin from non-atopic donors was transplanted onto SCID/beige mice. After xenotransplant reinnervation by mouse sensory nerve fibers had occurred, mixed autologous human Th2 CD4+ and Tc2 CD8+ T cells that had been pretreated in vitro with IL-2, IL-4, and LPS were injected intradermally into the xenotransplants without skin barrier disruption. RESULTS: Injected non-atopic xenotransplants rapidly developed a morphological, functional, and immunological phenocopy of human AD lesions regarding skin barrier defects, immunopathology including intraepidermal eosinophils, mast cell activation, increase of thymic stromal lymphopoietin, eotaxin-1 and type 2 cytokine circuits, and even showed characteristic neuroimmunological abnormalities such as ß2-adrenergic receptor downregulation. The experimentally induced AD lesions in human skin responded to standard AD therapy (topical dexamethasone or tacrolimus; systemic anti-IL-4Rα antibody [dupilumab]), and relapsed when neurogenic skin inflammation was induced by exposing mice to perceived stress. CONCLUSIONS: This new animal model uniquely mimics the complexity of human AD and its clinical response to standard therapy and psychoemotional stressors in vivo, and shows that Th2-polarized lymphocytes associated with excessive IL-4Rα-mediated signaling suffice to induce human AD skin lesions, while atopy and epidermal barrier disruption are dispensable.
Assuntos
Dermatite Atópica , Humanos , Camundongos , Animais , Camundongos SCID , Pele/patologia , Citocinas/metabolismo , Linfócitos T CD8-PositivosRESUMO
Transplanting aged human skin onto young SCID/beige mice morphologically rejuvenates the xenotransplants. This is accompanied by angiogenesis, epidermal repigmentation, and substantial improvements in key aging-associated biomarkers, including ß-galactosidase, p16ink4a, SIRT1, PGC1α, collagen 17A, and MMP1. Angiogenesis- and hypoxia-related pathways, namely, vascular endothelial growth factor A (VEGF-A) and HIF1A, are most up-regulated in rejuvenated human skin. This rejuvenation cascade, which can be prevented by VEGF-A-neutralizing antibodies, appears to be initiated by murine VEGF-A, which then up-regulates VEGF-A expression/secretion within aged human skin. While intradermally injected VEGF-loaded nanoparticles suffice to induce a molecular rejuvenation signature in aged human skin on old mice, VEGF-A treatment improves key aging parameters also in isolated, organ-cultured aged human skin, i.e., in the absence of functional skin vasculature, neural, or murine host inputs. This identifies VEGF-A as the first pharmacologically pliable master pathway for human organ rejuvenation in vivo and demonstrates the potential of our humanized mouse model for clinically relevant aging research.
RESUMO
BACKGROUND: Psoriasis is characterized by aberrant activation of several pro-inflammatory circuits as well as abnormal hyperproliferation and dysregulated apoptosis of keratinocytes (KCs). Most currently available therapeutic options primarily target psoriasis-associated immunological defects rather than epidermal abnormalities. OBJECTIVE: To investigate the efficacy of the histone deacetylase (HDAC) inhibitor, Vorinostat, in targeting hyperproliferation and impaired apoptosis in psoriatic skin. METHODS: Vorinostat effect was investigated in primary KCs cell cultures using cell cycle analysis by flow cytometry, apoptosis assays (Annexin V-FICH and caspase-3/7) and antibody arrays, qRT-PCR and immunohistochemistry. Vorinostat impact on clinical manifestations of psoriasis was investigated in a chimeric mouse model. RESULTS: Vorinostat was found to inhibit KCs proliferation and to induce their differentiation and apoptosis. Using a chimeric mouse model, vorinostat was found to result in marked attenuation of a psoriasiform phenotype with a significant decrease in epidermal thickness and inhibition of epidermal proliferation. CONCLUSIONS: Our results support the notion that vorinostat, a prototypic HDAC inhibitor, may be of potential use in the treatment of psoriasis and other hyperproliferative skin disorders.
Assuntos
Inibidores de Histona Desacetilases , Psoríase , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Camundongos , Psoríase/tratamento farmacológico , Vorinostat/farmacologia , Vorinostat/uso terapêuticoRESUMO
Mouse models for atopic dermatitis (AD) are an indispensable preclinical research tool for testing new candidate AD therapeutics and for interrogating AD pathobiology in vivo. In this Viewpoint, we delineate why, unfortunately, none of the currently available so-called "AD" mouse models satisfactorily reflect the clinical complexity of human AD, but imitate more "allergic" or "irriant" contact dermatitis conditions. This limits the predictive value of AD models for clinical outcomes of new tested candidate AD therapeutics and the instructiveness of mouse models for human AD pathophysiology research. Here, we propose to initiate a rational debate on the minimal criteria that a mouse model should meet in order to be considered relevant for human AD. We suggest that valid AD models should at least meet the following criteria: (a) an AD-like epidermal barrier defect with reduced filaggrin expression along with hyperproliferation, hyperplasia; (b) increased epidermal expression of thymic stromal lymphopoietin (TSLP), periostin and/or chemokines such as TARC (CCL17); (c) a characteristic dermal immune cell infiltrate with overexpression of some key cytokines such as IL-4, IL-13, IL-31 and IL-33; (d) distinctive "neurodermatitis" features (sensory skin hyperinnervation, defective beta-adrenergic signalling, neurogenic skin inflammation and triggering or aggravation of AD-like skin lesions by perceived stress); and (e) response of experimentally induced skin lesions to standard AD therapy. Finally, we delineate why humanized AD mouse models (human skin xenotransplants on SCID mice) offer a particularly promising preclinical research alternative to the currently available "AD" mouse models.
Assuntos
Dermatite Atópica/induzido quimicamente , Dermatite Atópica/fisiopatologia , Modelos Animais de Doenças , Animais , Biomarcadores , Calcitriol/análogos & derivados , Dermatite Atópica/genética , Dermatite Atópica/patologia , Haptenos , Humanos , Camundongos , Ovalbumina , Fenômenos Fisiológicos da PeleRESUMO
The C3H/HeJ model has long dominated basic alopecia areata (AA) in vivo research and has been used as proof-of-principle that Jak inhibitors are suitable agents for AA management in vivo. However, its histologic features are not typical of human AA, and it is questionable whether it is sufficiently clinically predictive for evaluating the therapeutic effects of candidate AA agents. Instead, the humanized mouse model of AA has been used to functionally demonstrate the role of key immune cells in AA pathogenesis and to discover human-specific pharmacologic targets in AA management. Therefore, we advocate the use of both models in future preclinical AA research.
Assuntos
Alopecia em Áreas/tratamento farmacológico , Alopecia em Áreas/patologia , Modelos Animais de Doenças , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos , Camundongos Endogâmicos C3HRESUMO
This current review explores selected and as yet insufficiently investigated frontiers in current alopecia areata (AA) pathobiology research, with an emphasis on potential "new" players in AA pathobiology that deserve more systematic exploration and therapeutic targeting. Indeed, new evidence suggests that CD8+ T cells, which have long been thought to be the central players in AA pathobiology, are not the only drivers of disease. Instead, subsets of natural killer (NK) and so-called "unconventional" T cells (invariant NK T cells, γδ T cells, classic NK cells, and type 1 innate lymphoid cells), all of which can produce large amounts of IFN-γ, might also drive AA pathobiology independent of classical, autoantigen-dependent CD8+ T-cell functions. Another important new frontier is the role of regulatory lymphocyte subsets, such as regulatory T cells, γδ regulatory T cells, NKT10 cells, and perifollicular mast cells, in maintaining physiologic hair follicle immune privilege (IP); the extent to which these functions are defective in patients with AA; and how this IP-protective role could be restored therapeutically in patients with established AA. Broadening our AA research horizon along the lines suggested above promises not only to open the door to innovative and even more effective immunotherapy strategies for AA but will also likely be relevant for other autoimmune disorders in which pathobiology, ectopic MHC class I expression, and IP collapse play an important role.
Assuntos
Alopecia em Áreas/imunologia , Doenças Autoimunes/imunologia , Imunidade Inata , Alopecia em Áreas/patologia , Autoantígenos/imunologia , Doenças Autoimunes/patologia , Pesquisa Biomédica , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/patologiaRESUMO
Activated T cells are pathological in various autoimmune and inflammatory diseases including Psoriasis, and also in graft rejection and graft-versus-host-disease. In these pathological conditions, selective silencing of activated T cells through physiological receptors they express remains a clinical challenge. In our previous studies we found that activation of dopamine receptors (DRs) in resting human T cells activates these cells, and induces by itself many beneficial T cell functions. In this study, we found that normal human T cells express all types of DRs, and that expression of D1R, D4R and D5R increases profoundly after T cell receptor (TCR) activation. Interestingly, DR agonists shift the membrane potential (Vm ) of both resting and activated human T cells, and induces instantaneous T cell depolarization within 15 seconds only. Thus, activation of DRs in T cells depolarize these immune cells, alike activation of DRs in neural cells. The skin of Psoriasis patients contains 20-fold more D1R+ T cells than healthy human skin. In line with that, 25-fold more D1R+ T cells are present in Psoriasis humanized mouse model. Highly selective D1-like receptor agonists, primarily Fenoldopam (Corlopam) - a D1-like receptor agonist and a drug used in hypertension, induced the following suppressive effects on activated T cells of Psoriasis patients: reduced chemotactic migration towards the chemokine SDF-1/CXCL12; reduced dramatically the secretion of eight cytokines: tumor necrosis factor-α, interferon-γ, interleukin-1ß (IL-1ß), IL-2, IL-4, IL-6, IL-8 and IL-10; and reduced three T cell activation proteins/markers: CD69, CD28 and IL-2. Next, we invented a novel topical/dermal Fenoldopam formulation, allowing it to be spread on, and providing prolonged and regulated release in, diseased skin. Our novel topical/dermal Fenoldopam: reduced secretion of the eight cytokines by activated human T cells; reduced IL-1ß and IL-6 secretion by human lipopolysaccharide-inflamed skin; eliminated preferentially >90% of live and large/proliferating human T cells. Together, our findings show for the first time that both resting and activated T cells are depolarized instantaneously via DRs, and that targeting D1-like receptors in activated T cells and inflamed human skin by Fenoldopam, in Psoriasis, and potentially in other T cell-mediated diseases, could be therapeutic. Validation in vivo is required.
Assuntos
Fenoldopam/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Psoríase/imunologia , Receptores Dopaminérgicos/imunologia , Pele/imunologia , Linfócitos T/imunologia , Adulto , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos CD28/imunologia , Citocinas/imunologia , Feminino , Humanos , Lectinas Tipo C/imunologia , Masculino , Pessoa de Meia-Idade , Psoríase/patologia , Pele/patologia , Linfócitos T/patologiaRESUMO
Alopecia areata (AA) is understood to be a CD8+/NKG2D+ T cell-dependent autoimmune disease. Here, we demonstrate that human AA pathogenesis of is also affected by iNKT10 cells, an unconventional T cell subtype whose number is significantly increased in AA compared to healthy human skin. AA lesions can be rapidly induced in healthy human scalp skin xenotransplants on Beige-SCID mice by intradermal injections of autologous healthy-donor PBMCs pre-activated with IL-2. We show that in this in vivo model, the development of AA lesions is prevented by recognized the iNKT cell activator, α-galactosylceramide (α-GalCer), which stimulates iNKT cells to expand and produce IL-10. Moreover, in pre-established humanized mouse AA lesions, hair regrowth is promoted by α-GalCer treatment through a process requiring both effector-memory iNKT cells, which can interact directly with CD8+/NKG2D+ T cells, and IL-10. This provides the first in vivo evidence in a humanized model of autoimmune disease that iNKT10â¯cells are key disease-protective lymphocytes. Since these regulatory NKT cells can both prevent the development of AA lesions and promote hair re-growth in established AA lesions, targeting iNKT10â¯cells may have preventive and therapeutic potential also in other autoimmune disorders related to AA.
Assuntos
Alopecia em Áreas/imunologia , Imunoterapia Adotiva/métodos , Células T Matadoras Naturais/imunologia , Transplante de Pele , Pele/patologia , Adulto , Animais , Autoimunidade , Células Cultivadas , Modelos Animais de Doenças , Feminino , Galactosilceramidas/imunologia , Humanos , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Ativação Linfocitária , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Células T Matadoras Naturais/transplante , Transplante HeterólogoRESUMO
We have previously reported that the topical application of erythropoietin (EPO) to cutaneous wounds in rats and mice with experimentally induced diabetes accelerates their healing by stimulating angiogenesis, reepithelialization, and collagen deposition, and by suppressing the inflammatory response and apoptosis. Aquaporins (AQPs) are integral membrane proteins whose function is to regulate intracellular fluid hemostasis by enabling the transport of water and glycerol. AQP3 is the AQP that is expressed in the skin where it facilitates cell migration and proliferation and re-epithelialization during wound healing. In this report, we provide the results of an investigation that examined the contribution of AQP3 to the mechanism of EPO action on the healing of burn wounds in the skin of pigs with experimentally induced type 1 diabetes. We found that topical EPO treatment of the burns accelerated their healing through an AQP3-dependent mechanism that activates angiogenesis, triggers collagen and hyaluronic acid synthesis and the formation of the extracellular matrix (ECM), and stimulates reepithelialization by keratinocytes. We also found that incorporating fibronectin, a crucial constituent of the ECM, into the topical EPO-containing gel, can potentiate the accelerating action of EPO on the healing of the burn injury.
Assuntos
Indutores da Angiogênese/administração & dosagem , Aquaporina 3/metabolismo , Queimaduras/tratamento farmacológico , Eritropoetina/administração & dosagem , Cicatrização/efeitos dos fármacos , Cicatrização/genética , Administração Tópica , Animais , Queimaduras/genética , Colágeno/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Matriz Extracelular/genética , Fibronectinas/administração & dosagem , Ácido Hialurônico/biossíntese , Queratinócitos/metabolismo , Neovascularização Fisiológica , Reepitelização/genética , Pele/metabolismo , SuínosRESUMO
BACKGROUND: Fat grafting is an increasingly popular method of augmentation/reconstruction of soft tissue defects. However, the clinical unpredictability and high resorption rates of the grafts remain problematic. Cellular stress from the harvest and the ensuing ischemic episode may be the cause of this. Cellular stress activates the p38 mitogen-activated protein kinase (MAPK) signaling pathway. In response to cellular stress, the p38 pathway can lead to apoptosis and can negatively regulate cell proliferation. Inhibition of p38 in ex vivo experiments has been shown to promote the expansion of human cord blood hematopoietic stem cell and improve the adipogenesis process through its upstream regulator, Shp2. Because of its wide-ranging cell regulation and antiinflammatory properties, large-scale clinical trials using p38 inhibitors are also currently being performed, especially for therapeutic effect in chronic obstructive pulmonary disease and asthma. The rationale for our study was that the treatment of fat grafts with p38 inhibitor would (a) prevent apoptosis of adipose-derived stem cells in the fat grafts, (b) increase adipose-derived stem cells proliferation, and (c) stimulate the release of several angiogenic factors and promote revascularization. METHODS: Clinical and histological testing was performed on 5 fat-transplanted (1 mL) CD-1 nude mice compared with the test group of 5 mice, which were injected with a p38 MAPK inhibitor at 1, 3, 6, and 9 days after the fat transplantation. RESULTS: The weights and volumes of the control group grafts were significantly higher than those of the p38 MAPK inhibitor-treated grafts. Average volume resorption was 36% in the control group and 92% in the test group. Histological evaluation of the grafts revealed significantly improved integration, with a significant reduction of fibrosis and inflammation in the control group versus the treated group. CONCLUSIONS: This preliminary study suggests that as opposed to our hypothesis, inhibition of p38 significantly increases fat graft resorption. The dramatic effects observed in our study may suggest that p38 may act differently on the numerous cell types that constitute the fat graft, and further investigation is necessary.
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In Xenopus, animal cap (AC) cells give rise to ectoderm and its derivatives: epidermis and the central nervous system. Ectoderm has long been considered a default pathway of embryonic development, with cells that are not under the influence of vegetal Nodal signaling adopting an ectodermal program of gene expression. In the present study, we describe the involvement of the animally-localized maternal transcription factor myocyte enhancer factor (Mef) 2D in regulating the identity of AC cells. We find that Mef2D is required for the formation of both ectodermal lineages: neural and epidermis. Gain and loss of function experiments indicate that Mef2D regulates early gastrula expression of key ectodermal/epidermal genes in the animal region. Mef2D controls the activity of zygotic bone morphogenetic protein (BMP) signaling known to dictate the epidermal differentiation program. Exogenous expression of Mef2D in vegetal blastomeres was sufficient to induce ectopic expression of ectoderm/epidermal genes in the vegetal half of the embryo, when Nodal signaling was inhibited. Depletion of Mef2D caused a loss of AC cell adhesion that was rescued by the expression of E-cadherin or bone morphogenetic protein 4. In addition, expression of Mef2D in the prospective endoderm caused unusual aggregation of vegetal cells with animal cells in vitro and inappropriate segregation to other germ layers in vivo. Mef2D cooperates with another animally-expressed transcription factor, FoxI1e. Together, they regulate the expression of genes encoding signaling proteins and the transcription factors that control the regional identity of animal cells. Therefore, we describe a new role for the animally-localized Mef2D protein in early ectoderm specification, which is similar to that of the vegetally-localized VegT in endoderm and mesoderm formation.