RESUMO
Skin models mimicking features of psoriasis-related inflammation are needed to support the development of new drugs in dermatology. Reconstructed skin models lack tissue complexity, including a fully competent skin barrier, and presence and/or diversity of immune cells. Here, we describe InflammaSkin®, a novel human Th17-driven ex vivo skin inflammation model. In this model, skin-resident T cells are in situ activated by intradermal injection of anti-CD3 and anti-CD28 antibodies and Th17 cell polarization is sustained by culture in a chemically defined medium supplemented with IL-1ß, IL-23 and TGF-ß for seven days. The acquired Th17 signature is demonstrated by the sustained secretion of IL-17A, IL-17AF, IL-17F, IL-22, IFN-γ, and to some degree IL-15 and TNF-α observed in the activated ex vivo skin inflammation model compared with the non-activated skin model control. Furthermore, expression of S100A7 and Keratin-16 by keratinocytes and loss of epidermal structure integrity occur subsequently to in situ Th17cell activation, demonstrating cellular crosstalk between Th17 cells and keratinocytes. Finally, we demonstrate the use of this model to investigate the modulation of the IL-23/IL-17 immune axis by topically applied anti-inflammatory compounds. Taken together, we show that by in situ activation of skin-resident Th17 cells, the InflammaSkin® model reproduces aspects of inflammatory responses observed in psoriatic lesions and could be used as a translational tool to assess efficacy of test compounds.
Assuntos
Dermatite/imunologia , Ativação Linfocitária , Modelos Biológicos , Células Th17/imunologia , Anti-Inflamatórios/uso terapêutico , Anticorpos , Betametasona/análogos & derivados , Betametasona/uso terapêutico , Antígenos CD28/imunologia , Complexo CD3/imunologia , Comunicação Celular , Meios de Cultura , Dermatite/tratamento farmacológico , Humanos , Interferon gama/metabolismo , Interleucina-15/metabolismo , Interleucina-17/metabolismo , Interleucinas/metabolismo , Queratina-16/metabolismo , Queratinócitos/metabolismo , Inibidores da Fosfodiesterase 4/uso terapêutico , Proteína A7 Ligante de Cálcio S100/metabolismo , Células Th17/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interleucina 22RESUMO
BACKGROUND: Epidermolysis bullosa simplex is a skin-blistering disorder caused by mutations in keratin (K)14 or K5. Treatment with nuclear factor (erythroid-derived 2)-like 2 inducer sulforaphane ameliorated skin blistering in Krt14-null mice, correlating with induction of K17. To be therapeutically useful for epidermolysis bullosa simplex, topical broccoli sprout extract (BSE), enriched for sulforaphane, would ideally induce the expression of homologous keratins (eg, K6, K17, K16) in the basal layer of human epidermis without impacting expression of defective keratins (K5/K14). OBJECTIVE: The purpose of this 1-week, randomized, split-body, single-blinded, placebo-controlled trial was to assess the impact of BSE on keratin expression. METHODS: Five subjects (34-71 years old) applied BSE (500 nmol of sulforaphane/mL) or vehicle alone to the inner aspect of the arm daily. Expression of keratin, nuclear factor (erythroid-derived 2)-like 2, and other markers was assessed using reverse transcription-polymerase chain reaction and indirect immunofluorescence. RESULTS: One subject (age 71 years) was excluded a posteriori because of poor tissue quality. Topical BSE activated nuclear factor (erythroid-derived 2)-like 2 and up-regulated K17 in the epidermis of all subjects, had variable effects on K16 and K6 expression, and did not alter expression of K14 or K5. LIMITATIONS: Small sample size is a limitation. CONCLUSION: BSE represents an attractive therapeutic candidate for K14-associated epidermolysis bullosa simplex.
Assuntos
Brassica , Epidermólise Bolhosa Simples/tratamento farmacológico , Epidermólise Bolhosa Simples/metabolismo , Queratinas/metabolismo , Fitoterapia , Extratos Vegetais/uso terapêutico , Administração Cutânea , Adulto , Idoso , Humanos , Queratina-14/genética , Queratina-14/metabolismo , Queratina-16/genética , Queratina-16/metabolismo , Queratina-17/genética , Queratina-17/metabolismo , Queratina-5/genética , Queratina-5/metabolismo , Queratina-6/genética , Queratina-6/metabolismo , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/metabolismo , Extratos Vegetais/administração & dosagem , RNA Mensageiro/metabolismo , Plântula , Método Simples-Cego , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate dermal remodeling effects of crystal-free microdermabrasion on photodamaged skin. DESIGN: Biochemical analyses of human skin biopsy specimens following microdermabrasion treatment in vivo. SETTING: Academic referral center. PARTICIPANTS: Volunteer sample of 40 adults, aged 50 to 83 years, with clinically photodamaged forearms. Intervention Focal microdermabrasion treatment with diamond-studded handpieces of varying abrasiveness on photodamaged forearms and serial biopsies at baseline and various times after treatment. MAIN OUTCOME MEASURES: Quantitative polymerase chain reaction, immunohistochemistry, and enzyme-linked immunosorbent assay were used to quantify changes in inflammatory, proliferative, and remodeling effectors of normal wound healing. Type I and type III procollagen served as the main outcome marker of dermal remodeling. RESULTS: Coarse-grit microdermabrasion induces a wound healing response characterized by rapid increase in induction of cytokeratin 16 and activation of the AP-1 transcription factor in the epidermis. Early inflammation was demonstrated by induction of inflammatory cytokines, antimicrobial peptides, and neutrophil infiltration in the dermis. AP-1 activation was followed by matrix metalloproteinase-mediated degradation of extracellular matrix. Consistent with this wound-healing response, we observed significant remodeling of the dermal component of the skin, highlighted by induction of type I and type III procollagen and by induction of collagen production enhancers heat shock protein 47 and prolyl 4-hydroxylase. Dermal remodeling was not achieved when microdermabrasion was performed using a medium-grit handpiece. CONCLUSIONS: Microdermabrasion using a coarse diamond-studded handpiece induces a dermal remodeling cascade similar to that seen in incisional wound healing. Optimization of these molecular effects is likely the result of more aggressive treatment with a more abrasive handpiece.
Assuntos
Citocinas/metabolismo , Terapia com Luz de Baixa Intensidade/métodos , Regeneração/fisiologia , Envelhecimento da Pele/patologia , Centros Médicos Acadêmicos , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha , Estudos de Coortes , Colágeno Tipo III/metabolismo , Citocinas/genética , Feminino , Humanos , Imuno-Histoquímica , Queratina-16/metabolismo , Masculino , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , Biologia Molecular , Probabilidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/metabolismo , Envelhecimento da Pele/efeitos da radiação , Resultado do TratamentoRESUMO
OBJECTIVE: To quantitatively examine the epidermal and dermal cellular and molecular changes that occur after photodynamic therapy of photodamaged human skin. DESIGN: Serial in vivo biochemical and immunohistochemical analyses after photodynamic therapy using topical 5-aminolevulinic acid (5-ALA) and pulsed-dye laser treatment. SETTING: Academic referral center, Department of Dermatology, University of Michigan, Ann Arbor. PATIENTS: A volunteer sample of 25 adults, 54 to 83 years old, with clinically apparent photodamage of the forearm skin. INTERVENTIONS: Three-hour application of 5-ALA followed by pulsed-dye laser therapy using non-purpura-inducing settings to focal areas of photodamaged forearms and serial biopsy specimens taken at baseline and various times after treatment. MAIN OUTCOME MEASURES: Immunohistochemical analysis was used to assess levels of markers of epidermal proliferation (Ki67), epidermal injury (cytokeratin 16), and photodamage (p53), as well as various markers of dermal collagen production (including prolyl 4-hydroxylase and heat shock protein 47, and type I procollagen). Real-time reverse transcriptase-polymerase chain reaction technology was used to quantify type I and type III collagen. Type I procollagen protein was quantified with enzyme-linked immunosorbent assay. RESULTS: Epidermal proliferation was stimulated as demonstrated by increases in Ki67 (more than a 5-fold increase; P < .05) and epidermal thickness (more than a 1.4-fold increase; P < .05). Epidermal injury was produced with increased cytokeratin 16 levels demonstrated (to nearly 70-fold of baseline levels; P < .05). Upregulation of collagen production was demonstrated with increases in procollagen I messenger RNA (2.65-fold; P < .05), procollagen III messenger RNA (3.32-fold; P < .05), and procollagen I protein (2.42-fold; P < .05) levels detected. The baseline epidermal p53 level correlated with cytokeratin 16 levels at acute time points, and the latter were found to correlate with peak collagen production. CONCLUSIONS: Photodynamic therapy with the specific treatment regimen employed produces statistically significant quantitative cutaneous molecular changes (eg, production of types I and III collagen) that are associated with improved appearance of the skin. Baseline epidermal p53 immunostaining levels may be predictive of dermal responses to this therapy. Comparison with historical data using pulsed-dye laser therapy alone suggests that use of the photosensitizer may enhance dermal remodeling. The quantitative in vivo molecular data presented herein are in keeping with an evolving model to potentially predict the efficacy of new techniques for the treatment of photoaging.