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Desenvolvimento de um modelo de epiderme 3D inflamada, in vitro, para avaliar a eficácia de compostos anti-inflamatórios
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Resumo(s)
A principal função da pele relaciona-se com o revestimento externo do corpo, protegendo-o contra a
ação de patogénios e agressões mecânicas e químicas. Anatomicamente, é composta por duas camadas
principais, a derme e a epiderme. Os queratinócitos são o tipo celular predominante na epiderme e estão
organizados em diferentes camadas, estando constantemente sujeitos à ação de estímulos ambientais.
Atualmente, sabe-se que as funções dos queratinócitos vão para além do seu caráter estrutural, sendo
células imunologicamente ativas que funcionam como sentinelas capazes de reconhecer moléculas e de
produzir mediadores inflamatórios, dos quais se destacam as citocinas. Em condições homeostáticas, os
níveis de expressão destes mediadores pelos queratinócitos são bastante reduzidos. Contudo, na
presença de vários estímulos, como é o caso da exposição à radiação ultravioleta (UV) ou a fatores
tumorais, os queratinócitos tornam-se células ativadas que produzem e libertam vários tipos de citocinas.
Consequentemente, tornam-se mais proliferativos e adquirem um fenótipo migratório, caracterizado por
alterações na composição do citoesqueleto, começando a expressar queratinas que não são expressas
normalmente (ex. queratina 6 (K6) e queratina 17 (K17)). Devido à crescente pressão ética e legal sobre
a utilização de animais para fins académicos ou industriais, os modelos tridimensionais (3D) in vitro
têm-se tornado ferramentas imprescindíveis. Comparativamente aos modelos bidimensionais (2D), os
modelos 3D permitem recapitular melhor a fisiologia da pele nativa, possibilitando a interação celular e
a comunicação com a matriz extracelular. Neste projeto, o principal objetivo consistiu no
desenvolvimento de um modelo tridimensional epidérmico que simulasse uma resposta inflamatória por
parte dos queratinócitos. Foram utilizados queratinócitos epidérmicos humanos neonatais (HEKn) e
queratinócitos da linha celular N/TERT-1, os quais foram semeados em matrizes comerciais porosas de
policarbonato. Para induzir a resposta inflamatória explorou-se a ação de diferentes intensidades de
radiação UV-B, ao fim de 48 e 96 horas, e também de diferentes concentrações do fator de necrose
tumoral alfa (TNF-), após 24 e 48 horas. Na ausência de estímulos inflamatórios, foi possível obter
modelos com HEKn com organização estrutural semelhante à verificada in vivo, tendo esta sido
confirmada através da expressão de marcadores epidérmicos. Os ensaios com radiação UV-B parecem
indicar que os HEKn efetivamente adotam um fenótipo ativado, no qual existe um aumento da
proliferação celular e da expressão da K6 e da K17, bem como da libertação de citocinas inflamatórias
(IL-6 e IL-1), 48 horas após a exposição. Ao fim de 96 horas os HEKn parecem permanecer num estado
ativado, já que foi possível identificar aumentos nos níveis de citocinas relativamente à situação
controlo. O TNF- parece ter provocado um aumento na expressão da K6 e da K17, tendo este aumento
sido verificado para ambos os tempos de pós-exposição. Por outro lado, a linha celular N/TERT-1 não
se mostrou tão predisponível para recriar a arquitetura multicamada da epiderme nativa, não tendo sido
observados sinais de proliferação, diferenciação ou estratificação celular. A espessura dos cortes
histológicos, a estabilidade da solução de vitamina C e o tempo de exposição à interface ar-líquido (airliquid interface, ALI) foram estudados como possíveis fatores que influenciassem estes resultados, não
se tendo verificado uma relação de causalidade. Uma vez otimizados, os modelos poderão ser utilizados
em testes de avaliação de fármacos de ação anti-inflamatória, permitindo prever de forma mais
aproximada à realidade o comportamento dos mesmos na pele humana nativa.
The main function of the skin is related to the external covering of the body, protecting it against the action of pathogens and mechanical and chemical aggressions. Anatomically, it is composed of two main layers, the dermis and the epidermis. Keratinocytes are the predominant cell type in the epidermis and are organized in different layers, being constantly subject to the action of environmental stimuli. Currently, it is known that the functions of keratinocytes go beyond their structural character, being immunologically active cells that function as sentinels capable of recognizing molecules and producing inflammatory mediators, of which cytokines stand out. Under homeostatic conditions, the expression levels of these mediators by keratinocytes are greatly reduced. However, in the presence of various stimuli, such as ultraviolet radiation (UV) or tumour factors, keratinocytes become activated cells that produce and release various types of cytokines. Consequently, they become more proliferative and acquire a migratory phenotype, characterized by changes in the composition of the cytoskeleton, starting to express keratins that are not normally expressed (e.g., keratin 6 (K6) and keratin 17 (K17)). Due to increasing ethical and legal pressure on the use of animals for academic or industrial purposes, threedimensional (3D) in vitro models have become essential tools. Compared to two-dimensional models (2D), 3D models allow a better recapitulation of the physiology of the native skin, enabling cellular interaction and communication with the extracellular matrix. In this project, the main goal was to develop a 3D epidermal model that simulates an inflammatory response by keratinocytes. For this purpose, neonatal human epidermal keratinocytes (HEKn) and keratinocytes from the N/TERT-1 cell line were seeded in commercial porous polycarbonate matrices. To induce the inflammatory response, we analyse the action of different intensities of UV-B radiation, 48 and 96 hours post irradiation, and of different concentrations of tumour necrosis factor alpha (TNF-α), 24 and 48 hours after treatment. In the absence of inflammatory stimuli, it was possible to obtain HEKn models with structural organization like that observed in vivo, which was confirmed through the expression of epidermal markers. Assays with UV-B radiation seem to indicate that HEKn adopt an activated phenotype, in which there was an increase in cell proliferation and expression of K6 and K17, as well as the release of inflammatory cytokines (IL-6 and IL-1), 48 hours after exposure. After 96 hours, HEKn appears to remain in an activated state, as it was possible to identify increases in cytokine levels, in comparison to the control situation. TNF-α seemed to have caused an increase in the expression of K6 and K17, and this increase was observed for both post-exposure times. On the other hand, the N/TERT-1 cell line was not able to recreate the multilayer architecture of the native epidermis, and no signs of cell proliferation, differentiation or stratification were observed. The thickness of the histological sections, the stability of the vitamin C solution and the time of exposure to the air-liquid interface (ALI) were studied as possible factors that contributed to these results. However, no causal relationship was found. Once the models are optimized, they can be used in assays to evaluate drugs with anti-inflammatory action, allowing predicting more closely to reality their behaviour in native human skin.
The main function of the skin is related to the external covering of the body, protecting it against the action of pathogens and mechanical and chemical aggressions. Anatomically, it is composed of two main layers, the dermis and the epidermis. Keratinocytes are the predominant cell type in the epidermis and are organized in different layers, being constantly subject to the action of environmental stimuli. Currently, it is known that the functions of keratinocytes go beyond their structural character, being immunologically active cells that function as sentinels capable of recognizing molecules and producing inflammatory mediators, of which cytokines stand out. Under homeostatic conditions, the expression levels of these mediators by keratinocytes are greatly reduced. However, in the presence of various stimuli, such as ultraviolet radiation (UV) or tumour factors, keratinocytes become activated cells that produce and release various types of cytokines. Consequently, they become more proliferative and acquire a migratory phenotype, characterized by changes in the composition of the cytoskeleton, starting to express keratins that are not normally expressed (e.g., keratin 6 (K6) and keratin 17 (K17)). Due to increasing ethical and legal pressure on the use of animals for academic or industrial purposes, threedimensional (3D) in vitro models have become essential tools. Compared to two-dimensional models (2D), 3D models allow a better recapitulation of the physiology of the native skin, enabling cellular interaction and communication with the extracellular matrix. In this project, the main goal was to develop a 3D epidermal model that simulates an inflammatory response by keratinocytes. For this purpose, neonatal human epidermal keratinocytes (HEKn) and keratinocytes from the N/TERT-1 cell line were seeded in commercial porous polycarbonate matrices. To induce the inflammatory response, we analyse the action of different intensities of UV-B radiation, 48 and 96 hours post irradiation, and of different concentrations of tumour necrosis factor alpha (TNF-α), 24 and 48 hours after treatment. In the absence of inflammatory stimuli, it was possible to obtain HEKn models with structural organization like that observed in vivo, which was confirmed through the expression of epidermal markers. Assays with UV-B radiation seem to indicate that HEKn adopt an activated phenotype, in which there was an increase in cell proliferation and expression of K6 and K17, as well as the release of inflammatory cytokines (IL-6 and IL-1), 48 hours after exposure. After 96 hours, HEKn appears to remain in an activated state, as it was possible to identify increases in cytokine levels, in comparison to the control situation. TNF-α seemed to have caused an increase in the expression of K6 and K17, and this increase was observed for both post-exposure times. On the other hand, the N/TERT-1 cell line was not able to recreate the multilayer architecture of the native epidermis, and no signs of cell proliferation, differentiation or stratification were observed. The thickness of the histological sections, the stability of the vitamin C solution and the time of exposure to the air-liquid interface (ALI) were studied as possible factors that contributed to these results. However, no causal relationship was found. Once the models are optimized, they can be used in assays to evaluate drugs with anti-inflammatory action, allowing predicting more closely to reality their behaviour in native human skin.
Descrição
Tese de Mestrado, Biologia Humana e do Ambiente, 2022, Universidade de Lisboa, Faculdade de Ciências
Palavras-chave
Queratinócitos Modelo 3D epidérmico Radiação UV-B TNF-x Citocinas Teses de mestrado - 2023