Estudo ex vivo e in vitro da modulação da imunidade inata e adaptativa por queratinócitos displásicos em leucoplasia oral e leucoplasia verrucosa proliferativa
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Data
2020-03-13
Autores
Fernandes, Darcy [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O carcinoma espinocelular (CEC) representa mais de 95% de todas as neoplasias malignas que acometem a cavidade oral e muitas vezes estes tumores são precedidos por alterações clínicas que apresentam um evidente potencial de transformação maligna, as quais são chamadas de desordens potencialmente malignas orais (DPMOs). Dentre estas, a leucoplasia oral (LO) possui taxa de transformação maligna que varia de 0,2% até 17,5%; contudo, uma outra DPMO conhecida como leucoplasia verrucosa proliferativa (LVP) apresenta um comportamento persistente e progressivo para malignidade, com taxa de transformação maligna maior que 70%. Diferente da LO, fator de risco como tabaco, álcool e noz de areca não parecem estar associados com o desenvolvimento da LVP. Adicionalmente, a LVP frequentemente apresenta resposta inadequada a todas as modalidades de tratamento, sofre rápida disseminação pelos sítios orais e muitas vezes demonstra recorrência. Estudos recentes sugerem que o infiltrado inflamatório associado às lesões leucoplásicas de paciente com LVP pode estar relacionado a etiologia e/ou comportamento clínico agressivo desta DPMO. Assim, foi realizada uma análise comparativa entre amostras de LO e LVP que consistiu em: (1) avaliar a porcentagem e identificar os subtipos de linfócitos T auxiliares e estado de ativação de linfócitos T citotóxicos, (2) avaliar a densidade e estado de ativação das células dendríticas, e (3) determinar o efeito de produtos solúveis de células displásicas na modulação da imunidade inata e/ou adaptativa e se tal modulação confere uma vantagem para a progressão do tumor. Para alcançar estes objetivos, a expressão imuno-histoquímica de marcadores linfócitos T e B, para células dendríticas (CD) de Langerhans, CD submucosas imaturas e maturas, CD plasmocitóides, foram utilizados. Adicionalmente, ensaios com cultura de células foram realizados para acessar o potencial modelador de fatores solúveis produzidos por culturas celulares derivadas de PVL sobre células mononucleares do sangue periférico (PBMCs). Estes ensaios incluíram a avaliação das taxas de apoptose e imunofenotipagem dos PBMCs após contato com meios de cultura condicionados de diferentes culturas celulares, incluindo queratinócitos normais (HOK), displásicos (DOK e DOK-PVL) e tumorais (SCC-25). Os resultados da análise imuno-histoquímica mostraram uma redução global de células T e B acompanhada de uma inibição da ativação de células Th17 e consequente indução de uma resposta imune mediada por células Treg na LVP, parece favorecer o comportamento mais agressivo desta DPMO em relação a LO. Adicionalmente, CD de Langerhans, maturas e plasmocitóides estão reduzidas nos compartimentos epitelial e subepiteliais na LO e LVP em relação ao controle. Por outro lado, CD imaturas encontram-se aumentadas nos compartimentos epitelial e subepiteliais destas duas DPMOs. Juntos, estes achados favorecem uma apresentação inadequada dos antígenos pré-tumorais facilitando a tolerância imunológica. Os experimentos com cultura de células revelaram ainda, que os produtos solúveis de queratinócitos displásicos tem capacidade de alterar o fenótipo dos PBMCs favorecendo a transformação maligna de DPMOs. Assim, os resultados deste estudo indicam que os produtos solúveis dos queratinócitos derivados de LVP são capazes de modular de forma distinta a resposta imunológica desta DPMO em relação à LO, sendo esta modulação acompanhada do ganho de vantagens que favorecem o processo de tumorigênese.
Oral squamous cell carcinoma (SCC) represents more than 95% of all malignant neoplasms that affect the oral cavity and these tumors are often preceded by clinical changes that have an evident potential for malignant transformation, which are called potentially malignant oral disorders (OPMDs). Among these, oral leukoplakia (OL) has a malignant transformation rate that varies from 0.2% to 17.5%; however, another OPMD known as proliferative verrucous leukoplakia (PVL) has a persistent and progressive behavior for malignancy, with a malignant transformation rate greater than 70%. Unlike OL, a risk factor such as tobacco, alcohol and areca nut does not seem to be associated with the development of PVL. Additionally, PVL often presents an inadequate response to all treatment modalities, suffers rapid dissemination through oral sites and often shows recurrence. Recent studies suggest that the inflammatory infiltrate associated with leukoplastic lesions in a patient with PVL may be related to the etiology and / or aggressive clinical behavior of this OPMD. Thus, a comparative analysis was performed between OL and PVL samples that consisted of: (1) evaluating the percentage and identify subtypes of auxiliary T lymphocytes and activation status of cytotoxic T lymphocytes, (2) evaluating the density and activation status of dendritic cells, and (3) determine the effect of soluble products of dysplastic cells on the modulation of innate and / or adaptive immunity and whether such modulation confers an advantage for progression of the tumor. To achieve these goals, immunohistochemical expression of markers for Langerhans dendritic cells (DC), immature and mature submucous DC, plasmacytoid DC, T and B lymphocytes were used. In addition, cell culture assays were performed to access the potential modeling of soluble factors produced by PVL-derived cell cultures on peripheral blood mononuclear cells (PBMCs). These trials included the assessment of apoptosis and immunophenotyping rates of PBMCs after contact with conditioned culture media from different cell cultures, including normal keratinocytes (HOK), dysplastic (DOK and DOK-PVL) and tumor (SCC-25). The results of the immunohistochemical analysis showed a global reduction in T and B cells accompanied by an inhibition of Th17 cell activation and the consequent induction of an immune response mediated by Treg cells in PVL, seems to favor the more aggressive behavior of this OPMD in relation to OL. In addition, Langerhans DC, matures and plasmacytoids are reduced in the epithelial and subepithelial compartments in the OL and PVL in relation to the control. In contrast, immature DCs are increased in the epithelial and subepithelial compartments of these two OPMD. Together, these findings favor an inadequate presentation of pre-tumor antigens, facilitating immunological tolerance. The experiments with cell culture also revealed that the soluble products from dysplastic keratinocytes may to change the PBMCs phenotype favoring the malignant transformation of OPMD. Thus, the results of this study indicate that soluble product from PVL-derived keratinocytes are capable of differently modulating the immune response of this DPMO in relation to OL, and this modulation is accompanied by the gain of advantages that favor the tumorigenesis process.
Oral squamous cell carcinoma (SCC) represents more than 95% of all malignant neoplasms that affect the oral cavity and these tumors are often preceded by clinical changes that have an evident potential for malignant transformation, which are called potentially malignant oral disorders (OPMDs). Among these, oral leukoplakia (OL) has a malignant transformation rate that varies from 0.2% to 17.5%; however, another OPMD known as proliferative verrucous leukoplakia (PVL) has a persistent and progressive behavior for malignancy, with a malignant transformation rate greater than 70%. Unlike OL, a risk factor such as tobacco, alcohol and areca nut does not seem to be associated with the development of PVL. Additionally, PVL often presents an inadequate response to all treatment modalities, suffers rapid dissemination through oral sites and often shows recurrence. Recent studies suggest that the inflammatory infiltrate associated with leukoplastic lesions in a patient with PVL may be related to the etiology and / or aggressive clinical behavior of this OPMD. Thus, a comparative analysis was performed between OL and PVL samples that consisted of: (1) evaluating the percentage and identify subtypes of auxiliary T lymphocytes and activation status of cytotoxic T lymphocytes, (2) evaluating the density and activation status of dendritic cells, and (3) determine the effect of soluble products of dysplastic cells on the modulation of innate and / or adaptive immunity and whether such modulation confers an advantage for progression of the tumor. To achieve these goals, immunohistochemical expression of markers for Langerhans dendritic cells (DC), immature and mature submucous DC, plasmacytoid DC, T and B lymphocytes were used. In addition, cell culture assays were performed to access the potential modeling of soluble factors produced by PVL-derived cell cultures on peripheral blood mononuclear cells (PBMCs). These trials included the assessment of apoptosis and immunophenotyping rates of PBMCs after contact with conditioned culture media from different cell cultures, including normal keratinocytes (HOK), dysplastic (DOK and DOK-PVL) and tumor (SCC-25). The results of the immunohistochemical analysis showed a global reduction in T and B cells accompanied by an inhibition of Th17 cell activation and the consequent induction of an immune response mediated by Treg cells in PVL, seems to favor the more aggressive behavior of this OPMD in relation to OL. In addition, Langerhans DC, matures and plasmacytoids are reduced in the epithelial and subepithelial compartments in the OL and PVL in relation to the control. In contrast, immature DCs are increased in the epithelial and subepithelial compartments of these two OPMD. Together, these findings favor an inadequate presentation of pre-tumor antigens, facilitating immunological tolerance. The experiments with cell culture also revealed that the soluble products from dysplastic keratinocytes may to change the PBMCs phenotype favoring the malignant transformation of OPMD. Thus, the results of this study indicate that soluble product from PVL-derived keratinocytes are capable of differently modulating the immune response of this DPMO in relation to OL, and this modulation is accompanied by the gain of advantages that favor the tumorigenesis process.
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Palavras-chave
Leucoplasia, Sistema imunitário, Neoplasias bucais, Técnicas de cultura de células, Leukoplakia, Immune System, Mouth Neoplasms, Cell Culture Techniques