Estudo da progressão tumoral e perfil de expressão de citocinas em modelos tumorais associados ao HPV-16 em camundongos / Cytokine expression profile in HPV16 associated tumors in C57Bl/6 mice

RESUMO

O papilomavírus humano (HPV) é o principal agente etiológico do carcinoma de colo uterino. Citocinas e quimiocinas produzidas pelas células neoplásicas, assim como pelas células do sistema imunológico, podem direcionar à tolerância ou à uma resposta imune efetiva. A presença do HPV16 pode influenciar o perfil de citocinas e quimiocinas expressas durante o desenvolvimento dos tumores, sendo o objetivo geral deste trabalho avaliar o perfil de expressão de citocinas na resposta imune contra tumores TC-1 em camundongos C57Bl/6 ... Um achado interessante foi a expressão exclusiva de IL-10 pelo infiltrado leucocitário, que nos levou a investigar o papel desta citocina em nosso modelo. IL-10 tem papel supressor da resposta celular mediada por linfócitos TCD8+, uma vez que observamos maior quantidade de infiltrado de linfócitos TCD8+ específicos para um peptídeo de E7 de HPV16 nos animais deficientes para IL-10. Além disso, na ausência de IL-10 os tumores TC-1 crescem mais lentamente, e apresentam quantidade reduzida de linfócitos T regulatórios nos linfonodos dos animais. A presença de CCL2 e MCSF foi identificada após análise de mRNA e proteína. A elevada expressão de CCL2 pelas células tumorais nos levaram a investigar o papel desta quimiocina no recrutamento de células mielóides em resposta às células HPV-16 positivas. Em ensaio de migração in vitro observamos que CCL2 possui papel importante no recrutamento de monócitos CD11b+, sendo este efeito específico de células que expressam as proteínas oncogênicas E6 e E7 de HPV-16. Os resultados apresentados neste trabalho ilustram o complexo painel de citocinas e quimiocinas que estão envolvidos na inibição da resposta imune anti-tumoral e podendo influenciar o fenótipo de células do sistema imune recrutadas para o local da lesão.

ABSTRACT

Infection with Human Papillomavirus (HPV) is the main cause of cervical cancer. Cytokines and chemokines are key molecules in the local immune response owing to their immunomodulators activities. Great varieties of these molecules are produced by tumor cells as well as by the tumor infiltrate that lead to the recruitment of leukocytes associated to tumor progression. Due to the importance of local immune response against tumor establishment we proposed to evaluate the expression of cytokines, chemokines and their receptors involved in the inflammatory response in HPV associated tumors. Previous data showed us that TC-1 tumors were infiltrated mainly by CD45+/CD11b+F4/80+ macrophages with M2-like phenotype, and virtually no lymphocytes. In this study, our results showed high levels of MCSF-R and MMP-9 expression by the infiltrate. We also observed TGFß, IL-1ß, IL-6, VEGF, CCL2 and CCL5 expression by tumor cells as well as by the infiltrating leukocytes. Our in vitro studies showed that CCL2 is responsible for myeloid cell recruitment by HPV16 E6/E7 expressing cells. Interestingly, we observed high expression of IL-10 and TNFα by the infiltrate. Moreover, we tested the hypothesis that IL-10 produced by the tumor inflammatory infiltrate has a role in suppression of T cell anti-tumor response. In IL-10 deficient mice or wild type mice treated with neutralizing antibodies against IL-10 and IL-10R, tumor growth was inhibited in parallel with an increase in tumor infiltration by HPV16 E7 specific TCD8+ lymphocytes. At the same ime, we observed that upon antigen stimulation less regulatory T cells were observed in lymph node cultures which IL-10 effect was neutralized. Our data indicate that IL-10 produced by infiltrating leukocytes inhibits anti-tumor T cell responses in this model. In summary, we observed expression of inflammatory cytokines like TNFα and IL-1 in TC-1 tumors, which are usually associated to chronic inflammation leading to tissue damage and transformation. We also detected suppressor cytokines like IL-10 and TGFß, which, as demonstrated have a role in suppression of anti-tumor T cell responses. Our data describes a tumor environment where tumor growth is facilitated by the absence of cellular responses and possibly by the inflammation.