Estabelecimento de um modelo humanizado em camundongos NOD-SCID IL-2Rγnull (NSG) com potencial translacional para avaliação de novos alvos terapêuticos

Abstract

Human studies are limited by ethical and technical issues. Thus, the use of mice as in vivo and ex vivo models has provided great advances in research, but they are still not enough. Thus, the development of humanized mice (hu-mice) and their use becomes relevant where there is no other suitable animal model available for research. Hu-mice are defined as mice reconstituted with cells or tissues of human origin and represent an innovative in vivo study tool of the role played by the human immune system during the pathophysiology of different conditions, as well as a new approach to the study of the human immune response with translational potential and evaluation of new therapeutic targets, especially regarding to pathogens whose tropism is exclusive to human cells, such as the dengue virus. Therefore, the aim of this study was to establish a human immune system model in NOD-scid IL-2Rγnull (NSG) mice to evaluate new therapeutic targets against dengue. Therefore, three different sources of human cells for the humanization of animals were tested and evaluated: 1) CD34+ progenitor stem cells from human fetal liver; 2) CD34+ progenitor stem cells from a leukopheresis pouch enriched with GM-CSF (granulocyte and macrophage colony stimulating factor) and 3) human PBMC (peripheral blood mononuclear cells) from healthy donors and their respective non-injected controls. The first two strategies did not result in satisfactory results, which made the continuation of the experiments unfeasible. Thus, the next source of cells was human PBMC's and, with this model, we continued with the experiments. NSG animals were injected with PBMC's and the presence of human cells in the animals' peripheral blood was evaluated weekly by flow cytometry. Our results demonstrate that animals grafted with human PBMC’s presented an average percentage of human CD45+ cell reconstitution higher than 40%, a criterion used to consider humanization satisfactory. The evaluation was done in blood, spleen and liver and T cells were the predominant cell type found in these sites. Histopathological evaluation of the liver demonstrated a cell recruitment composed of human cells. Furthermore, our data demonstrated that humanization from PBMC's was not associated with weight loss, death or changes in blood parameters of animals. Once the average frequency of 40% of circulating human cells was reached, the humanized mice, hereinafter called hu-mice, were infected by DENV-2 (dengue virus-2) and the inflammatory, histopathological, hematological and clinical parameters were monitored. Infection of mice hu was not associated with weight loss or death. However, the evaluation of blood parameters showed that there was a decrease in monocytes and an increase in platelets in the blood of infected animals when compared to non-infected ones. However, there was no significant difference in the percentage of hematocrit and circulating levels of lymphocytes and neutrophils in relation to the infected versus non-infected groups. Additionally, it was possible to observe an increase in the production of INFγ (interferon gamma) only in the TCD4 cells of the spleen of humanized infected animals. The evaluated histopathological parameters revealed that there was a recruitment of cells to the tissue and that these cells were human. In general, this model seems to be a good strategy for evaluating new therapeutic targets against viral diseases with tropism for human cells, in particular human lymphocytes, and will be further explored in our laboratory in the future, which may expand our investigations to test other pathogens with tropism for human cells.