Diferenciação e maturação de células dendríticas da medula óssea de camundongos BALB/c: um modelo para estudos imunoterapêuticos experimentais

Abstract

Dendritic cells (DCs) represent an essential link between the innate and adaptive immune systems. These cells are known for their high capacity to capture, process, and present antigens, in addition to secreting pro-inflammatory cytokines that direct naïve T lymphocyte polarization. This study aimed to establish an in vitro model of DC differentiation and maturation from the bone marrow of BALB/c mice, with GM-CSF supplementation and activation with an TLR3 agonist, Poly I:C, to evaluate its applicability in experimental immunotherapeutic studies. For this purpose, BALB/c mice were euthanized for bone marrow collection, from which hematopoietic stem cells were isolated, purified, and cultured in RPMI medium supplemented with GM-CSF. After seven days of differentiation, the cells were treated with Poly I:C (200 µg/mL) for 48 hours and subjected to morphological, phenotypic, and functional analyses. Morphological analysis showed progressive changes consistent with immature DCs and, after treatment, with mature DCs—including elongation of cytoplasmic extensions and increased structural complexity. Phenotypic analysis indicated a significant increase in CD11c expression, especially in the group treated with Poly I:C. Gene expression analysis showed a significant increase in TLR6 transcription after treatment with Poly I:C. These data suggest possible cross-regulation of Toll-like receptors, even though the agonist used is specific for TLR3. Additionally, ELISA assays revealed that differentiation with GM-CSF elevated levels of the cytokine IL-1β, associated with the functional activation of DCs. Treatment with Poly I:C did not promote an additional increase in cytokine production, indicating a possible limitation of the soluble form of the agonist for inducing a robust inflammatory response. It is concluded that the developed protocol is efficient for obtaining DCs from myeloid precursors, allowing their differentiation and activation in vitro with morphological and functional characteristics compatible with those described in the literature. Poly I:C-induced maturation confirmed the responsiveness of DCs to immune stimulation, demonstrating the model's potential for application in immunotherapeutic studies. Thus, the present work contributes to the standardization of a reproducible, viable, and functionally relevant experimental model for the study of therapeutic strategies based on the modulation of dendritic cells.