Modulação da migração de macrófagos e células dendríticas por leishmania: alterações funcionais e moleculares em ambiente tridimensional e no contexto da infecção in vivo

Abstract

Leishmaniases can present with different clinical manifestations, ranging from localized skin lesions to visceral disease. The dissemination of infected cells is essential for disease establishment, but the mechanisms driving cell migration after infection remain poorly understood. Studies with macrophages infected by L. amazonensis have shown reduced 2D migration following infection; however, the process of transendothelial migration of macrophages involves regulatory signals within a 3D environment. Additionally, infection with L. braziliensis induces inflammatory mediators that enhance host cell recruitment, but the in vivo effects on bone marrow cell activation and their influence on the migration and response of macrophages remain unclear. In this study, we investigated the migration of macrophages and dendritic cells (Dds) in a three-dimensional environment, as well as the alterations induced in the bone marrow of mice infected with L. braziliensis and their potential role in modulating the migration of bone marrow-derived macrophages (BMMΦ). BMMΦ from mice infected with L. braziliensis were isolated after 2, 5, and 10 weeks of infection. These macrophages, along with DCs, were subjected to 2D and 3D migration assays in transwell systems, and adhesion complex formation was evaluated by immunostaining for pFAK and pPaxillin. Actin cytoskeleton dynamics were analyzed using Phalloidin, Rac1, Cdc42, RhoA, and gelsolin, while podosome formation was assessed by talin and vinculin staining. Analyses were performed by confocal microscopy, and the expression of matrix metalloproteinases (MMPs) was measured by a Luminex-based assay. To identify differentially expressed proteins, samples were characterized by liquid chromatography coupled with mass spectrometry (LC-MS/MS). It was observed that human and murine macrophages exhibit reduced migratory capacity in 3D environments, associated with decreased adhesion complex formation and alterations in actin dynamics after infection with Leishmania spp. In contrast, DCs showed increased migration in 3D environments following L. infantum infection, accompanied by enhanced adhesion complex formation and increased actin dynamics. Furthermore, a significant reduction in both 2D and 3D migration of BMMΦ was observed after in vivo infection with L. braziliensis, together with decreased expression of pFAK and pPaxillin at all evaluated time points. Rac1 and Cdc42 expression increased at weeks 2 and 5 but decreased after 10 weeks, while RhoA expression was consistently reduced throughout the study period. Vinculin expression increased at week 2 and decreased at week 5. MMP expression was elevated at week 2 and reduced at week 5. Proteomic analysis identified 26 differentially expressed proteins after 2 weeks of infection, 11 proteins after 5 weeks, and 6 proteins after 10 weeks. Understanding how Leishmania infection affects parasite dissemination and the recruitment of bone marrow cells in vivo is essential for elucidating the pathogenesis of leishmaniasis.