Leishmaniases are neglected diseases caused by protozoa of the genus Leishmania, with the cutaneous form being the most prevalent. Although macrophage infection is well characterized in 2D models, studies using 3D models ‒ which better mimic the in vivo environment ‒ are still scarce. Understanding invasion mechanisms in these models is essential to advance the identification of therapeutic strategies. To assess the adhesion index of Leishmania braziliensis in macrophages, as well as the involvement of actin and the early endosome marker Rab5 in the endocytosis process, comparing different infection times and 2D versus 3D models. RAW 264.7 macrophages were infected with L. braziliensis promastigotes for 5 and 24 hours in 2D models (coverslips) and 3D models (type I collagen matrix). Infection was analyzed by immunofluorescence and confocal microscopy, evaluating the adhesion index, actin organization, and Rab5 recruitment. The t-test was used for statistical analysis, considering differences statistically significant when p<0.05. In the 2D model, the adhesion index was significantly higher than in the 3D model at both time points (p=0.0164 and 0.0039 for 5h and 24h, respectively). In 3D, adhesion decreased at 24h compared with 5h (p=0.0173). Rab5 staining was intense in 2D as early as 5h, but absent in 3D at that time point, appearing only at 24 h around infected cells. Actin was observed in all infected groups, with reorganization around the parasite, suggesting active participation in invasion, especially in the 3D model. Invasion mechanisms of Leishmania Brasiliense’s, involving Rab5 recruitment and actin cytoskeleton reorganization, are modulated by the experimental environment and infection time. The 3D model-imposed barriers to parasite adhesion, showing lower adhesion compared with 2D and an additional reduction over time, possibly reflecting progressive internalization of parasites. These findings reinforce the importance of three-dimensional models for studying host–pathogen interactions in more realistic contexts and contribute to guiding new therapeutic strategies aimed at preventing parasite entry and survival in macrophages.
Brígida et al. (Sun,) studied this question.