Host-Parasite Interface: Exploring the Interaction of Trypanosoma cruzi trypomastigotes forms Y strain Extracellular Vesicles (EVs) with Human Monocytes and Macrophage

Introduction: The protozoan Trypanosoma cruzi causes Chagas disease (CD). CD is classified as a neglected tropical disease (NTD) and is estimated to kill 14,000 people across Latin America every year.  The disease's treatment, diagnosis, and epidemiological control remain challenging.  T. cruzi delivers EVs at various stages of its life cycle, which aid in communication with cells and modulation of the host's immune response. EVs may be useful as CD biomarkers and targets for new therapeutic strategies. The goal is to investigate to how T. cruzi-released EVs influence the profile and behavior of primary monocytes and macrophages, with a focus on activation markers.

Methods: Nanoparticle Tracking Analysis (NTA) was used to determine the concentration and size of T.cruzi EVs. Following that, the EVs were labeled with PKH26 Dye Solution (SIGMA). Flow cytometry (FC) and confocal microscopy (CM) were used to assess the interaction of the EVs with THP-1 and differentiated THP-1 after 5, 15, 30 minutes, 1 h, 24 h, and 48 h. After isolating mononuclear cells from whole blood using Ficoll-Paque (SIGMA), human monocytes were isolated using a negative selection strategy according to the commercial Pan Monocyte Isolation Kit, human (Miltenyi Biotec). Monocytes were incubated with EVs that contained the following markers: CD14-V450 (BD Biosciences), CD16-PE-CY7 (ExBio), CD80-FITC (BD Biosciences), CD86-APC (BD Biosciences), CD69-PE (ExBio), HLA-DR-V500 (BD Biosciences), TexasRed (BD Biosciences), and FC.

Results: Our results show that internalization of T. cruzi EVs was uptake using confocal microscopy after only 5 minutes of incubation with human THP-1 cells. Flow cytometry (FC) also confirmed this uptakeand cells interactions. Furthermore, after 24 hours of incubation, significant changes in monocyte morphology were observed. Furthermore, we found a significant increase in the expression of key surface markers on monocytes, which could be important in their differentiation into macrophages.

Conclusion: The T.cruzi EVs interact with host cells, resulting in observable changes in cellular morphology and suggesting the potential to alter the host's immune response profile. These findings provide insight on the quick internalization dynamics of T. cruzi EVs, their effect on cellular morphology, and the implications for monocyte differentiation.