Unleashing the immune modulatory potential of Leishmania amazonensis-derived extracellular vesicles in American Cutaneous Leishmaniasis

Bruna Eugénia de Freitas^1,2Maria Armanda Rodrigues^1*Joana Palma-Marques¹Juliana Inês Weber¹Ana Valério-Bolas¹Rodrigo Pedro Soares³Ana Claudia Torrecilhas⁴Micheli Ferla¹Munira Muhammad Abdel Baqui⁵Raul Alexander Gonzáles Córdova⁵Graça Alexandre-Pires⁶Isabel Pereira Fonseca⁶Helida Monteiro de Andrade²Gabriela Santos-Gomes^1*

• ¹Institute of Hygiene and Tropical Medicine, New University of Lisbon, Lisbon, Portugal
• ²Instituto de Ciências Biológicas, ICB, Universidade Federal de Minas Gerais, Fundação Osvaldo Cruz, Belo Horizonte, Brazil
• ³Biotecnology applied to pathogens group (BAP) Instituto René Rachou, Fiocruz, Belo Horizonte, Brazil
• ⁴Insituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de S. Paulo (UNIFESP), São Paulo, Brazil
• ⁵Department of Cellular and Molecular Biology and Pathogenic Bioagents, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
• ⁶Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisboa, Lisboa, Portugal

American cutaneous leishmaniasis (ACL) constitutes a neglected skin disease that causes severe disability and significant social stigma for millions of people each year. This parasitic infection is caused by several species of the protozoan Leishmania, including Leishmania amazonensis. There is an urgent need to develop effective new tools to control ACL, which is exacerbated by the growing burden of the disease and its social impact. In recent years, scientific research has focused on extracellular vesicles (EVs), which are lipid-enclosed nanostructures that carry macromolecules to recipient cells. The role of Leishmania-derived EVs in host pathogenesis has attracted considerable attention among researchers, with studies suggesting that EVs may play a key role in modulating the host immune response. This study examined the immunogenicity and protein cargo of EVs shed by L. amazonensis, exploring their effect on immune activation in the murine macrophages (MΦs). Nanoparticle tracking analysis, microscopy, proteomic methodologies, colorimetric assays, serological immune methods, PCR, and multiparametric flow cytometry were employed. EVs derived from L. amazonensis cultured promastigotes contain key components, such as the 63 kDa surface glycoprotein, intracellular heat shock protein 70, and alpha-type proteasome subunit, which may be involved in parasite survival. Moreover, EVs are recognized by mouse-and human-specific antibodies, indicating that they have the potential to elicit humoral immune responses and can be inactivated by host-specific antibodies. Depending on the concentration, EVs can drive MΦs to express MHC molecules that are essential for antigen presentation to T lymphocytes, being able to promote cellular immune response. EVs favor IL-1beta+MΦs contraction, promote low nitric oxide production, and activate the arginase pathway to produce urea along with the generation of proinflammatory cytokines. This MΦs' modulation may support parasite control through the specific activation of T cells while preserving skin homeostasis, reducing the pathology associated with L. amazonensis infection, leading to the development of chronic disease. Thus, this study's findings suggest that although L. amazonensis-derived EVs can trigger MΦs activation, favoring a pro-inflammatory immune response, they also have the potential to ensure parasite survival while limiting host pathogenesis. This can be advantageous for parasite transmission and essential for completing the parasite life cycle.