Impact of Bacterial Membrane Vesicles on Cellular Responses in Leishmania amazonensis ‐Infected Macrophages In Vitro

Leishmania amazonensis is an intracellular protozoan parasite and etiological agent of Localised Cutaneous Leishmaniasis (LCL). A challenge in managing LCL is the frequent occurrence of secondary bacterial co-infections caused by opportunistic bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. This study aimed to evaluate the effects of Bacterial Membrane Vesicles (BMVs) from these bacteria on macrophages infected with L. amazonensis. BMVs were characterised by Nanoparticle Tracking Analysis (NTA), and their effects on macrophage viability, infection dynamics, and cytokine production were assessed. Macrophages infected with L. amazonensis and subsequently treated with BMVs exhibited reduced parasite burden and cell viability compared to infected controls. The production of cytokines was variably modulated depending on the vesicle source. TNF-α levels were higher in macrophages infected with L. amazonensis and treated with BMVs than in those infected with the parasite alone. Additionally, elevated levels of IL-1β were observed in co-cultures treated with P. aeruginosa membrane vesicles (POMVs), suggesting a stronger inflammatory profile than that induced by S. aureus extracellular vesicles (SEVs). In contrast, IL-4 and IL-6 levels were not significantly affected by either POMVs or SEVs. These results indicate that uninfected and L. amazonensis-infected macrophages respond distinctly to POMVs and SEVs, particularly through enhanced secretion of IL-1β and TNF-α. In conclusion, macrophages exposed to both L. amazonensis and bacterial vesicles simulate a host-parasite-bacteria interaction, activating production of cytokines. Given their immunogenic properties, BMVs may represent promising targets for the development of therapeutic strategies associated with LCL.