Evaluation of the effects of Leishmania braziliensis exosomes in the production of Inflammatory mediators by macrophages

Abstract

Exosomes are 30-100nm organelles secreted by various cell types. They occur when the endosomal membrane invaginates into multivesicular bodies (MVB) and are secreted through the melding of MVBs with the plasma membrane (PM). Leishmania exosomes are composed by several proteins such as heat shock proteins, annexins, GP63, proteins with signaling activity and also contain mRNAs and miRNA. Over the past 10 years it has been reported that these vesicles actively manipulate host signaling and immune cell functions, due to the enrichment by Leishmania exosomes of virulence factors such as GP63. Studies demonstrate that Leishmania donovani exosomes activate tyrosine-phosphatases, downregulating IFN-γ and inhibiting the expression of microbicidal molecules such as TNF and NO, which creates a microenvironment that favors parasite proliferation. Despite of not having immunological memory, studies suggest that after a first stimuli mononuclear phagocytes can get “trained”, passing through epigenetic changes, and responding more effectively against a second stimuli. That way, the sensitization of macrophages with L. braziliensis exosomes, prior to its infection by this pathogen, may be associated with higher inflamatory cytokines production as well as inflammasome activation; once the pathways involved in this process would already be activated. Methods and Results: Healthy subjects’ macrophages were sensitized for 24 hours with L. braziliensis exosomes. Afterwards, these cells were infected with L. braziliensis and cultured for 24 hours. We observed higher levels of IL-1β and IL-6 in cultures that were sensitized before infection than in cells only infected. Furthermore, stimulation with L. braziliensis exosomes induced production of IL-1β, IL-6, IL-10 & TNF by macrophages. We inhibited the secretion of exosomes by L. braziliensis prior to macrophage infection and observed that cells infected with those parasites induced less production of IL-1β, IL-6, IL-10 & TNF and cultures presented lower infection rate than cells that were infected with regular Leishmania. Exosome stimulation also induced the consumption of NLRP3 inflammasome in macrophages and the blocked of these receptors resulted in lower levels of IL-6, TNF and IL- 1β. Our results suggest that L. braziliensis exosomes stimulate macrophages leading to inflammation and those effects might be NLRP3 dependent.