Estudo da necroptose associada ao eixo Ripk1-Ripk3-Mlkl durante a infecção de neutrófilos por Leishmania Infantum

Abstract

INTRODUCTION: Leishmaniasis is an infectious-parasitic disease caused by the protozoan of the genus Leishmania and it is considered a public health problem in Brazil and in the world. It presents a wide spectrum of clinical forms, with Visceral Leishmaniasis (LV) being the most severe. In Brazil, the agent of VL is the species Leishmania infantum, and it is characterized by high fever, weight loss, hepatosplenomegaly, anemia, leukopenia and haemorrhage in the most severe cases. Neutrophils have been shown to play an important role in the immunopathogenesis of Leishmaniasis, especially in LV. It is known that different cell death pathways can be activated in infected neutrophils, influencing the course of infection by different pathogens. Necroptosis, a regulated death pathway, is canonically activated in the absence of caspase 8, has proinflammatory characteristics and has been described in the context of infectious and non-infectious diseases. OBJECTIVE: The general objective of this work was to evaluate the effect of the necrotic death pathway induction on human and murine neutrophil infection by L. infantum. MATERIALS and METHODS: Initially, we analyzed serum levels of lactate dehydrogenase (LDH), an enzyme characteristic of cellular / tissue damage, in patients with VL, and observed high levels compared to healthy controls. Using the in vitro system of infection of human and murine neutrophils with caspase inhibitors, we demonstrated the activation of the necroptosis death pathway with the involvement of RIPK1, RIPK3 and MLKL. Next, the role of necroptosis in the parasite load of human neutrophils was tested in vitro. RESULTS: Pre-treatment with caspase inhibitors, followed by infection, increased LDH levels, RIPK3 and MLKL expression in human neutrophils, while decreasing the viability of the parasites. This effect was reversed by Necrosulfonamide (NSA), a pharmacological inhibitor of MLKL. In murine neutrophils infected by L. infantum, we also observed the involvement of necrotic pathway molecules, RIPK1 and RIPK3 in the control of parasite viability. In these groups, neutrophils pretreated with caspase inhibitors showed high levels of Reactive Oxygen Species (ROS) and LDH, suggesting the participation of these proinflammatory molecules in the control of infection. Pharmacological inhibition of RIPK1 by Nec-1 and RIPK3 by GSK'872 reversed the death of parasites. Finally, transmission electron microscopy (MET) revealed morphological features associated with necroptosis in neutrophils infected with L. infantum when these were pre-treated with caspase inhibitors, whereas cells infected in the presence of necroptosis pathway inhibitors showed no structural changes. CONCLUSION: These data suggest that inhibition of caspase 8 on neutrophils may contribute to control of L. infantum infection, possibly by stimulating an inflammatory response associated with necroptotic death.