The Role of Nitric Oxide and Reactive Oxygen Species in the Killing of Leishmania braziliensis by Monocytes from Patients with Cutaneous Leishmaniasis.

Pedro Paulo Carneiro,Jacilara Conceição,Viviane Magalhães,Edgar M Carvalho,Olivia Bacellar,Michael Macedo,Simona Stäger

doi:10.1371/journal.pone.0148084

Abstract

Human cutaneous leishmaniasis (CL) caused by Leishmania braziliensis, presents an exaggerated Th1 response that is associated with ulcer development. Macrophages are the primary cells infected by Leishmania parasites and both reactive oxygen species (ROS) and nitric oxide (NO) are important in the control of Leishmania by these cells. The mechanism involved in the killing of L. braziliensis is not well established. In this study, we evaluate the role of ROS and NO in the control of L. braziliensis infection by monocytes from CL patients. After in vitro infection with L. braziliensis, the oxidative burst by monocytes from CL patients was higher when compared to monocytes from healthy subjects (HS). Inhibition of the ROS pathway caused a significant decrease in the oxidative burst in L. braziliensis infected monocytes from both groups. In addition, we evaluated the intracellular expression of ROS and NO in L. braziliensis-infected monocytes. Monocytes from CL patients presented high expression of ROS after infection with L. braziliensis. The expression of NO was higher in monocytes from CL patients as compared to expression in monocytes from HS. A strong positive correlation between NO production and lesion size of CL patients was observed. The inhibition of ROS production in leishmania-infected monocytes from CL patients allowed the growth of viable promastigotes in culture supernatants. Thus, we demonstrate that while production of ROS is involved in L. braziliensis killing, NO alone is not sufficient to control infection and may contribute to the tissue damage observed in human CL.

Highlights

Leishmaniasis is caused by protozoan parasites of the genus Leishmania transmitted by sandfly vectors
We first assessed the oxidative burst in monocytes from cutaneous leishmaniasis (CL) patients after infection with L. braziliensis compared with the production by monocytes from healthy subjects (HS)
We evaluated whether or not L. braziliensis infection was able to induce the oxidative burst in monocytes from CL patients, and the production and role of reactive oxygen species (ROS) and nitric oxide (NO) in parasite killing

Summary

Introduction

Leishmaniasis is caused by protozoan parasites of the genus Leishmania transmitted by sandfly vectors. IFN-γ has been shown to synergize with TNF, to activate inducible nitric oxide synthase (iNOS or NOS2) to produce nitric oxide (NO) resulting in eradication of intracellular parasites [11, 12]. Several studies have shown iNOS is present in lesions of patients with cutaneous leishmaniasis caused by Leishmania braziliensis and Leishmania tropica [5, 13, 14], [15, 16]. In monocytes from humans unexposed to L.braziliensis, we have shown that both promastigote and amastigote forms of L. braziliensis induce an oxidative burst but control of parasite replication is dependent on ROS [17]. Patients with CL due to L. braziliensis produce high amounts of IFN-γ and TNF, but despite an environment adverse to leishmania proliferation, the infection progresses to disease. NO production, rather than control parasite growth, is associated with pathology

Material and Methods

Evaluation of the oxidative burst

Results

Discussion