Review ArticleLeishmania–macrophage interactions: Insights into the redox biology
Introduction
Widespread in 22 countries in the New World and in 66 in the Old World, leishmaniasis is primarily found in Southeast Asia, East Africa, and Brazil. Human infections occur in 16 countries in Europe, including France, Italy, Greece, Malta, Spain, and Portugal. The disease forms of Leishmania are remarkably diverse. In most cases, the outcome is divided into three major clinical forms: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL). CL causes nonfatal, disfiguring skin lesions and is primarily caused by Leishmania major, L. tropica, and L. aethiopica in the Old World or L. mexicana, L. amazonensis, L. braziliensis, L. panamanesis, and L. guyanensis in the New World. VL, which is fatal when not treated properly, is responsible for thousands of deaths each year and is caused by L. donovani and L. infantum in the Old World or L. chagasi in the New World. MCL is mainly caused by L. braziliensis and occasionally by L. panamensis or L. guyanensis. About 90% of the MCL cases occur in Bolivia, Brazil, and Peru. This form of leishmaniasis is characterized by metastasis of skin lesions to mucous tissues by lymphatic or hematogenous dissemination.
Leishmania species are transmitted by a phlebotome sandfly vector [1]. Most of the leishmaniases are zoonoses, although anthroponotic forms, in which humans are the sole reservoir, exist as well. In contrast to the invertebrate vector, the reservoir hosts are various mammal species including canines, rodents, and marsupials and are responsible for the long-term maintenance of Leishmania in nature [2], [3]. Most reservoir hosts are well adapted and develop subclinical or only mild infections that may persist for years. An important exception is the dog, which may develop a generalized and fatal disease [4].
Leishmania organisms have a relatively simple life cycle, characterized by two principal stages: the flagellated mobile promastigotes living in the gut of the sandfly vector and the immobile amastigotes within phagolysosomal vesicles of the vertebrate host macrophages [2], [4] (Fig. 1). Infected female sandflies transmit the disease by inoculating the promastigote form into the skin during their blood meal. In the vertebrate host, the parasites are phagocytosed by macrophages and dendritic cells in the dermis. After uptake and internalization of promastigotes into a phagosome, fusion with lysosomes proceeds as normal and the parasites survive in the phagolysosome. During this process, the promastigotes rapidly transform into amastigotes within 12–24 h and continue to grow and divide within the phagolysosomal compartment [5]. When a sandfly takes a blood meal from an infected vertebrate host, it ingests amastigote-containing macrophages and monocytes. The amastigotes are released into the sandfly midgut where they develop into flagellated promastigotes. These go through a process called metacyclogenesis, in which the dividing, noninfective procyclic form acquires virulence capabilities and is transformed into a nondividing, infective metacyclic form [6]. The metacyclic promastigotes migrate into the pharynx and buccal cavity, ready for transmission during a next blood meal [7].
The various life-cycle stages have different sensitivities to reactive oxygen species (ROS) and provoke different oxidative responses of the macrophage. After recognition of Leishmania spp., macrophages are activated and become so-called “effector cells” that can phagocytose and destroy the unwanted guest. Various cellular processes start after macrophage activation, including production of phagolysosomal degradation enzymes (e.g., proteases, nucleases, phosphatases, lipases, and esterases), oxidative burst generation, and nitric oxide (•NO) production.
The production of lysosomal enzymes induces a direct toxic effect and acidification of the environment. The oxidative burst provoked by the enzyme NADPH oxidase is a result of the dramatic increase in oxygen consumption that is typical of the phagocytosis process. After macrophage activation, increased concentrations of various cytokines such as IFN-γ and TNFα enhance NADPH oxidase activity and subsequently production of ROS, such as superoxide radical (O2•−). The production of O2•− leads to the spontaneous or enzymatic formation of hydrogen peroxide (H2O2), hydroxyl radical (HO•), hypochlorite (OCl−), and peroxynitrite (ONOO−) [8], [9]. The increased •NO and •NO-metabolite levels in activated macrophages are the result of iNOS (inducible nitric oxide synthase or NOS2) activation. l-Arginine acts as a nitrogenous donor of •NO, which spontaneously converts to nitrite and nitrate. Parasite persistence within the macrophages is determined by a balance between the ability of the immune response to sufficiently activate Leishmania-infected macrophages and the ability of the parasite to resist cytotoxic mechanisms of macrophage activation [10].
The production of these cytokines, ROS, and •NO derivatives normally leads to destruction of the phagocytosed microorganism, but Leishmania spp. are one of the few protozoa that can survive and even replicate in this hostile environment [11]. Understanding this survival process may lead to important information for the research on and the development of new antileishmanial drugs. The existing treatment options for Leishmania are limited and far from satisfactory, thereby endorsing the need for the development of new drugs or therapies.
In this review, the role of ROS in parasite survival, macrophage defense, and treatment is discussed. First, the sensitivity of the various parasite stages to exogenous ROS is presented, followed by an in-depth study of the production of and resistance against the various ROS produced by macrophages as a function of specific phases in the life cycle. Next, the antioxidant defense system of the parasite is discussed, to conclude with an overview of the role of ROS in the treatment of Leishmania.
Section snippets
Susceptibility of Leishmania parasites to exogenous reactive oxygen species
O2•− and •NO are two key players in the macrophage defense system against parasites. In addition to their own toxicity, they give rise to the production of various metabolites such as H2O2 and ONOO−. This section discusses the in vitro and in vivo susceptibility of Leishmania to exogenous (which means donated by a donor or exogenously added enzymes and substrates) free radicals.
Phagocytosis of Leishmania: role of ROS
Promastigotes released during the bite of an infected sandfly are phagocytosed by macrophages. Similarly, amastigotes released after the burst of their host macrophage are rapidly phagocytosed by new macrophages. It is well described that phagocytosis of microbes leads to a burst of O2•− production [8], [39] through activation of NADPH oxidase [40]. Despite its susceptibility to exogenous ROS and •NO, the Leishmania parasite can survive phagocytosis. How the parasite escapes this detrimental
Trypanothione/trypanothione reductase
Mammalian cells depend on glutathione (GSH)/glutathione reductase (GR) to control their intracellular thiol redox state. ROS and oxidized cell components can be efficiently reduced by GSH forming oxidized glutathione disulfide (GSSG). GSH can be regenerated from GSSG by GR [79]. Leishmania does not express this system, but its redox metabolism relies on the glutathione conjugate N1,N8-bis(l-γ-glutamyl-l-hemicystinylglycyl) spermidine, also known as trypanothione (T(SH)2) [80]. Trypanothione
Antileishmanial drugs affecting macrophage or Leishmania redox signaling
Treatment of Leishmania is based on the administration of a few drugs that are suffering from side effects and increasing resistance. Most of the drugs need to be administered parenterally. The target for chemotherapy is the intracellular amastigote that survives and divides in tissue macrophages, thereby rendering the task of antileishmanial therapy difficult.
Antileishmania drug research asks for very specific experimental conditions. The in vitro drug susceptibility differs between log-phase
Future perspectives and conclusion
From the moment the Leishmania parasite enters the macrophage, it protects itself against the macrophage's oxidative burst. The parasite achieves this “passively” through the expression of antioxidant enzymes and proteins, as well as “actively” by the inhibition of O2•− and •NO production in the macrophage. Interestingly, promastigotes and amastigotes exert different inhibiting effects. Whereas the promastigotes reduce the O2•− production only locally in the phagosome, amastigotes cause a
Acknowledgment
Paul Cos is supported by the Fund for Scientific Research–Flanders (Belgium).
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These authors contributed equally to this work.