Leishmaniasis is a parasitic disease caused by the Leishmania spp. protozoa, transmitted to the skin of a mammalian host during the bite of an infected female sand fly vector. Infections range in severity from asymptomatic to disfiguring forms of tegumentary and potentially fatal visceral leishmaniasis [1, 2]. American tegumentary leishmaniasis (ATL) presents a spectrum of clinical manifestations characterized by cutaneous (CL), mucosal (ML), disseminated (DL) and diffuse cutaneous leishmaniasis (DCL). The major Leishmania species that cause ATL in the New World are L. (V.) braziliensis, L. (V.) guyanensis, L. (L.) amazonensis and L. (L.)mexicana. The typical clinical manifestation of American CL is a single ulcerated lesion with elevated borders, frequently located on the inferior limbs [3]. Mucosal leishmaniasis (ML) is a destructive disease that predominantly affects the nasopharyngeal mucosa. The disease is most common in areas of L. (V.) braziliensis transmission and usually occurs months or years after cutaneous leishmaniasis [4]. Disseminated leishmaniasis (DL) has been reported almost exclusively in northern and northeastern Brazil. DL is characterized by the appearance of multiple pleomorphic lesions in two or more noncontiguous areas of the body [5].

Leishmania is a digenetic protozoan with an extracellular flagellated promastigotes form which replicates and matures to the infectious metacyclic form in the gut of the sand fly vector. The promastigotes is transmitted to a mammalian host during the bite of an infected sand fly. Promastigotes undergo facilitated phagocytosis by a macrophage and convert to the obligate intracellular amastigote life stage [6, 7]. Amastigotes survive in macrophage phagolysosomes, a hostile environment for many microbes. Leishmania spp. must undergo profound biochemical and morphological adaptations to survive successfully and multiply within macrophages [7]. The mechanisms through which the parasite resists killing within the toxic environment of the phagolysosome remain incompletely defined.

Leishmaniasis is controlled through cell-mediated immune defenses [8]. Murine models have illustrated that macrophages produce IL-12, which induces CD4+ T cells and NK cells to release interferon gamma (IFN-γ), polarizing the immune response toward a type 1 (Th1 type) phenotype [9, 10]. In murine systems, IFN-γ has been shown to synergize with another macrophage derived cytokine, tumor necrosis factor alpha (TNF-α), activating nitric oxide synthase (iNOS or NOS2) to produce nitric oxide (NO^•) with resultant in killing of intracellular parasites [11–13]. NO^• is generated from the oxidation of the terminal guanidine nitrogen atoms of the L-arginine by NADPH dependent enzyme nitric oxide synthase (NOS) [14]. In murine models of leishmaniasis, NO^•-dependent mechanisms have been shown to be critical for control of Leishmania infection[15, 16]. The role of NO^• in leishmanicidal activity of human macrophages, has been debated [17]. However, recent data suggest that NO^• plays a role in the response of human macrophages to intracellular infections, but the nature of this role is not yet clear [18, 19].

Putative NO^•-mediated leishmanicidal actions in eukaryotic cells include inhibition of mitochondrial respiration, inactivation of peroxidases, increasing susceptibility to oxidant damage, inhibition of glycolysis, mutation of DNA, inhibition of DNA repair and synthesis, S-nitrosylation, ADP-ribosylation, tyrosine nitration of proteins, disruption of Fe-S clusters, zinc fingers or heme groups, and peroxidation of membrane lipids [20, 21]. The Leishmania spp. possesses unique antioxidant mechanisms and enzymes. Notably, they convert their abundant GSH stores to trypanothione (TSH) and use TSH reductase/oxidase systems for redox cycling [22]. The protozoa express an iron superoxide dismutase (SOD) but not a manganese SOD, and they have peroxidoxins for handling oxidative stress [23–26]. Nonetheless, oxidant resistance in these parasites are inducible [27], and one expects these systems are susceptible to inactivation by oxidant species similar to other eukaryotes.

Resistance to nitric oxide has been described in E. coli and M. tuberculosis. Resistant isolates have been associated with a more severe outcome of disease than that caused by non-resistant strains [28]. However, natural NO^• resistance in Leishmania spp. isolates has not previously been described. In the present study, we evaluated the effect of NO^• generated from NaNO₂ (pH 5.0) on the viability of L. (V.) braziliensis and L. (L.) amazonensis promastigotes. NO^• resistant Leishmania amastigotes multiplied significantly better than nitric oxide-susceptible parasites during infection of human macrophages. Furthermore, NO^• resistance was directly associated with lesion size, a clinical measure of disease severity.

In the current study we demonstrated for the first time that some isolates of both L. (V.) braziliensis and L. (L.) amazonensis promastigotes are resistant to killing by nitric oxide. Additionally, we showed that the amastigotes from two resistant isolates survived and multiplied better than susceptible isolates in resting human macrophages in vitro. Macrophages play a pivotal role in Leishmania infection. After phagocytosis, Leishmania promastigotes enter a parasitophorous vacuole within which the macrophage can provide a safe haven for the parasite to transform into amastigotes and proliferate in a naïve host [6, 31]. In an immune host, macrophages can be activated by inflammatory cytokines to produce toxic metabolites that result in intracellular killing of Leishmania [32], or their microbicidal capacity can be dampened or abrogated by suppressive cytokines, leading to disease symptoms [33]. Specifically, TNF-α and IFN-γ elaborated by macrophages or T cells synergize to up-regulate iNOS and the NADH oxidase, with the resultant production of reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI), respectively, that mediate killing of intracellular Leishmania [32–36]. Macrophages can alternatively produce IL-10 and TGF-β that inhibit leishmanicidal activity. Both of these cytokines enable the parasite to grow locally and disseminate to distant sites [37–39].

Many prior studies focus on the host immune response during leishmaniasis and the ability of host cells/cytokines to influence the outcome of Leishmania infection. During the current study we focused instead on the innate susceptibility of the parasite to leishmanicidal molecules, and their ability to resist to a host microbicidal response. We found that human L. (V.) braziliensis and L. (L.) amazonensis isolates differ in their innate susceptibility to killing by RNI in vitro, falling into two groups based on their resistance to nitric oxide. Susceptible isolates of both Leishmania species were nearly totally killed by 8 mM of acidified NaNO₂, whereas NO^•-resistant isolates remained viable even in 16 mM NaNO₂. These divisions were biologically relevant, in that patients with NO^•-resistant cutaneous isolates produced significantly larger cutaneous ulcers than NO^•-susceptible Leishmania spp. isolates. Other clinical parameters were not different between NO^•-resistant and NO^•-susceptible Leishmania, arguing against a spurious association. These clinical data suggest that NO^•-resistance may lead to more aggressive forms of clinical disease. Differences in the time of storage in liquid nitrogen or in the length of in vitro promastigote cultivation could not explain our observations, since both NO^•-resistant and NO^•-susceptible parasites had similar time of storage and were expanded in growth medium only after selection to the present study.

Interestingly, a higher proportion of L. (L.) amazonensis isolates than L. (V.) braziliensis isolates were NO^•-resistant (73% versus 18%, respectively). A comparable published study showed that promastigotes and amastigotes of L. (L.) enriettii were more sensitive to NO^• than L. (L.) major [21]. In conjunction with our data, this suggests that there are inter- and intra-species variations in susceptibility to toxic nitrogen products.

Isolate-specific differences in NO^• susceptibility are consistent with the observed high degree of DNA polymorphism between isolates of L. (V.) braziliensis from several endemic areas of Brazil, documented in literature reports. Techniques used to discern these polymorphic sequences include multilocus enzyme electrophoresis (MLEE) and internal transcribed spacers (ITS) between the small and large subunits of the rRNA gene and polymorphic DNA amplification (RAPD) [40–42]. Utilizing polymorphic DNA amplification (RAPD) we reported DNA polymorphisms in L. (V.) braziliensis isolates from Corte de Pedra (CP), Bahia, the same location from which the current patient isolates were derived. In addition to finding polymorphism among the L. (V.) braziliensis isolates, we published that certain genotypes are associated with specific forms of leishmaniasis [42]. The current study extends these observations to suggest that there are biological differences between L. (V.) braziliensis and L. (L.) amazonensis isolates that correlate with the clinical course of disease. Murine resistance to Leishmania infections depends at least in part on NO^•-mediated intracellular killing of parasites through the action of iNOS [type 2 NO^•-synthase (NOS2)]. However, the contribution of iNOS to parasite killing in human macrophages remains debated. Some reports claim a role for nitric oxide in killing of intracellular M. tuberculosis by human alveolar macrophages [18, 43]. Our group and another has published evidence for a role of nitric oxide in macrophage microbicidal activity toward L. (L.) chagasi/infantum [19, 44]. Nonetheless prior studies have reported difficulty in demonstrating NO^• production by human macrophages [17].

In addition to NO^• derived from the macrophage, the Leishmania spp. themselves are able to produce NO^• [45–47]. It is likely that the parasite additionally has innate mechanisms for NO^• resistance in order to avoid toxicity from endogenous NO^•. As such, the toxic effects of exogenous NO^• generated by macrophages or added experimentally would be expected to represent the sum of NO^• generated by the parasite plus exogenous NO^•, minus the amount of NO^• scavenged or inactivated by innate parasite defense mechanisms. We hypothesize that such anti-NO^• defenses may be utilized by the parasite for anti-oxidant defense in human infections. This hypothesis is supported by our observation that the degree of NO^• resistance correlates with the severity of lesion.

We have previously reported that L. chagasi isolates from Brazilians with visceral leishmaniasis are susceptible to killing by NO^• [19]. We showed in the current study that Leishmania isolates obtained from of humans with CL differ in their susceptibility to NO^•. NO^•-resistant Leishmania isolates were found to enter macrophages at a similar rate as susceptible strains, but they resisted intracellular killing by 72 to 96 h after infection. The timing of intracellular killing is consistent with the kinetics of iNOS induction, which acts 48–72 hours after infection [19]. We hypothesize that NO^• resistance is one of the mechanisms enhancing parasite survival. Alternatively or additionally, NO^•-resistant parasite isolates could inhibit NO^• production by macrophages, or other killing mechanisms such as ROI. Importantly, it has been shown that M. bovis inhibits NO^•-mediated killing by murine macrophages [48], as do Cryptococcus neoformans [49], Trypanosoma cruzi [50], as well, as L. (L.) amazonensis infection [51]. Other studies have reported that amastigote surface enzymes can inhibit NO^• production and thereby reduce leishmanicidal activity [52, 53]. Furthermore, the LPG-associated kinetoplastid membrane protein 11 has been reported to suppress iNOS activity, because it contains monomethylarginine residues, a structural analog of L-arginine, a known inhibitor of iNOS [31, 54].

The meaning of differences in NO^• susceptibility amongst different Leishmania spp. isolates is not entirely straightforward. The finding that NO^•-resistant Leishmania exhibit improved survival within human macrophages may indicate evasion of iNOS catalyzed toxicity as in murine macrophages and a role for iNOS in control of cutaneous leishmaniasis. Alternatively, since NO^• can also play a role in signaling within the infected cell, it is possible that NO^• resistant isolates are changing the intracellular signaling, or resistant to alternate microbicidal effector molecules not tested here. It seems likely that NO^• resistance may contribute to the apparent increased virulence of these parasites in a human host, based on the differences in severity of the clinical parameters evaluated in the present study (significantly larger lesion size, and trend toward more resistant isolates from ML compared to CL patients). Most certainly, factors other than NO^• resistance determine in part the differences in lesion size. Such factors could include the magnitude of parasite inoculum, the host immune response, and the effect of saliva. Nonetheless, it is quite interesting that the NO^• resistance correlates with disease severity in our small study. Future studies are needed to better determine the clinical effects of NO^• resistance on human infection and response to therapy.

These data suggest that nitric oxide-resistance of Leishmania isolates confers a survival benefit for the parasites inside the macrophage, and possibly exacerbates the clinical course of human leishmaniasis.