Leishmania amazonensis fails to induce the release of reactive oxygen intermediates by CBA macrophages

Summary CBA mouse macrophages effectively control Leishmania major infection, yet are permissive to Leishmania amazonensis. It has been established that some Leishmania species are destroyed by reactive oxygen species (ROS). However, other species of Leishmania exhibit resistance to ROS or even down-modulate ROS production. We hypothesized that L. amazonensis–infected macrophages reduce ROS production soon after parasite–cell interaction. Employing a highly sensitive analysis technique based on chemiluminescence, the production of superoxide () and hydrogen peroxide (H2O2) by L. major- or L. amazonensis-infected CBA macrophages were measured. L. major induces macrophages to release levels of 3·5 times higher than in uninfected cells. This production is partially dependent on NADPH oxidase (NOX) type 2. The level of accumulated H2O2 is 20 times higher in L. major-than in L. amazonensis-infected cells. Furthermore, macrophages stimulated with L. amazonensis release amounts of ROS similar to uninfected cells. These findings support previous studies showing that CBA macrophages are effective in controlling L. major infection by a mechanism dependent on both production and H2O2 generation. Furthermore, these data reinforce the notion that L. amazonensis survive inside CBA macrophages by reducing ROS production during the phagocytic process.


INTRODUCTION
Leishmania are obligate intracellular parasites that cause either visceral or cutaneous leishmaniases. To understand the mechanisms involved in host response to Leishmania, studies using several mouse strains have been carried out (1). The outcome of Leishmania infection is determined by the early events occurring during innate immune response. The main initial events in Leishmania-macrophage interaction are recognition, followed by parasite internalization (2)(3)(4). Parasite recognition may induce macrophages to release reactive oxygen species (ROS), such as superoxide (O ÁÀ 2 ). O ÁÀ 2 production is dependent on the recruitment of NADPH oxidase (NOX) subunits to the membrane of nascent phagosome, resulting in NOX assembly.
O ÁÀ 2 and nitric oxide (NO) are key molecules known to be involved in the macrophage-mediated innate host defence against protozoan parasites (5)(6)(7). O ÁÀ 2 can be produced by macrophages even without any previous activation during the early contact of parasites with the host cell (5). On the other hand, NO is a molecule produced only by activated macrophages. Depending on Leishmania species both O ÁÀ 2 and NO play a crucial role in controlling infections (8)(9)(10). In addition to its own toxicity, O ÁÀ 2 is precursor of other ROS, such as hydrogen peroxide (H 2 O 2 ), hydroxyl radical (HǑ .), hypochlorite (HOCl ) ) (5,6,11). These molecules can combine with NO to produce peroxynitrite (ONOO ) ) that exhibited a high toxic effect against Leishmania parasites (11). A recent in vitro study has demonstrated that there is an association between high levels of O ÁÀ 2 production and the significant leishmanicidal capacity of host cells (12). Nonetheless, some Leishmania species adopt various defence mechanisms to cope with oxidative stress, such as decrease in O ÁÀ 2 production, inhibition of NOX assembly, as well as by expression of antioxidant molecules (11,(13)(14)(15).
CBA mice, while known to be resistant to Leishmania major, are susceptible to Leishmania amazonensis. This model allows the trigger mechanisms involved in Leishmania infection to be identified because of the static genetic background of the host (16). Additionally, CBA macrophages control L. major infection, while they are permissive to L. amazonensis infection (17). We have previously shown that interferon-gamma (IFN-c)-stimulated CBA macrophages produce similar amounts of NO in response to L. major or L. amazonensis infection (17). However, using this model, NO produced in response to IFN-c only played a role in controlling L. major infection, which suggests that L. amazonensis modulates or is resistant to factors that control L. major infection. We hypothesized that L. amazonensis modulates the production of microbicidal molecules other than NO, such as ROS, soon after infection, allowing parasites to survive inside CBA macrophages.
A comparative study endeavouring to evaluate the ability of macrophages to release distinct levels of ROS in response to two distinct Leishmania species has not been previously performed. As the O ÁÀ 2 production at early stages of infection can be crucial to efficient intracellular parasite killing (12), we aimed to characterize ROS production by measuring the levels of O ÁÀ 2 released and H 2 O 2 generated by CBA mouse peritoneal, thioglycolate-elicited macrophages in response to L. major or L. amazonensis stimulation. The data herein show that CBA macrophages exposed to L. major produced high levels of ROS, yet in response to L. amazonensis very low levels of ROS were generated during the phagocytic process.

Parasites
L. amazonensis (MHOM ⁄ Br88 ⁄ Ba-125) and L. major (MHOM ⁄ RI ⁄ ) ⁄ WR-173) parasites were provided by Dr. Aldina Barral (CPqGM ⁄ FIOCRUZ). L. major and L. amazonensis promastigotes were maintained in Schneider's medium plus 10% FBS for up to six passages and were expanded for 3-5 days in Schneider's medium plus 10% FBS to reach the stationary phase, then washed with a saline solution as previously described (16) and finally adjusted to a ratio of ten parasites per macrophage (10 : 1 ratio).

Thioglycolate-elicited peritoneal macrophages
All experiments were performed accordingly to the standards of the Ethics Committee on Animal Experimentation at the Oswaldo Cruz Foundation (CPqGM ⁄ FIOCRUZ). Macrophages were harvested from the 4-day thioglycolateelicited peritoneal cavity of CBA mice as previously described (17). Briefly, macrophages were cultivated in DMEM medium at a concentration of 5 · 10 5 cells ⁄ mL and then plated in 35-mm Petri dishes at 37°C in 5% CO 2 ⁄ 95% humidified air. After 4 h, the nonadherent cells were removed and the cell cultures were incubated overnight.

ROS production by Leishmania-stimulated macrophages
The ROS production by peritoneal inflammatory macrophages response to Leishmania stimulation was estimated using a photon-counting device monitoring chemiluminescence (CL) incorporating a gallium arsenide photomultiplier tube (Hamamatsu R943, Hamamatsu Photonics K.K., Hamamatsu City, Japan). CL emissions from sample dishes, incubated at 37°C in a sealed chamber, were reflected and focused onto the photomultiplier tube. The emitted signal was fed directly to a frequency counter unit, and data were collected in units of photon counts per second (8).
Macrophage cultures were set aside for 3 min to allow for temperature stabilization before sampling. The O ÁÀ 2 production and H 2 O 2 formation were measured using CL. To quantify O ÁÀ 2 production, thioglycolate-elicited peritoneal CBA macrophages (5 · 10 5 cells ⁄ mL) were stimulated with L. major or L. amazonensis promastigotes (10 : 1 ratio) during the first 30 min of parasite-host cell interaction at 37°C in the presence of lucigenin (25 lM). Macrophage cultures were maintained for 30 min at 37°C in the presence of lucigenin (25 lM) to evaluate basal O ÁÀ 2 production (negative control). Opsonized zymosan particles (10 : 1 ratio) were used as positive (18), and latex beads (0AE9 lm; 10 : 1 ratio) as negative controls. The rapid decay values of photon emission in response to the addition of SOD (2AE5 UI ⁄ mL) were verified at the end of each assay, confirming that photon released was as a result of O ÁÀ 2 production. For H 2 O 2 measurement, CBA macrophages were incubated with luminol (25 lM) and immediately exposed to L. major or L. amazonensis (10 : 1 ratio) at 37°C. After 30 min, cell supernatants were collected, and the supernatants were stored at -20°C, centrifuged at 200·g for 3 min prior to peroxide determination using a luminol-dependent CL assay (19). Briefly, luminol (25 lM) was added to cell supernatants, followed by microperoxidase (80 nM). The microperoxidase-dependent H 2 O 2 decay was determined for the next 2 min.

NOX inhibition using apocynin
Apocynin acts as an inhibitor of O ÁÀ 2 production by blocking the phosphorylation and translocation of the p47 phox and p67 phox subunits of NOX to phagosome membrane, resulting in inhibition of NOX assembly (20). To evaluate the role NOX plays in O ÁÀ 2 production induced by Leishmania spp. promastigotes, macrophage cultures (5 · 10 5 cells ⁄ mL) were treated with apocynin (500 lM) for 18 h at 37°C and then infected with L. major or L. amazonensis promastigotes at a 10 : 1 ratio. O ÁÀ 2 production by L. major-and L. amazonensis-infected cells treated with apocynin was measured for 10 min at 37°C in the presence of lucigenin (25 lM). Apocynin treatment (250-1000 lM) did not alter macrophage viability for 48-h culture (data not shown).

Sequential phagocytosis assays
To test whether the parasite-induced effect on O ÁÀ 2 production is an active and specific L. amazonensis-induced mechanism, a sequential stimulation assay was used and Leishmania-infected macrophages were incubated with a second stimulus. Macrophages were initially incubated with L. major or L. amazonensis promastigotes for 30 min. Next, the parasite stimuli were switched, and the cells were incubated for a second 30-min period, with either L. amazonensis or L. major promastigotes (10 : 1), respectively. These sequential stimulations were performed in the presence of lucigenin (25 lM) at 37°C and O ÁÀ 2 production was measured by determining photon counts emitted by stimulated cells.

Data presentation and statistical analyses
O ÁÀ 2 release is represented as the average level of ROS production (n = 11 experiments) by inflammatory macrophages following the addition of L. major or L. amazonensis promastigotes. O ÁÀ 2 production by infected and control cells were also expressed as R max , which represents average of the highest CL response from stimulated cells. H 2 O 2 accumulation is illustrated by a representative experiment (one of five identical experiments). The equation R = R max ⁄ (T max ) T i ) was used to estimate the amount of H 2 O 2 detected in culture supernatants of Leishmaniainfected cells (21). R max = average of the highest CL response from stimulated cells, T max = the point in time (seconds) at which the maximum number of photons is emitted by cells and T i = the time point (seconds) at which cells begin to emit photons. All statistical tests were performed using GRAPHPAD PRISM 4AE00 (San Diego, CA, USA), and analyses used were Student's t-test with Welch's correction, Mann-Whitney U-test or one-way ANO-VA with Newman-Keuls post-test. Differences with P < 0AE05 were considered statistically significant.

RESULTS AND DISCUSSION
L. major but not L. amazonensis induces O ÁÀ 2 production in CBA macrophage cultures.
The present study aimed to evaluate ROS production by macrophages in response to different stimuli. Uninfected macrophages released very low levels of O ÁÀ 2 that ranged from 10 to 122 photon counts (n = 10) and were similar to those detected in macrophage cultures stimulated with latex beads (38AE4-99AE10 photon counts) (n = 1). By contrast, the positive control cultures stimulated with zymosan particles released high levels of O ÁÀ 2 (581AE2-7072AE2, n = 11). Kinetics analysis of O ÁÀ 2 production shows an increase in the O ÁÀ 2 amount when L. major promastigotes were added to cells (Figure 1a). By contrast, L. amazonensis promastigotes fail to induce the release of significant amounts of O ÁÀ 2 ( Figure 1a) which was similar to levels in control nonstimulated macrophages or stimulated with latex beads (data not shown). When dead L. major promastigotes were added to macrophage cultures, no increase in photon counts was observed (Figure 1a), which supports the notion that O ÁÀ 2 production is dependent on L. major viability. The addition of SOD (2AE5 UI ⁄ mL) at the end of each assay confirms that photon released is as a result of O ÁÀ 2 production (Figure 1b). Next, the participation of NOX assembly in O ÁÀ 2 production was evaluated in cells pretreated with apocynin (500 lM). First, pretreatment of L. major-infected cells with apocynin was performed and induced a partial reduction on O ÁÀ 2 production (n = 4, P = 0AE02, Mann-Whitney; Figure 1c). This partial inhibition of O ÁÀ 2 production by apocynin indicates that L. major-induced release of O ÁÀ 2 production is dependent on NOX2 and also on a different NOX, such as NOX4. NOX4 is an NADPH-dependent oxidase that is not inhibited by apocynin (20). It is highly expressed in numerous cell types including endothelial cells (22) and embryonic stem cells (23). Although it has been described that NOX4 is involved in other cell functions (24,25), its role in innate immunity has been suggested (26), so it is possible that this oxidase also participates in the O ÁÀ 2 production involved in the control of Leishmania infection. Then, L. amazonensis-infected macrophages were pretreated with apocynin that did not modify O ÁÀ 2 production by these cells (data not shown). This finding suggests that the O ÁÀ 2 production by CBA macrophages detected during the assay was not dependent upon NOX.
The average value of the maximum number of lucigeninderived photons released (R max ) by macrophages in response to L. major was then calculated and shown to be 276AE10 € 98AE08 photon counts, a value 3AE5 times higher (P < 0AE05; n = 6; Kruskal-Wallis) than the R max detected in uninfected macrophage cultures (40AE70 € 10AE39 photon counts; P > 0AE05; n = 6, Kruskal-Wallis). In addition, the R max values of lucigenin-derived photons in macrophage cultures stimulated with L. amazonensis (177AE30 € 73AE54 photon counts) was not statistically different (P > 0AE05; n = 6, Kruskal-Wallis) from those in control macrophages. These findings show that, different from L. major, L. amazonensis did not trigger O ÁÀ 2 production during phagocytosis. Next, we hypothesized that L. amazonensis inhibits O ÁÀ 2 production in response to L. major infection. To test this hypothesis, sequential phagocytic assays were then performed by incubating cells with L. amazonensis promastigotes for 30 min, followed by a 30-min period of incubation with L. major. As expected, macrophages uniquely infected with L. amazonensis produced very low levels of O ÁÀ 2 (Fig-ure 2). The addition of L. major promastigotes to L. amazonensis-stimulated cells reverted the relatively low levels of O ÁÀ 2 production, which were increased to levels similar to those produced by cells uniquely stimulated with L. major ( Figure 2). Thereafter, cells were primarily stimulated with L. major promastigotes for 30 min, followed by a 30-min period of incubation with L. amazonensis. Interestingly, the addition of L. amazonensis promastigotes to macrophages previously stimulated with L. major did not reverse the L. major-induced enhancement of O ÁÀ 2 production. O ÁÀ 2 levels remained similar to those produced by macrophages which were exclusively stimulated with L. major (Figure 2), showing that L. amazonensis promastigotes did not additionally stimulate O ÁÀ 2 production by macrophages, even when NOX complex was already assembled in response to L. major stimulation. In sum, these findings suggest that the events, regarding O ÁÀ 2 production in response to L. major and lack of production in response to L. amazonensis, are independent of each other.
The mechanism involved in the failure of O ÁÀ 2 production in L. amazonensis-infected cells remained to be elucidated. It is possible that L. amazonensis alters ROS production by host cells, using one of the mechanisms that have been previously described for several microbes: (i) Leishmania donovani promastigotes delay O ÁÀ 2 production by preventing NOX assembly and phagosome maturation (5,13,14), subsequent to maintenance of a periphagosomal (a) (b) (c) Figure 1 Leishmania major promastigotes induce NOX-dependent O ÁÀ 2 production. Thioglycolate-elicited peritoneal macrophages were incubated with L. major or L. amazonensis promastigotes at a 10 : 1 ratio at 37°C for 30 min in the presence of lucigenin (25 lm). Control cells were incubated with dead L. major or dead L. amazonensis promastigotes, as well as zymosan, under the same conditions. O ÁÀ 2 production was measured using lucigenin-based chemiluminescence (CL), expressed in photon counts. L. major promastigotes induce the release of significantly higher amounts of O ÁÀ 2 in comparison with L. amazonensis (n = 11, P < 0AE001, One-way anova and Newman-Keuls), but these levels did not differ significantly from those produced by control macrophage cultures stimulated with dead parasites (a). Lucigenin-based CL decreased in stimulated cell cultures in response to superoxide dismutase (SOD). SOD (2AE5 U ⁄ mL) was added at the end of each assay, which confirms that photon released in response to L. major or zymosan is dependent on O ÁÀ 2 production (one representative experiment out of eight similar experiments) (b). NOX inhibition by apocynin cause partial reduction in lucigenin-based CL. L. major-infected cells were pretreated for 18 h with apocynin (500 lm) prior to the addition of parasites. O ÁÀ 2 production was detected at 37°C for 10 min and was partially reduced by apocynin. Results are expressed as the percentage of the number of photons emitted by apocynin-treated cells (ranging from 57AE6 to 193AE9 photons) in relation to untreated macrophages considered as 100% (ranging from 126AE2 to 318AE7 photons) (n = 4, P = 0AE02, Mann-Whitney U-test) (c). coat of F-actin (5,13,14,27); (ii) Leishmania pifanoi amastigotes avoid O ÁÀ 2 production by inducing an increase in haeme degradation. This results in blockage of the maturation of gp91 phox subunit of NOX, and, subsequently, prevents assembly of the NOX complex (15); (iii) Salmonella typhimurium reduces O ÁÀ 2 production by removing cytochrome b 558 subunit from the phagosomal membrane of infected macrophages (16,28); and (iv) Helicobacter pylori recruits to nascent phagosomes cytochrome b 558 , yet does not efficiently acquire or retain p47 phox or p67 phox components of NOX. This results in disruption of NOX, lack of ROS accumulation inside phagosomes and O ÁÀ 2 release into the cytoplasm (29).

L. major induces H 2 O 2 accumulation in macrophage cultures
ROS generation is a process involving a cascade of events that begins with O ÁÀ 2 production, which dismutates into H 2 O 2 either spontaneously, especially at low pH levels, or via a mechanism dependent on SOD (30,31). In vitro experiments demonstrated a dose-dependent leishmanicidal effect of H 2 O 2 against L. donovani, Leishmania tropica and Leishmania chagasi promastigotes (11). Using the phenol red method, we described previously that L. amazonensis induced the accumulation of half as much H 2 O 2 as was accumulated in L. major-infected macrophages (17). To confirm this, we measured peroxide levels using the more sensitive luminol-based CL method to determine microperoxidase-induced decay of H 2 O 2 (19). The highest amount of H 2 O 2 was detected in supernatants from live L. major-infected macrophages (Figure 3). To illustrate the differences in H 2 O 2 accumulation between L. major-and L. amazonensis-infected cells, the maximal oxidative responses for a specific time interval were calculated using the equation R = R max ⁄ (T max ) T i ) (21). Figure 3(b) illustrates the R values corresponding to H 2 O 2 accumulation in supernatants of L. major-or L. amazonensis-stimulated macrophages. These findings reveal that H 2 O 2 accumulation in supernatants of L. major-stimulated macrophages was 20 times greater than in L. amazonensis-stimulated cells (P = 0AE04, Student's t-test with Welch's correction; Figure 3b).  The luminol-microperoxidase method was used to distinguish H 2 O 2 accumulation from the production of others ROS, which are also detected by luminol-based CL, such as O ÁÀ 2 (32). In addition to the highest levels of O ÁÀ 2 produced by L. major-infected macrophages, it is also conceivable that the initial cellular production of O ÁÀ 2 , followed by subsequent dismutation into H 2 O 2 , was likely responsible for the elevated levels of ROS detected in supernatants ( Figure 3). The data presented herein do not rule out the possibility that other ROS besides H 2 O 2 are released in L. major-infected cultures. In fact, there is evidence that inside macrophages, H 2 O 2 can be converted in a variety of other ROS, such as • OH, HOCl ) (33) and ONOO ) . As ONOO ) exhibited a great toxic effect against Leishmania, it is possible that this compound plays a crucial role in L. major killing inside macrophages from CBA mice (6). Furthermore, the cellular and molecular mechanisms whereby ROS exert their cytotoxic activities are not yet fully described for Leishmania (11). Regarding O ÁÀ 2 and H 2 O 2 microbicidal activity (30,34), we suggest that these molecules may contribute to intracellular events resulting in L. major killing inside CBA macrophages.

CONCLUDING REMARKS
Previous study using the phenol red method showed that L. amazonensis induced the accumulation of half as much H 2 O 2 as was accumulated in L. major-infected inflammatory macrophages (17). These data are in accordance with this study which employed comparative and real-time CL assay, a high-sensitive approach that evaluates ROS production in cell cultures (35,36). A recent study has demonstrated that Leishmania mexicana, a parasite species closely related to L. amazonensis, diminished ROS production in PMA-stimulated macrophages from both BALB ⁄ c and C57BL ⁄ 6 mice (37). Nonetheless, this study is the first report, which demonstrates that two distinct species of Leishmania markedly triggered the production of different levels of ROS in macrophages from a unique mouse strain. The fact that L. amazonensis-infected cells release lower amounts of ROS, in comparison with either uninfected or L. major-infected macrophages, suggests that the inability of CBA macrophages to destroy L amazonensis parasites (7) depend, at least partially, on inefficient ROS production. It has been recently demonstrated by Khouri et al. (12) that exposition of Leishmania braziliensis-or L. amazonensisinfected cells to increasing levels of O ÁÀ 2 induced a severe reduction in the number of intracellular parasites, demonstrating an effective role for O ÁÀ 2 in intracellular parasite killing. Other authors have shown that a low ROS production by Leishmania-infected macrophages is a result of the parasite antioxidative response for ROS production (38,39). However, we present evidence against this idea, because L. major and L. amazonensis parasites did not exhibit any O ÁÀ 2 production and H 2 O 2 formation when incubated alone with lucigenin or luminol, respectively (data not shown) and also did not exhibit any anti-oxidative responses when incubated with O ÁÀ 2 and H 2 O 2 donors (data not shown). Alternatively, the inability of CBA macrophages to kill L. amazonensis may depend on interactions between parasite surface molecules and macrophage receptors (38)(39)(40), which may lead to the modulation of host-cell signalling pathways (41) and a macrophage deficiency in the activation of parasite innate killing mechanisms (42). Also viable parasites can express different surface molecules able to interact with macrophage's surface receptors necessary to induce ROS production. We showed that the genetic background of the host determines the relative degree in which the parasite could be modulating the oxidative response, but further experiments need to be performed to determine the exact mechanism involved in the impairment of ROS production in L. amazonensis-infected CBA macrophages.