The Modulation of NADPH Oxidase Activity in Human Neutrophils by Moroccan Strains of Leishmania major and Leishmania tropica Is Not Associated with p47phox Phosphorylation

Polymorphonuclear neutrophils (PMNs) are the first phagocyte recruited and infected by Leishmania. They synthetize superoxide anions (O2−) under the control of the NADPH oxidase complex. In Morocco, Leishmania major and L. tropica are the main species responsible for cutaneous leishmaniasis (CL). The impact of these parasites on human PMN functions is still unclear. We evaluated the in vitro capacity of primary Moroccan strains of L. major and L. tropica to modulate PMN O2− production and p47phox phosphorylation status of the NADPH oxidase complex. PMNs were isolated from healthy blood donors, and their infection rate was measured by microscopy. O2− production was measured by superoxide dismutase–inhibitable reduction of cytochrome C. P47phox phosphorylation was analyzed by Western blot using specific antibodies against Ser328 and Ser345 sites. Whereas we did not observe any difference in PMN infectivity rate, our results indicated that only L. tropica promastigotes inhibited both fMLF- and PMA-mediated O2− production independently of p47phox phosphorylation. Leishmania soluble antigens (SLAs) from both species significantly inhibited O2− induced by fMLF or PMA. However, they only decreased PMA-induced p47phox phosphorylation. L. major and L. tropica modulated differently O2− production by human PMNs independently of p47phox phosphorylation. The inhibition of ROS production by L. tropica could be a mechanism of its survival within PMNs that might explain the reported chronic pathogenicity of L. tropica CL.


Introduction
Leishmania are protozoan parasites causing leishmaniases with 350 million people at risk in about 98 countries or territories, and an incidence of approximately 2 million cases

Ethics Statements
This work was conducted according to the principles specified in the Declaration of Helsinki and under the local ethical guidelines (Ethics Committee for Biomedical Research, Faculty of Medicine and Pharmacy, University Hassan II of Casablanca, Casablanca, Morocco; International Review Board 00002504). The strains used in this study were isolated from the dermal lesions of two patients recruited at the Department of Dermatology (Ibn Rochd University Hospital of Casablanca, Casablanca, Morocco). Patient consent (for adults) or from parents (for minors under the age of 18 years) was obtained by the dermatologist. Oral consent was obtained before samplings. In 2010, oral consent was the sole requirement imposed by the Ethics Committee in Morocco for research purposes. Blood was obtained from healthy adult donors (Établissement Français du Sang, Paris, France). All donors signed informed consent allowing the use of their blood for research purposes.  [23].

Soluble Leishmania Promastigote Antigens (SLAs) Preparation
SLAs were prepared from stationary phase promastigotes of our L. major and L. tropica strains as described by Dey R et al. [24]. Briefly, the promastigotes were washed 3 times in cold sterile phosphate-buffered saline (PBS) and then adjusted to 10 8 promastigotes/mL in PBS. The parasites were disrupted by 10 cycles of freezing (−80 • C) and thawing (37 • C) followed by ultra-sonication (20 times for a period of 30 s). SLAs were aliquoted and stored at −80 • C until use. The protein concentration was determined by the Bradford protein assay (kit Protein Assay, BioRad, Marnes-la-Coquette, France).

Human PMN Isolation
PMNs were isolated from the blood of healthy adult donors under LPS-free conditions by dextran (Millipore SIGMA, Lyon, France) sedimentation, followed by Ficoll density gradient centrifugation at 500× g for 30 min (GE Healthcare Life Sciences, Marcy-l'Étoile, France). Red blood cells were removed by hypotonic lysis.

In Vitro Infection of Human PMNs by L. major and L. tropica Promastigote Strains
The PMNs (10 6 cells) were infected with stationary phase of L. major and L. tropica promastigote strains at a 5:1 ratio (5 parasites per PMN) and incubated for 20 min at 37 • C. Subsequently, PMNs were washed four times with PBS in order to remove non-ingested parasites and then re-suspended in PBS (pH 7.2). Cytospin smears from the cell suspension were stained with 10% Giemsa solution and examined microscopically to determine the PMN infection rate and intracellular parasite load. The rate of infected PMNs was calculated as follows: (Number of infected neutrophils/Total number of neutrophils) × 100. The intracellular parasite load was estimated by the mean number of intracellular amastigotes in 100 cells. The number of infected neutrophils was determined by microscopic analysis (×1000) coupled with a photographic system. The slides were read by two independent experimenters.

Neutrophils Viability
We also assessed the impact of L. major and L. tropica SLAs (5 µg/mL and 10 µg/mL) on PMNs viability. For this purpose, 2 × 10 6 cells were incubated with different concentrations of L. major and L. tropica SLAs for 20 min at 37 • C. Cells viability was evaluated using the Trypan blue exclusion assay.

Superoxide Anion Scavenging Assay
The superoxide anion scavenging potential effect of SLAs was examined in a cell-free xanthine/xanthine oxidase assay system by following the reduction of cytochrome C at 550 nm. The reaction mixture prepared in HBSS contained 0.02 U of xanthine oxidase, and 100 µM of cytochrome C in the presence or absence of increasing SLA concentrations. Superoxide production was initiated by adding 500 µM of xanthine and the absorbance increase at 550 nm was recorded for 10 min at 37 • C in a spectrophotometer (UVIKON ® 860spectrophotometer, KONTRON Instruments, Augsburg, Germany). SOD (75 U) was used to determine reaction specificity.

Statistical Analysis
Results are expressed as the mean ± SEM of at least three independent experiments. The protein bands in Western blots were quantified using Image J 1.43 software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Before performing the analysis, a Gaussian distribution by two different tests of Normality: the Shapiro-Wilk test and the Kolmogorov-Smirnov test attesting the exclusion of non-Gaussian distribution if the p-value was found to be higher than 0.05; the distribution of quantiles along the Henri line in Gaussian "qqplot" (quantile-quantile plot) was also verified graphically for comparison. These verifications were performed in an R software environment version 3.5.3. One-way ANOVA analysis of variance with the Tukey test for multiple comparisons was implemented using GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA). For any experiment design with comparison, factor decomposition was investigated by two-way ANOVA analysis (aov R function) with corresponding dot-and-wisker plots by ggplot2 graph definition in ggpubr R-package.

Infection of Human PMNs by L. major and L. tropica Primary Strains
The L. major (MHOM/MA/2010/L02) and L. tropica (MHOM/MA/2010/L112) strains were isolated from patients living in Casablanca, a non-endemic area for CL. They were infected during a summer stay in a known endemic foci of CL due to L. tropica or L. major. The patients presented with different clinical phenotypes in terms of the number of dermal lesions (simple vs. multiple) and lesion healing time after treatment [15].
We evaluated the infection rate of PMNs in the presence of either L. major or L. tropica primary strains. At 20 min of infection, we observed an average of 6-9 amastigotes per cell ( Figure 1A). The percentages of infected PMNs were of 36.35% ± 6.27 and 38.50% ± 6.65 for L. major and L. tropica strains, respectively. These percentages reached 60.75% ± 8.11 and 64.49% ± 10.13 at 1 h post-infection ( Figure 1B). Thus, no significant difference in PMN infectivity was observed with the two strains of Leishmania used and it was confirmed by two-way ANOVA factorial analysis (Supplemental Figure S1A, species effect p = 0.6092357, with significant time effect p = 7.325 × 10 −4 ).

Impact of L. major and L. tropica Promastigote Strains on O 2 − Production by Human PMNs
PMNs infected with stationary phase promastigotes were stimulated or not with either the bacterial formyl peptide (fMLF, 10 −7 M) or the direct PKC activator (PMA, 100 ng/mL). O 2 − production was measured by an end-point SOD-inhibitable cytochrome C reduction method. Figure 2 shows that neither L. major nor L. tropica promastigotes induced O 2 − production. Next, we evaluated whether they modulated O 2 − production in cells stimulated with fMLF ( Figure 2A) or PMA ( Figure 2B), which was respectively confirmed by two-way ANOVA factorial analyses for each treatement: fMLP (Supplemental Figure S1B, treatment effect: p < 2.2 × 10 −16 ), PMA (Supplemental Figure S1C: treatment effect: p < 2.2 × 10 −16 ). Herein, we observed that L. major significantly potentiated fMLF-induced O 2 − production by human PMNs (p < 0.01), whereas L. tropica significantly inhibited this production (p < 0.01) (Figure 2A). L. tropica also significantly inhibited PMA-induced O 2 •production (p < 0.01), whereas L. major had no effect ( Figure 2B). These results demonstrate that primary L. major and L. tropica strains differently modulated superoxide anion production in human PMNs.  We next evaluated whether soluble Leishmania promastigote antigens (SLAs) modulated O 2 • production. For this purpose, PMNs were incubated with several concentrations of L. major or L. tropica SLAs for 20 min, and then stimulated or not with fMLF ( Figure 3A,C) or PMA ( Figure 3B,D). The superoxide anion production was measured by the SODinhibitable cytochrome C reduction method. Our data showed that neither L. major nor L. tropica SLAs induced O 2 − production in PMNs. However, L. major and L. tropica SLAs significantly inhibited superoxide anion generation induced by fMLF ( Figure 3A,C) or PMA ( Figure 3B,D). Factorial analyses by two-way ANOVA with decomposition of treatment and doses factors in case of SLA-L. major stimulation confirmed significant treatment effect for fMLP (Supplemental Figure S1D, p-value = 6.671 × 10 −7 ) and also significant treatment effect for PMA (Supplemental Figure S1E, p-value = 5.732 × 10 −12 ). The same was found in the case of SLA-L. tropica stimulation factorial analyses, which confirmed a significant treatment effect for fMLP (Supplemental Figure S1F, p-value = 6.079 × 10 −12 ) and for PMA (Supplemental Figure S1G, p-value = 6.007 × 10 −13 ). PMNs (10 6 /mL) were infected at 5:1 ratio for 20 min at 37 • C. The cells were incubated in the presence or absence of fMLF (10 −7 M) or PMA (100 ng/mL). As a control, uninfected and nonstimulated PMNs (Resting), and uninfected and stimulated PMNs (Control) were used. Superoxide anion production was measured by the SOD-inhibitable cytochrome C reduction assay by the end-point method. The superoxide anion production was expressed as nmole/min/10 6 cells in the presence or absence of fMLF (A), or PMA (B). Results are expressed as mean ± SEM from three independent experiments, each performed in duplicate. Data were analyzed using a one-way ANOVA test with Tukey's post-hoc test for multiple comparisons. ** p < 0.01.
Because previous studies have reported that L. donovani has a high effective capacity in scavenging hydroxyl radicals and superoxide anion [27][28][29], we then determined whether the inhibitory effect of L. major and L. tropica SLAs on human PMNs superoxide anion production was related to potential O 2 − scavenging properties. We quantified the production of superoxide anion in a cell-free xanthine/xanthine oxidase assay in the presence or absence of SLAs. The results showed that L. major and L. tropica SLAs had no scavenging effect at the highest concentration used (10 µg/mL) ( Figure 4A). Furthermore, this inhibition was not related to PMN death as assessed by a trypan blue dye exclusion assay ( Figure 4B). Thus, our results showed that fMLF and PMA, in inducing O 2 − production by PMNs, were differently modulated by promastigotes and SLAs. Superoxide anion production was measured by the SOD-inhibitable cytochrome C reduction assay. Results are expressed as mean ± SEM from 4 independent experiments, each performed in duplicate. Data were analyzed using one-way ANOVA test with Tukey's post-hoc test for multiple comparisons. * p < 0.05; ** p < 0.01; *** p < 0.001.

Impact of L. major and L. tropica Promastigotes on the Phosphorylation Status of p47 phox in Human PMNs
Several studies have previously shown that p47 phox cytosolic subunit phosphorylation is a crucial event for NADPH oxidase activation [25,30], notably on Ser345 and Ser328, which are involved in NADPH oxidase priming and activation, respectively [22]. We then examined the impact of L. major and L. tropica on the phosphorylation status of p47 phox ( Figure 5). PMNs were first infected with Leishmania promastigotes, and then stimulated with or without either fMLF or PMA. The phosphorylation status of p47 phox on Ser345 ( Figure 5A) and on Ser328 ( Figure 5B) was analyzed by Western blot, using specific antiphospho-Ser345 and anti-phospho-Ser328 antibodies, as previously reported [25,31]. Our results showed that phosphorylation of p47 phox on Ser345 in freshly unstimulated PMNs was unchanged. fMLF and PMA stimulation had no effect on p47 phox phosphorylation on Ser345, as has already been shown [25]. Furthermore, our results indicated that L. major and L. tropica promastigote infection had no impact on p47 phox phosphorylation on Ser345 after fMLF or PMA stimulation ( Figure 5A), being confirmed by factorial analysis with decomposition of the species and treatment factors (Supplemental Figure S2A, species effect p-value = 0.9013 and treatment effect p-value = 0.7902). On the contrary, we observed that fMLF and PMA stimulation induced a p47 phox phosphorylation on Ser328 ( Figure 5B), which was confirmed by factorial analysis with decomposition of the species and treatment factors (Supplemental Figure S2B, specie effect p-value = 0.20316 and treatment effect p = 0.01772). We also observed that both Leishmania strains induced Ser328 phosphorylation of p47 phox in the absence of stimulation ( Figure 5B). However, nor L. major or L. tropica promastigotes significantly inhibited p47 phox phosphorylation on Ser328 ( Figure 5B); thus, our results indicate that L. major and L. tropica promastigotes modulated O 2 − production induced by fMLF or PMA independently of p47 phox phosphorylation.

Impact of SLAs on p47 phox Phosphorylation in Human PMNs
We then examined the impact of SLAs on the phosphorylation status of the p47 phox on Ser345 ( Figure 6A) and Ser328 ( Figure 6B,C). PMNs were incubated with SLAs (5 and 10 µg/mL), then stimulated or not with either fMLF or PMA. Our results indicated that SLAs from both Leishmania strains had no effect on Ser345 phosphorylation ( Figure 6A), which was confirmed by two-way ANOVA factorial analysis with species and treatment effects (Supplemental Figure S2C, specie: p-value = 0.5424 and treatment: p = 0.8990). Contrary to the promastigote's infection, SLAs did not induce the phosphorylation of p47 phox on Ser328 in PMNs. Furthermore, we observed that SLAs decreased the levels of p47 phox phosphorylated form on Ser328 mediated by PMA, a PKC activator ( Figure 6B,C). Thus, at the dose of 10µg/mL, L. tropica SLAs reduced by 75% this amount, whereas this is reduced by only 50% in the presence of L. major SLAs. Interestingly, we observed no modulation of the p47 phox phosphorylated form on Ser328-induced by fMLF. Altogether, our results indicated that SLAs from Leishmania modulated the phosphorylation on Ser328 of p47 phox , which is important for NADPH oxidase activation, which depends on the PKC pathway. For testing the effect of SLA on P-ser328 p47phox, two-way ANOVA factorial analyses performed with treatment and doses confirmed significant effect of treatments in the case of stimulation with L. major SLA (Supplemental Figure S2D, p-value = 9.545 × 10 −10 ) or with L. tropica SLA (Supplemental Figure S2E, p-value =3.889 × 10 −7 ). Cell lysates were subjected to SDS-PAGE and analyzed by Western blot using specific rabbit polyclonal antibodies (A) anti-phospho-Ser345 p47 phox and (B) anti-phospho-Ser328 p47 phox . The blots were re-probed with rabbit anti-p47 phox antibody as a loading control. The ratio of phospho-p47 phox to the total amount of p47 phox was quantified using Image J.Values. Results are expressed as Mean ± SEM from 5 independent experiments. Data were analyzed using a One-way ANOVA test with Tukey's post-hoc test for multiple comparisons. * p < 0.05; ** p < 0.01. Figure 6. Effect of L. major and L. tropica SLAs on the p47 phox phosphorylation on Ser328 and Ser345. PMNs (4 × 10 6 ) were incubated with SLAs at 10µg/mL (A), 5 µg/mL and 10µg/mL (B,C) and then stimulated with fMLF (10 −7 M) and PMA (100 ng/mL). PMNs were also incubated with SLA in the absence of further stimulation. As controls, PMNs were incubated in medium alone (Resting), PMNs were non infected and stimulated (−). Cell lysates were subjected to SDS-PAGE and analyzed by Western blotting using specific rabbit polyclonal antibodies (A) anti-phospho-Ser345 p47 phox and (B,C) antiphospho-Ser328 p47 phox (B): L. major SLA and C: L. tropica SLA). The blots were probed again with a rabbit anti-p47 phox antibody to assess loading. The ratio of phospho-p47 phox to the total amount of p47 phox was quantified using Image J 1.43u software. Values are expressed as Mean ± SEM from 6 independent experiments. Data were analyzed using one-way ANOVA test with Tukey's post-hoc test for multiple comparisons. * p < 0.05; *** p < 0.001.

Discussion
In Morocco, CL is a major public health problem with two main causative entities: the zoonotic form due to L. major and the anthroponotic form due to L. tropica [32]. Although macrophages are the definitive refuge for Leishmania into the host, neutrophils are considered by many as a transitional refuge for parasites that are able to survive the toxic extracellular environment [33,34]. Furthermore, it has been shown that PMN recruitment at the infection site contributes to host susceptibility or resistance to Leishmania infection [34,35]. Despite the early recruitment of PMNs following Leishmania infection, the impact of the parasites on human PMN functions, especially ROS production is still unclear. In this study, we evaluated whether primary strains of L. major and L. tropica and their respective SLAs modulated superoxide anion (O 2 − ) production by human PMNs and analyzed the phosphorylation status of the p47 phox involved in the activation of the NADPH oxidase [25,30]. We demonstrated that both promastigote strains modulated fMLF and PMA-induced O 2 − production in a different manner. Whereas L. major potentiated fMLF-induced O 2 − production or had no effect on the levels of O 2 − production induced by PMA, L. tropica promastigotes inhibited both. Our results also indicated that SLAs derived either from L. major or L. tropica had no impact on the basal levels of O 2 − production but inhibited O 2 − production induced by fMLF or PMA in a concentration-dependent manner. By analyzing the phosphorylation status of the subunit p47 phox , our results indicated that SLAs reduced the levels of the p47 phox phosphorylated form on Ser328 but had no effect on Ser345 phosphorylation mediated by fMLF or PMA. This effect was more pronounced when using SLAs derived from L. tropica compared to those derived from L. major. This inhibition was only observed in the presence of PMA indicating a PKC-dependent pathway effect. Altogether, our results suggested that L. tropica is more potent in inhibiting PMN O 2 − production than L. major but the precise mechanisms associated with NADPH oxidase modulation need to be further explored.
It is well known that phosphorylation of p47 phox on several serine residues, especially Ser328, is crucial for NADPH oxidase assembly and activation [36]. Although we observed a phosphorylation on Ser328 of p47 phox , upon PMN infection with promastigotes, this was not associated with O 2 − production, suggesting that other processes are probably missing for full NADPH oxidase activation following infection with live parasites. Furthermore, previous studies have shown that phosphorylation of p47 phox on Ser345 is a key event for the priming process, as it allows prolylisomerase Pin1 binding, which then acts as a molecular switch to facilitate the assembly of NADPH oxidase in human PMN [25,31]. However, in the present study, L. major promastigote did not affect Ser345 phosphorylation on p47 phox . Thus, it may induce NADPH oxidase priming through other mechanisms such as an increase in intracellular calcium or lipids messengers, such as arachidonic acid or phosphatidic acid, thereby favoring the assembly of the enzyme [21]. This effect could also include phosphorylation of p47 phox on other sites, translocation of p47 phox and/or p67 phox to the membrane, fusion of specific granules to the phagosome, or activation of the small G protein Rac 2. Moreover, only live parasites were able to induce p47 phox phosphorylation since their respective SLAs did not.
Regarding L. major, our observation is consistent with previous studies indicating that L. major interaction with PMNs did not elicit the production of reactive oxygen species [37]. This inability to induce NADPH oxidase activation was related to the lack of fusion of tertiary and specific granules of neutrophils with Leishmania-containing phagosomes. These granules indeed contain gp91 phox and p22 phox which are involved in superoxide anion generation. On the contrary, other studies have shown that L. major promastigotes are able to induce ROS production in human PMNs [34,35]. Thus, RicciAzevedo et al. observed an increase in ROS production in L. major infected human PMNs compared to uninfected cells [33]. In these studies, ROS production was measured by luminolamplified chemiluminescence, a more sensitive approach than the cytochrome C reduction assay used in our study. This may account for the observed difference. Discrepancies may be related to Leishmania strains, since we used primary isolates of patients living in Morocco, and therefore differences as interspecific and intraspecific polymorphisms of Leishmania proteins were described [38,39].
In addition, SLAs from both strains were neither able to inhibit fMLF-induced p47 phox phosphorylation, although they inhibited PMA-induced p47 phox phosphorylation. It is known that p47 phox could be phosphorylated by several PKC isoforms, and that fMLF and PMA differentially activate these isoforms [30,40]. The difference observed between SLA versus live parasite could be consistent with a general observation that antigens released such as exosomes, differ in terms of promastigote properties [41], that have been reported to be increased following a temperature shift mimicking the parasite's entry into mammalian cells. Similarly, live and dead parasites induced distinct outcomes in infected host cells [42,43]. Therefore, the difference observed between L. major and L. tropica could be related to a difference in exosome release between these two Leishmania strains. However, this cannot exclude the role of additional factors such as lipophosphoglycan (LPG), that strongly inhibits the activity of PKC [44], and is therefore more potent to inhibit PMA than fMLF-mediated p47 phox phosphorylation. Because LPG is polymorphic, particularly LPG from L. tropica, which is the most complex structure [39], the role of LPG in modulating PMN activation should be further analyzed. ROS production is well-known to contribute in the control of intracellular parasites. Recently, Pitale DM et al. reported that ROS production mediated by L. major induces PMN autophagy, which could influence parasite clearance [45]. Our study showed that L. major primed ROS production while L. tropica impaired it in PMNs, a fact that could lead to a significant difference between the two strains regarding disease outcome. The lesions due to L. tropica are usually single, and progress slowly to healing, over several months. In contrast, lesions due to L. major are often multiple, and heal after four to six months [15]. A recent analysis of CL cases diagnosed in Casablanca (Morocco) showed heterogeneity in the appearance of lesions that was not significantly associated with either of the responsible Leishmania species, i.e., L. major and L. tropica [46]. This polymorphic clinical appearance of lesions depends on the immune status of the host, the parasite genotype and burden.
Human infection caused by L. tropica seems to be more insidious compared to L. major infection. Furthermore, there have been reports of the chronic tendency of L. tropica CL as well as the differences in disease progression [47]. In particular, by performing transcriptional profiles of lesions caused by either L. major or L. tropica, we found that the latter presents an aggravated inflammatory/cytotoxic profile [48]. Even if dermal lesions of CL heal spontaneously, the time of healing for L. tropica can be as long as 12 months or more. A we know that this species is mainly considered as anthroponotic and that it is responsible in some countries for Leishmaniasis recidivans or even sporadically for visceral leishmaniasis, it is important to understand the pathophysiology of CL.
The increase in ROS production dampens infection establishment while the impairment in ROS production would favor infection by allowing greater survival of the parasites within neutrophils, thus facilitating their subsequent transfer into macrophages [9]. Additionally, we have previously reported that L. tropica is able to inhibit NO production by macrophages [15], therefore suggesting a more global impact of L. tropica on innate immune cells. It seems that L. tropica has developed a more efficient escape mechanism of innate immunity than L. major. Thus, it is important to understand the pathophysiology of CL going through the study of innate cells and their microbicidal power for a better control of this infection. Presently, the first choice drugs for leishmaniasis chemotherapy are pentavalent antimonials that are significantly toxic and have reported drug resistance, thus reinforcing the need for new therapeutic alternatives.

Conclusions
To the best of our knowledge, this is the first report addressing the impact of primary moroccan Leishmania species on human PMN activity especially on superoxide anions production. ROS production by PMNs is important to control several pathogens, including Leishmania parasites. Overall, our results indicate that L. major and L. tropica modulated O 2 − production by human PMNs differently and independently from p47 phox phosphorylation.
Although the precise mechanism of this effect remains to be assessed, our findings may have potential implications concerning the pathogenesis of this parasite. Indeed, PMNs are among the first innate cells that are massively recruited at the bite site and take up the parasite. It is possible that there could be a threshold of infection level required to stimulate ROS production by PMNs, and/or that parasites may prevent ROS generation by some PMNs. Further study on the mechanisms involved in the PMNs responses triggered by these species need to be developed. Likewise, the effect of these species on NADPH activation also needs more investigation. Although this observation has not been extended to additional primary Leishmania strains, and the mechanism used by these species to inhibit O 2 − production need to be addressed, we believe that inhibition of PMN killing functions would be an escape mechanism and a strategy to persist into the host providing support for the chronicity and spreading of L. tropica in Morocco. Indeed, our previous work demonstrated that this L. tropica strain also inhibited NO production by macrophages. We hypothesize that infected PMNs responding to Leishmania strain phagocytosis with low or absent ROS production could promote the maintenance of a parasite subset that establishes infection. Furthermore, responses of some infected and bystander PMNs may contribute to a local inflammatory environment that is ineffective at parasite clearance.
Therefore, understanding the mechanisms used by some Leishmania spp. to block the neutrophil oxidative burst would be helpful in the development of therapeutic strategies against leishmaniasis.