In vivo involvement of polymorphonuclear neutrophils in Leishmania infantum infection

Background The role of lymphocytes in the specific defence against L. infantum has been well established, but the part played by polynuclear neutrophil (PN) cells in controlling visceral leishmaniasis was much less studied. In this report we examine in vivo the participation of PN in early and late phases of infection by L. infantum. Results Promastigote phagocytosis and killing occurs very early after infection, as demonstrated by electron microscopy analyses which show in BALB/c mouse spleen, but not in liver, numerous PN harbouring ultrastructurally degraded parasites. It is shown, using mAb RB6-8C5 directed against mature mouse granulocytes, that in chronically infected mice, long-term PN depletion did not enhance parasite counts neither in liver nor in spleen, indicating that these cells are not involved in the late phase of L. infantum infection. In acute stage of infection, in mouse liver, where L. infantum load is initially larger than that in spleen but resolves spontaneously, there was no significant effect of neutrophils depletion. By contrast, early in infection the neutrophil cells crucially contributed to parasite killing in spleen, since PN depletion, performed before and up to 7 days after the parasite inoculation, resulted in a ten-fold increase of parasite burden. Conclusions Taken together these data show that neutrophil cells contribute to the early control of the parasite growth in spleen but not in liver and that these cells have no significant effect late in infection in either of these target organs.


Background
The protozoan Leishmania (L.) infantum (or chagasi) and L. donovani are responsible for visceral leishmaniasis (VL) which remains a significant cause of mortality in many developing countries (OMS). The parasite is inoculated as a flagellated extracellular promastigote by a sandfly vector through the skin of a susceptible host, mainly dog (L. infantum) and human (L. infantum and L. donovani). Once in the host tissue, the parasite disseminates throughout the reticulo-endothelial system reaching its target organs -bone marrow, spleen and liver. The main cell target is the macrophage where parasites reside and multiply in parasitophorous vacuoles as obligate intracellular amastigotes. Progressive intracellular infection can be interrupted by generation of T cell dependent specific immunity that induces, mainly through IFN gamma secretion or regulation, macrophage activation, followed by Leishmania killing or by control of its growth.
In the liver of experimentally L. infantum or L. donovani infected BALB/c mice, intracellular amastigotes multiply rapidly during first weeks of infection and the granulomatous tissue response which then occurs, results in a clearing of the parasite [1][2][3][4][5][6][7][8][9]. By contrast, the spleen becomes a site of chronic infection, where parasites survive for the life of the host [8,10], with progressive destruction of the architecture and loss of follicular dendritic cells and germinal centres [11][12][13]. The role of cell-mediated immunity and particularly lymphocytes in the specific defence against L. infantum has been considerably studied, but the part played by polymorphonuclear cells (PMN) and more particularly the neutrophils (PN) in controlling the disease is much less well established. Previous in vitro or in vivo studies on other parasite species have demonstrated the presence of PMN in areas of L. amazonensis destruction during acute and chronic phases of the infection [14,15] and showed that PMN could ingest and kill L. donovani promastigotes in vitro [16][17][18][19]. Contrasted results have been obtained in studies on dermotropic L. major species. One study showed that susceptible and resistant mice depleted for neutrophils exhibited accelerated parasite spreading, increased footpad swelling and augmented parasite counts in the lesion draining lymph nodes as compared to non-depleted mice [19]. Another study reported that the transient depletion of PMN in BALB/c mice inhibited Th2 cell development and responses and led to a partial resolution of the footpad lesions [20]. In L. donovani infected C57BL/6 mice the depletion for neutrophils induced an important enhancement of parasite growth in both liver and spleen [21]. Only one report has been devoted to ex vivo study, showing that PMN from infected dogs can phagocytize and kill L. infantum promastigotes [22].
In the present report, we investigated PN as L. infantum phagocytic cell and its role in controlling the early and late stage of experimental disease using neutrophil-depleted L. infantum infected BALB/c mice.

In vivo ingestion of promastigotes by human PMN
When examining bone marrow for diagnosis, observation of L. infantum amastigotes localised in human polymorphonuclear cells is an infrequent finding. Figure 1 shows the presence of such intracellular amastigotes in a patient's bone marrow smear, clearly indicating that the ingestion of Leishmania by PMN occurs in human VL.

In mouse, early in vivo involvement of neutrophils occurs in spleen but not in liver
In order to study the early in vivo participation of PN in host defence, spleen and liver of mice were examined by electron microscopy 1 h, 2 h, 5 h and 24 h hours after L.  infantum inoculation. In liver, the parasites were readily observed already at 1 h and 2 h after the infection. All parasites were localised close to or inside macrophages and no Leishmania were detected inside neutrophil cells, at any time of observation. In contrast, in spleen tissue, parasites were much less abundant and several fields had to be screened in order to find Leishmania. A part of spleen neutrophils was found to contain at least one parasite in a more or less degraded state. Figure 2 shows that within one hour post infection, the intraneutrophil protozoan, although recognised by parietal tubules, flagellar pocket and longitudinal-cut flagella, generally lost its ultrastructural integrity. The electron microscopy images do not at all instances allow determining the precise intracellular parasite localisation, so a quantitative evaluation in spleen of the percentage of parasitised macrophages versus parasitised neutrophils would be non-reliable. Twenty-four hours after the infection, neutrophil influx was still observed in the spleen ( Figure 3) but no intraneutrophil parasites were detected. In the parasitised monocytes ( Figure 4) Leishmania appeared relatively intact as compared to that phagocytised by PMN.

Effect of mAb RB6-8C5 on cell counts in blood and spleen
To carry out depletion experiments, we used mAb RB6-8C5, which has been shown to bind and lyse neutrophils and eosinophils in vivo and in vitro [19,23,24]. Table 1 shows that in mouse blood, 48 h after a single intra peritoneal injection of the mAb, the neutrophil percentage was reduced 9-fold and lymphocyte percentage was increased two-fold while 6 days after the injection, the counts from depleted and control mice (which received isotype-matched mAb) were comparable. In spleen, flow cytometric analyses of cell suspensions were performed 22 days after the RB6-8C5 mAb injection. Although the total number of cells was notably increased [(105 ± 10) × 10 6 , (362 ± 60) × 10 6 , (545 ± 65) × 10 6 cells per spleen of control, infected RB6-8C5-untreated, infected RB6-8C5treated mice, respectively], the mAb treatment showed no effect on the percentage of B cells, macrophages, Mac1+ cells and CD4+ cells. The only sub-population affected was that of CD8+ cells, percentage of which was decreased two-fold ( Figure 5).

Effect of neutrophil depletion on parasite burden
In the first series of experiments, we studied the role of neutrophils in the early stage of infection. RB6-8C5 mAb was administered as a single injection to four groups of

Figure 4
Electron microscopy analysis of early parasite phagocytosis in liver of infected mice. Micrograph of liver, 1 h after L. infantum inoculation (magnification × 4000).  Figure 6 shows that whereas hepatic amastigote loads in depleted and control groups were similar, in the spleen the parasite load increased 12-fold in neutrophil depleted mice as compared to controls. The data shown in Fig.  6 relate to mice that were RB6-8C5-treated 5 h before Leishmania infection; the same enhancing effect of neutrophil depletion on parasite multiplication was observed in all four groups of mice treated with antineutrophil mAb at different times. These results indicate that early in infection, in the spleen but not in liver, the neutrophil cells play a role in controlling L. infantum burden, and this effect is well detectable even after the state of depletion wears off.
Next, to examine the effect of neutrophil depletion in the late stage of infection, mice infected 3 months earlier were treated with RB6-8C5 mAb twice a week, for 4 weeks (4 RB6-8C5-treated mice and 4 controls). No significant increase of amastigote load was found either in the spleen, or in the liver (not shown), indicating that neutrophils are not significantly involved in the host protection against L. infantum in chronically infected mice.

Discussion
Obligate intracellular micro-organisms such as Leishmania, have adopted many diverse strategies for their replication inside the host (reviewed in [25]) and the host resistance is thought to depend on the development of specific cell-mediated immunity acting through activation and expansion of CD4+ T lymphocytes of the Th1 subset [26,27]. These activated T cells secrete cytokines such as gamma interferon that activate the mononuclear phagocytes turning them into cells with potent leishmanicidal activity (reviewed in [2]). This activity of macrophages is non-specific in nature since it is also effective against heterotologous microorganisms with unrelated antigenic specificities  [16][17][18] have suggested that these cells might be involved in the inhibition of the parasite multiplication. However, only very few studies using neutrophil-depleted mice infected with L. major [19] or L. donovani [21] have provided direct evidence for the participation of PN in non-specific resistance to these infections and only one report [22], studying the ex vivo phagocytosis by PN from infected dogs, has suggested a possible role for these cells in the infection with L. infantum.  In this report we show in vivo that neutrophil granulocytes are effectively capable to ingest and kill L. infantum promastigotes. The in vivo implication of PN, occasionally observed in human bone marrow as illustrated in Fig  1, was examined in mouse spleen and liver. The very early engagement of neutrophils and their ability to efficiently eliminate L. infantum were confirmed in the spleen using electron microscopy analyses. Indeed, as early as one hour after the infection, many PN were found to harbour 1 or more parasites presenting a marked loss of ultrastructural integrity. By contrast, in the liver more numerous parasites and in a less degraded state were observable. In both organs, parasites appeared undegraded in monocytic cells.
To further characterise the role of PN, we next performed depletion experiments in early and late phases of L. infantum infection, using the mAb RB6-8C5 directed against mature mouse granulocytes. The apparent health status of mice was unaffected neither by a single mAb administration (which efficiently reduced the number of circulating PN for at least 48 h) nor by repeated injections. We showed that in the early phase of L. infantum infection, the neutrophil cells play a protective role in spleen, since early PN depletion resulted in marked increase of the parasite growth. At the time of the parasite load assessment, the subpopulation of CD8+ cells was significantly decreased in the spleen of PN depleted animals. This reduction of CD8+ cells could be due to their neutrophil-dependent recruitement as observed in some studies [31] but also may be related to a possible recognition of this cell subset by RB6-8C5 [32]. No significant effect of neutrophils depletion on the parasite load was evidenced in liver.
Very interesting results published recently [21], reported that the resistance of B cell-deficient C57/BL6 mice to L. donovani infection was accompanied by neutrophil dependent destructive pathology in liver. In C57/BL6 mice neutrophil depletion led to enhancement of parasite growth both in liver and in spleen of B-cell deficient and wild type animals. It is noteworthy that in this study [21], neutrophil depletion of BALB/c mouse caused in spleen the enhancement of parasite growth close to that reported here, and a much lower, but significant, increase of liver burden. Therefore, the effect of neutrophil depletion on liver parasite multiplication might depend both on the mouse strain as well as the infecting parasite species. The lack of effect on liver parasite load observed in our study might be related to the relatively weak neutrophil influx elicited by L. infantum infection to this organ, as compared to spleen. It is noteworthy that in mouse liver amastigote load is initially larger than that in spleen but it resolves spontaneously, while the spleen supports a sustained parasite load [33].
In chronically infected mice, long term administration of anti-neutrophil antibody, did not significantly enhance parasite counts in either liver or spleen. Therefore, neutrophil cells do not appear to be involved in the late phase of L. infantum infection, in contrast to the early phase where they crucially contribute to parasite killing. The precise mechanisms of Leishmania destruction remain to be established. Some reports [34][35][36] suggest that PN could lyse infected host cells. A second hypothesis is that PN kill extracellular parasite before they enter macrophage or even after they have been released by necrotic cells. In this case, as already hypothesised [21], reactive oxygen intermediates, which play an important role in the early regulation of parasite growth and which are among others produced by PN under the control of TNFα, might be responsible for the parasite destruction.
Finally, PN could function as immunoregulatory cells by releasing soluble mediators such as TNFα [37] that regulate the entry and intracellular multiplication of parasite in host cells.
We hypothesise, that the neutrophil-generated early destruction of Leishmania, although beneficient for the host in terms of the early control of parasite growth, might be also responsible for induction of inflammatory pathology in the spleen which subsequently contributes to the lack of parasite clearance and chronicity [33].

Parasites
L. infantum MON1 (MHOM/FR/94/LPN101), isolated from a patient with visceral leishmaniasis, was maintained by serial passages in Syrian hamsters. The promastigote form was cultured and prepared for infection experiments as described previously [8][9][10]. Alternatively, animals previously infected for three months were injected as above twice a week during four weeks and analysed 2 days after the last injection. The assessment of the amastigote burden in livers and spleens was carried as described previously [8][9][10]. The neutrophil depletion was assessed by microscopic examination of Giemsa-stained blood smears, or cell sorting analyses of mouse blood. Neutrophil depletion did not lead to any deterioration in apparent health status of mice.

Tissue processing for electron microscopy
Samples of livers and spleens were fixed for 1 h with 2.5 % glutaraldehyde (Merck) in PBS buffer and cut into pieces of approximately 1 mm3. The material was fixed for another 24 h washed in the buffer, postfixed in 1% osmium tetroxide in buffer for 2 h, washed in buffer, dehydrated with graded ethanol, and embedded in Epon (TAAB). Ultrathin sections were poststained with uranyl acetate and lead citrate, examined, and photographed using a Zeiss 1200EXII transmission electron microscope.