Leishmania braziliensis isolated from disseminated leishmaniasis patients downmodulate neutrophil function

Summary Aims The polymorphism observed in Leishmania braziliensis is associated with different clinical forms of leishmaniasis. Neutrophils (PMNs) participate in the pathogenesis of leishmania infection, and here, we evaluate neutrophil function after infection with isolates of L. braziliensis from cutaneous leishmaniasis (CL) or disseminated leishmaniasis (DL) patients. Methods and results Neutrophils from 30 healthy subjects (HS) were infected with isolates of L. (V.) braziliensis obtained from three CL and three DL patients. They were infected at the ratio of 3:1 parasites per neutrophil, and leishmania uptake was evaluated by microscopy. The neutrophil activation markers and oxidative burst by expression of dihidrorhodamine (DHR) were evaluated by flow cytometry and cytokine production by ELISA. The frequency of infected cells and the number of amastigotes were higher in neutrophils infected with CL isolates compared to DL isolates (P < 0.05). The DHR and CD66b expression after infection with DL isolate was lower than with CL isolates. There was no difference regarding chemokine production. Conclusion The L. (V.) braziliensis isolates of DL induced lower respiratory burst and neutrophils activation markers compared with CL isolates which may contribute to parasite survival and dissemination in DL patients.


| INTRODUC TI ON
The American tegumentary leishmaniasis (ATL) is caused predominantly by L. (V.) braziliensis, a parasite that is associated with different clinical forms of the disease as cutaneous, mucosal and disseminated cutaneous leishmaniasis (DL). 1,2 The cutaneous leishmaniasis (CL) is the most common presentation of the disease occurring in over 90% of the cases and is characterized by a well-limited skin ulcer with raised borders. Approximately 3% of CL patients develop concomitantly or months and sometimes years after the cutaneous disease, mucosal leishmaniasis (ML) that affect primarily the nasal mucosa. 3 DL is an emerging clinical form of ATL defined by the presence of ten up to more than 1000 acneiform, papular and ulcerated lesions in at least two parts of the body. 4 In the majority of the cases, DL patients present initially as a typical CL ulcer, and after 1 or 2 weeks or sometimes even during antimony therapy, patients suddenly develop multiple lesions over the body. There are several indicators of the relevance to study this atypical presentation of ATL. DL is an emergent form of leishmaniasis as its prevalence increased 20-fold from 1986 to 2012, 5 up to 40% of DL patients have ML 4,6 and the disease is associated with a high hate of failure to antimony therapy. 7 The pathogenesis of DL is not fully understood. Initial studies showed that DL patients have more negative leishmania skin delayed type hypersensitivity test (LST) and an impairment in lymphocyte proliferation and in the production of interferon-γ and TNF in supernatants of mononuclear cells stimulated with soluble leishmania antigen (SLA) as compared to CL patients. 8 However, more recently, we document that cell-mediated immune response at the lesion site of DL patients is similar to what is observed in CL ulcers. 6,9 We believe that there is no impairment in T-cell response in DL patients and raised the hypothesis that the poor T-cell response observed in cells from peripheral blood was due to the migration of the majority of to the antigen reactive cells to the great number of lesions observed in DL patients. 6 Moreover, these data suggest that parasite, more than host factors, may be involved in the pathogenesis of DL.
It is known that L. (V.) braziliensis is polymorphic and genotypic differences intraspecies of L. (V.) braziliensis are associated with different clinical forms of ATL. 10,11 Specifically, it has been shown that there are six haplotypes presented in four loci of the chromosome 28 of L. (V.) braziliensis that are associated with DL. 10 It has been also documented that some haplotypes in the chromosome 28 are also associated with failure to antimony therapy, indicating the importance of differences intraspecies not only in the presentation of the disease, but also in failure to therapy. 12,13 However, there is a lack of data about the biological behaviour of isolates from DL on phagocytic cells. Neutrophils migrate quickly to the sites of infection and are the main phagocytic cells in the initial phase of L. (V.) braziliensis infection. 14 Studies in experimental models of leishmaniasis have shown that interaction of neutrophils with macrophage may determine the control or progression of leishmania infection. 15 In humans, we have previously shown that neutrophils from CL produce higher levels of reactive oxygen species and pro-inflammatory cytokines than healthy subjects neutrophils upon L. (V.) braziliensis infection. 16 To determine whether the behaviour of L. (V.) braziliensis isolated from patients with DL differs from that observed with parasites from CL patients, we evaluate in the present study the ability of L. (V.) braziliensis isolates from DL and CL patients to penetrate or be up-taken by neutrophils from healthy subjects, as well as whether the genotypic differences among these isolates would influence the neutrophil activation and leishmania killing.

| Subjects
Venous blood was obtained from 30 healthy volunteers from a nonendemic area of leishmaniasis. They denied previous history of leishmaniasis, and none of them have a scar of CL. The group was composed of 17 males and 13 females, and the mean age was 27 ± 12 years. The participants did not have previous contact with Leishmania, and they did not present other infectious diseases and denied symptoms of viral infections and fever at the time of peripheral blood sample collection. The study was approved by Institutional Review Boards (IRBs) of the Federal University of Bahia (Ethical Committee), and informed consent was obtained from all participants.

| Isolation of human peripheral blood neutrophils
Peripheral blood was collected in EDTA, and cells were isolated

| Neutrophils infection
The isolates of 03 CL and 03 DL patients were used to infect PMNs

| Assessment of respiratory burst of neutrophils
The production of reactive oxygen species (

| Evaluation of CXCL8 and CXCL9 production
The supernatants of uninfected neutrophils, cells infected with different L. (V.) braziliensis isolates or stimulated with ionomycin/ PMA were obtained after 90 minutes and frozen at −20°C. The production of CXCL8 and CXCL9 was measured by enzymatic immune assay (ELISA) (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

| Statistical analysis
The Friedman's test was used to analyse the frequency of infected neutrophils as well as the parasite burden, obtained by count of two independent blind analysers. The frequency of cells expressing activation molecules (CD62L and CD66b) and the mean fluorescence intensity (MFI) were analysed by Kruskal-Wallis followed by Dunn's post-test. These tests were also used to evaluate the expression of DHR on CD15 + cells and IL-8 production. Analyses were performed using Prism GraphPad software. The P-value of <0.05 was considered significant.

| Frequency of infected Neutrophils and parasite load after infection with different isolates of L. (V.) braziliensis
The Figure 1A

| Reactive oxidants production by infected neutrophils with different L. (V.) braziliensis isolates
We also evaluated the capacity of different isolates of L. (V.) braziliensis to trigger oxidant production in neutrophils.
Fluorescence of DHR-123, an indicator of the abundance of cellular reactive oxidants, was measured by flow cytometry. The Figure 2 presents the MIF of CD15 + DHR + uninfected cells (medium) and

| Neutrophils activation induced by L. (V.) braziliensis infection
The CD62L and CD66b expressions are widely used to detect neutrophil activation. CD62L is an integrin shed from neutrophil  Figure 2A shows the MIF of CD62L on uninfected and infected neutrophils. Ionomycin plus PMA was used as positive control. There was no difference in the intensity of fluorescence expression of CD62L between neutrophils infected with DL isolate or CL isolate ( Figure 2B). However, the frequency of CD15 + CD62L + cells was higher on neutrophils infected with DL isolate (38 ± 16%) than that observed with CL isolates (19 ± 14%), P ≤ 0.05.
CD66b is neutrophil granule membrane proteins that migrate in the surface membrane upon granule exocytosis. 22 Neutrophils infected with DL isolates expressed less CD66b on cells surface than neutrophils infected with CL isolate, P ≤ 0.001 ( Figure 3C).

| IL-8 and CXCL9 production by PMNs after infection with different L. (V.) braziliensis isolates
The DL or CL patients (P > 0.05). Production of CXCL-9 was lower than IL-8 but follows the same pattern with no difference in supernatants of cells infected with DL or CL isolates (data not shown). There was no difference between IL-8 ( Figure 4A) and CXCL-9 ( Figure 4B) production in cultures infected with CL or DL isolates production of these cytokines were high and similar was observed in cells stimulated with Ion/PMA.

| D ISCUSS I ON
DL is an emerging and severe form of L. (V.) braziliensis infection associated with a high rate of ML and failure to antimony therapy. 4,7 Substantial variability among the leishmania at the subgenus level has been described, and there are evidences that genotypic differences intraspecies may influence the presentation of the disease 10 and response to therapy. 13  The phagocyted microbes are destroyed by oxidative and nonoxidative mechanisms inside the neutrophils cytoplasm. The uptake of microbes induces the oxidative burst, increases reactive oxygen species (ROS) production and this leads to parasites clearance. 27,28 Monocytes produce ROS after exposure to L. (V.) braziliensis, and in this cell, ROS contribute to control parasite multiplication. 29,30 In contrast to monocytes, the ROS generated by infected neutrophils prevents parasite multiplication but did not decrease the number of intracellular amastigotes. 16 This may explain why despite the previous study showed that the use of EDTA rather than heparin or citrate decreases neutrophil activation after stimulation with PMA. 31 However, as in all experiments of our study, EDTA was used the differences observed were related in the source of L. braziliensis isolates rather than methodological aspects.
Following infection or exposure to PMA neutrophils increases the CD66b expression and decreases CD62L expression, surface markers indicative of an activated phenotype. 19,20 The CD66b expression indicates exocytosis from specific granules, and the decreased expression of CD62L is indicative of an increased ability of the cell to migrate out of the circulation. 21 We have previously shown that neutrophils from both CL and HS infected with an isolate of L. (V.) braziliensis obtained from a CL patient are similarly activated. 16 Here, we showed that neutrophils infected with a DL isolate expressed less CD66b than cells infected with CL isolates.
While we cannot ruled out that the lower number of intracellular parasites observed in neutrophils infected with DL isolates may have influenced a decreasing in the expression of neutrophils activation markers and in the oxidative burst, these data clearly indicate that genotypic differences among L. (V.) braziliensis isolates modify neutrophil function.
The CD66b is endogenous in specific granules, and its increased appearance on the neutrophils surface indicates exocytosis from specific granules. 19 The release of proteolytic enzymes, defences and myeloperoxidase from intracellular granules into phagosomes independently cooperates with neutrophil function to enhance microbicidal activity. 14,32 Our observation that isolates from DL induced less CD66b expression on PMN than isolates from CL indicates that parasites from DL decrease the release of granules decreasing neutrophil function. The CD62L is a homing receptor that is cleaved from neutrophils surface upon activation, and its loss facilitates cell migration out of the circulation. 21 Neutrophils are the first cell to arrive in the leish- Neutrophils produce a variety of cytokines and chemokines including IL-12, CXCL-8, CXCL-9, CXCL-10, CCL-3, CCL-4, IL-23 and IFN-γ. 33 We have previously shown that neutrophils from CL patients upon L. (V.) braziliensis infection produce more CXCL-8 and CXCL-9 than neutrophils from HS. 16 Here using HS neutrophil, we observed that IL-8 and CXCL-9 production did not differ in super- show that isolates from DL behaved differently than CL isolates in neutrophils. This find gives support to the hypothesis that genotypically different isolates of the same leishmania species induce different immune responses which may influence disease expression.

ACK N OWLED G M ENTS
We thank Dr. Ricardo Oliveira and Dr. Luciana Cardoso for their support in the development of this work, and Cristiano Franco for his assistance in the preparation of the manuscript.

D I SCLOS U R E S
All authors of this manuscript deny any conflict of interest.

DATA AVA I L A B I L I T Y
The data that support the findings of this study are openly available