Background
Leishmania Viannia braziliensis is the primary cause of American Tegumentary Leishmaniasis (ATL) in Brazil. Cutaneous leishmaniasis (CL), the most common form of ATL, is characterized by the appearance of a single or a few ulcerated skin lesions. A fraction (3%) of CL patients develops mucosal disease, characterized by disfiguring lesions that usually involve the nasal or oropharyngeal mucosa [
1,
2]. CL and ML lesion biopsies show an intense inflammatory reaction, characterized by a predominance of lymphocytes and macrophages and few or no detectable parasites [
1]. In areas of endemic
Leishmania transmission, approximately 10% of individuals have a positive Montenegro skin test (MST) but lack disease pathology [
3‐
6]. These individuals are classified as subclinical (SC) or asymptomatic infection cases. The immunological response in CL and ML has been well-characterized. The type-1 immune response predominates in CL and ML, with an exaggerated production of IFN-γ and TNF-α that fails to be modulated either by IL-10 or TGF-β or by neutralizing antibodies to IL-12 and IL-15 [
7,
8]. Lymphocytes from ML patients proliferate more than those from CL patients when stimulated with parasite antigens and produce higher levels of IFN-γ and TNF-α [
7,
9]. This exacerbated type-1 immune response is unable to control the disease and contributes to pathological tissue damage. In contrast, SC patients produce only low levels of IFN-γ and TNF-α [
10].
Macrophages, the host cells for the intracellular parasite
Leishmania, are crucial for the outcome of disease. However, very little is known about macrophage behavior in human
Leishmania infection. In addition to presenting antigens to T cells, macrophages secret chemokines and cytokines involved in the inflammatory response, such as IL-1β and TNF-α in response to
Leishmania infection. Lesions from CL patients show increased expression of CCL2, CXCL9 and CXCL10 [
11]. CCL2 and CCL3 are known to increase the leishmanicidal ability of human macrophages to the same level induced by IFN-γ [
12]. However, molecules such CXCL9, CXCL10 and TNF-α, which are highly expressed in the tissue from CL and ML patients, participate in the inflammatory reaction that may lead to the tissue damage observed in CL and ML [
11,
13].
When
Leishmania parasites enter host macrophages, an oxidative burst occurs that is characterized by an increase in reactive oxygen species and reactive nitrogen intermediates, such as nitric oxide (NO). The mechanisms used by human macrophages to kill
Leishmania are not yet well established. While NO production plays a role in killing
Leishmania in mice, its role in humans is still questionable [
14‐
16].
The aim of the present study was to characterize the immune response of macrophages from CL, ML, and SC patients and from healthy subjects (HS) after exposure to Leishmania. We evaluated the susceptibility of macrophages to infection by L. braziliensis and the ability of these cells to kill Leishmania. Additionally, we investigated the role of NO and O2
- in killing the L. braziliensis parasite. We also evaluated the production of chemokines (CCL2, CCL3, CXCL8 and CXCL9) and TNF-α.
Methods
Subjects
This study included patients with CL (n = 22), ML (n = 11) or SC (n = 18) and HS controls (n = 14). All patients were seen at Corte de Pedra Health Post, located in the southeast region of the State of Bahia, Brazil, which is well known for its high rate of L. braziliensis transmission. Some experiments did not include samples from every patient because some of the experiments in this study required a larger number of cells than could be obtained from the patients. The diagnostic criteria were a clinical picture characteristic of CL or ML, parasite isolation or a positive delayed-type hypersensitivity (DTH) response to leishmania antigen, and histological features of CL or ML. Household contacts of ATL patients without past or current history of leishmaniasis with a positive DTH test and/or IFN-γ production in cultures stimulated with SLA were considered as having subclinical infection. The physical examination of these individuals did not show a typical scar of CL. Immunological analyses were performed prior to therapy for all patients. Healthy subjects were composed of students at the Federal University of Bahia.
This study was approved by the Ethical Committee of the Federal University of Bahia. Informed consent was obtained from all enrolled subjects.
Parasites
A
L. braziliensis (MHOM/BR/2003/LTCP15344) isolate obtained from a skin lesion on a CL patient was initially cultivated in biphasic medium (NNN). Following isolation, the parasites were cryopreserved in frozen nitrogen. The parasites selected for this study had not been previously passaged in liquid culture medium. After selection, the parasites were expanded in complete Schneider's medium. The isolate was identified as
L. braziliensis by multilocus enzyme electrophoresis [
17].
Soluble leishmania antigen (SLA)
The leishmania lysate (crude antigen) was prepared from a
L. braziliensis strain (MHOM/BR/2001) as previously described [
18].
Cell separation and macrophage culture
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood using Ficoll Hypaque (LSM; Organon, Durham, NC). Monocytes were separated by adherence. After 6 days of culture in complete RPMI 1640 (10% heat inactivated human AB serum, 100 U penicillin/ml, 100 μg streptomycin/ml) (GIBCO BRL, Grand Island, NY, USA), the adherent cells displayed characteristics of monocytes-derived macrophages.
More than 99% of the cells in culture were identified as macrophages by microscopic observation. To assess purity of macrophages, adherent cells were removed by harvesting and analyzed by flow cytometry. Macrophages were gated according to their characteristic forward- and side-scatter. Purity was assessed by analysis using the following conjugated mouse anti-human mabs: CD14-PE-Cy5 (macrophages), CD3-PE (T cells), CD19- FITC (B cells), and were confirmed to be CD14+, CD3-, CD19-. The T cell influence on the system was considered to be absent because the adherent cell population consisted of less than 1% CD3+ T cells (data not shown). We also evaluated the production of IFN-γ in supernatants of infected macrophages by ELISA and observed that the IFN-γ levels were very low or undetectable.
Macrophage infection by L. braziliensisand evaluation of chemokines and TNF-α production
PBMC-derived macrophages from HS and from SC, CL and ML patients were infected with L. braziliensis stationary phase promastigotes at a 2:1 ratio. Uninfected macrophages and LPS-stimulated macrophages (100 ng/ml) were used as controls. Infected macrophages were incubated at 37°C in 5% CO2 for 2 h. After incubation, the remaining extracellular parasites were removed by gentle washing. Cells were stained with Giemsa stain after 2, 48 and 96 h of infection, and the infection levels were determined by counting the percentage of infected cells and the number of amastigotes per 100 macrophages. This analysis was performed by two independent observers who were blinded to the experimental conditions. After each timepoint, supernatants were harvested and stored at -70°C for analysis of chemokines, TNF-α NO and O2
- production.
Determination of Nitric Oxide and superoxide production
Nitrite (NO
2
-) accumulation in the cell culture supernatants was used as an indicator of NO production and was determined by a standard Griess reaction [
19]. Superoxide production (O
2
-) was determined by adding hydroxylamine (0.5 mM) to cultures of infected macrophages [
20,
21]. Hydroxylamine converts superoxide into nitrite, which can be quantified by performing the Griess reaction, as described above. Background levels of nitrite generated by the release of NO were determined in parallel, without the addition of hydroxylamine.
Determination of chemokines and TNF-α production
The cell culture supernatants from HS, SC, CL and ML patients were assayed for CCL2, CCL3, CXCL8, CXCL9 and TNF-α after 2, 48 and 96 h of infection by ELISA (BD Pharmingen and R&D Systems, Minneapolis, MN). Purified cytokines were used to derive a standard curve. The sensitivity of the cytokine assays was 15.6 pg/ml (CXCL8), 15.6 pg/ml (CXCL9), 7.8 pg/ml (CCL2), 7.8 pg/ml (CCL3) and 15.6 pg/ml (TNF-α).
Statistical analysis
Kruskal-Wallis with Dunn's post-test was used to analyze the results from the macrophage infection experiments and to compare the levels of chemokines TNF-α, NO and O2
- produced by macrophages. The cut-off for statistical significance was set at p < 0.05. Statistical analysis was performed using Grand Prism 3.0 (GraphPad software, San Diego, CA, USA)
Discussion
Macrophages are important for Leishmania infection because they can host the parasite for a long period of time, act as antigen presenting cells and secrete molecules that induce inflammatory response and may kill Leishmania. In this study we compared the ability of L. braziliensis to infect and survive in macrophages from individuals with different clinical forms of L. braziliensis infection. Furthermore, we measured molecules secreted by these cells. Our data clearly show that individuals with SC L. braziliensis infection can control parasite growth in the absence of T cells or T cell products through the ability of their macrophages to kill Leishmania. Alternatively, macrophages from CL and ML patients allow Leishmania survival and produce high amounts of pro-inflammatory chemokines that attract neutrophils, monocytes and activated T cells that induce an inflammatory reaction and contribute to the pathology associated with the L. braziliensis infection.
Analyzing macrophage function in individuals with different clinical forms of
L. braziliensis fills a gap in our understanding of the pathogenesis of leishmaniasis. The Th1-type immune response controls parasite multiplication and dissemination in CL and ML, but it does not eradicate
Leishmania infection [
22]. Moreover, this exaggerated and non-modulated type-1 immune response observed in CL and ML is associated with the production of pro-inflammatory cytokines and tissue damage [
7]. In contrast, individuals with SC
L. braziliensis infection control parasite growth and do not develop the disease, despite producing only low levels of IFN-γ, the main cytokine that activates macrophages to kill
Leishmania. Therefore, it is likely that in SC individuals the innate immune response plays an important role in the control of parasite growth. Macrophages from sub-clinically infected individuals have been shown to be susceptible to infection by
L. panamensis than macrophages from patients with recurrent leishmaniasis [
23]. In the present study, all groups of macrophages infected with
L. braziliensis had equal numbers of amastigotes at two hours after infection. We conclude that penetration of
Leishmania into macrophages is independent of the clinical group from which the macrophage samples were obtained. However, while the percentage of infected cells and number of amastigotes decreased in macrophages from individuals with SC infection, the parasite load in CL and ML remained high, indicating that cells from SC patients have a greater ability to kill parasites than cells from CL and ML patients. The mechanisms used by human macrophages to kill
Leishmania are not yet well established. NO plays an important role in controlling established murine leishmaniasis infection [
24]. A recent study showed that NO production remained unchanged in a co-culture of human neutrophils with macrophages infected with
L. braziliensis [
25]. Our results show that macrophages infected with
L. braziliensis produced NO and O
2
-, although no differences were observed among the different groups, suggesting that macrophage-derived oxidants are not critical in controlling the macrophage infection of
Leishmaniasis patients by
L. braziliensis. Analyzing different isolates of
Leishmania that originated from patients with CL from an endemic area of
L. braziliensis transmission identified some isolates that were resistant to death by NO, which positively correlated with lesion size [
26]. These data suggest that NO production is not the main mechanism used by macrophages to kill
Leishmania. The interaction between NO and O
2
- leads to the formation of peroxynitrite (ONOO-ROS), which has been shown to have a greater toxic effect
in vitro in amastigotes when compared to NO [
27,
28]. Further studies are warranted to evaluate the role of peroxynitrite in the killing of
L. braziliensis.
Chemokines participate in the inflammatory reaction during
Leishmania infection. While CCL3 was produced in similar amounts by macrophages from all four groups, the production of CCL2, CXCL8 and CXCL9 after
L. braziliensis infection was significantly higher in macrophages from CL and ML patients than in those from SC patients. CXCL8 attracts neutrophils to the inflammatory site where they are phagocytized by macrophages. Depending on whether the neutrophils are apoptotic or necrotic, macrophages can control or allow parasite load growth [
25,
29]. Exogenous addition of CCL2 is a highly specific chemotactic factor for macrophages and T-lymphocytes that enhances
L. infantum killing by human macrophages [
12]. This chemokine showed increased expression in CL lesions compared with control biopsy samples [
30]. The expression of CCL2 and CXCL9 was higher in CL tissue cells than in diffuse cutaneous leishmaniasis tissue [
11]. CXCL9, previously called a monokine, is induced by IFN-γ (MIG) and has the ability to attract T cells to the site of inflammation. These observations suggest that different chemokines may influence the parasite multiplication, the inflammation and the clinical manifestations of leishmaniasis [
12,
30]. However, CL and ML macrophages secreted higher levels of CCL2 and CXCL9 but did not kill
Leishmania, making it more likely that these chemokines are involved more in the inflammatory reaction and tissue damage in CL and ML patients than in protecting them against
L. braziliensis infection. CXCL9 is secreted in high levels in chronic inflammatory and auto-immune diseases, such as human T cell lymphotropic virus infection and multiple sclerosis. In both cases, CXCL9 is associated with inflammation and the disease pathology [
31,
32].
TNF-α plays an important role in protecting humans and mice against leishmaniasis [
33,
34]. However, TNF-α can also mediate the ATL pathology [
35‐
39] Here, we show that macrophages from CL and ML patients produce TNF-α after exposure to
L. braziliensis infection. Macrophages from CL and ML kill fewer parasites than macrophages from SC, which implies that these cells contribute to the pathology of CL and ML by producing pro-inflammatory cytokines.
The explanation for why CL and ML groups produce higher levels of chemokines and TNF-α is now under evaluation. For instance genetic factors such as polymorphisms in TLR receptors may partly explain this difference. Alternatively, parasite factors may also stimulate the production of chemokines and TNF-α. Recent studies have shown a RNA virus that actives TLR-3 in some
Leishmania species associated with mucosal disease [
40]. This study builds on previous observations that individuals with SC infection can control the
L. braziliensis parasite despite a poor type-1 immune response [
10], while the exaggerated inflammatory response and T cell activation observed in CL and ML contribute to the development of ulcers [
35,
41]. In this research, we show that macrophages play a pivotal role in the pathogenesis of tegumentary leishmaniasis by decreasing the parasite load in subjects with subclinical
L. braziliensis infection, even in the absence of T cells and inducing pathology in CL and ML patients, by increasing pro-inflammatory chemokines and cytokines.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AG, CV, EMC and OB participated equally in the study design. AG, CV, LPC and TL participated in the human macrophages infection and NO detection experiments. AG, CV and CB participated in detecting the levels of chemokines and TNF-α. EMC and OB are the principal investigators of this work and followed the work from the beginning to the end and also participated in the writing of the manuscript. All authors read and approved the final manuscript.