|Home | About | Journals | Submit | Contact Us | Français|
Interleukin 17 (IL-17) plays a critical role in inflammation and autoimmunity. Very little is known about IL-17 in protozoa infection. Here we show that lymphocytes from mucosal leishmaniasis (ML) and cutaneous leishmaniasis (CL) produce higher levels of IL-17 than uninfected controls (UC) (p<0.01). There was a tendency for higher number of cells in tissue expressing IL-17 in ML than in CL and a direct correlation between number of cells expressing IL-17 and cellular inflammation at the lesion site (r2 = 0.86, p = 0.0001). This data gives support for the role of IL-17 in the pathogenesis of the inflammatory reaction in leishmaniasis.
IL-17 is a member of a newly identified cytokine family produced predominantly by Th17 cell . IL-17 enhances T cell priming and stimulates fibroblasts, endothelial cells, neutrophils, macrophages, and epithelial cells to produce multiple pro-inflammatory mediators, including IL-1, IL-6, TNF-α, NOS-2, metalloproteinases and chemokines . IL-17 may protect against bacterial, fungal and protozoa infection , but it has been linked predominantly to the pathogenesis of chronic inflammatory and autoimmune diseases. In humans, expression of IL-17 has been detected in sera and target tissues of patients with systemic rheumatic diseases, multiple sclerosis, inflammatory bowel disease and asthma. [4, 5]. In arthritis, IL-17 is directly involved in the destruction of cartilage and bone .
Cutaneous leishmaniasis (CL) is a disease caused by protozoa of the genus Leishmania, characterized by the development ulcerated skin lesions. In areas of Leishmania braziliensis transmission, a small percentage of patients develop mucosal leishmaniasis (ML), concomitantly or after CL. ML affects predominantly the nose, leading to tissue damage and occasionally disfiguring facial lesions. While diffuse cutaneous leishmaniasis is associated with impaired T cell responses against parasite antigen, patients with CL and ML have a strong type 1 immune response to Leishmania, with high production of IFN-γ and TNF-α . However, the infection is only partially controlled and the exaggerated T cell response participates in tissue damage observed in both CL and ML. High concentrations of TNF-α, IL-6, IFN-γ and NO are documented in peripheral blood and tissues and are characterized by an intense inflammatory infiltrate with few or no detectable parasites .
Only a few studies have assessed the role of IL-17 in human infectious diseases and it is not known whether this cytokine participates as a defense mechanism or in the pathology of these diseases. In this study, we showed that IL-17 is produced during L. braziliensis infection. The direct correlation between TNF-α and IL-17 and the correlation between the number of cells expressing IL-17 and the intensity of the inflammatory infiltration indicate that IL-17 may be involved in the pathogenesis of leishmaniasis.
This study included 15 patients with ML and 30 CL patients matched by age (± 5 years) and sex with the mucosal patients. All patients were seen at the health post of Corte de Pedra, Bahia state, Brazil a well known area of L. braziliensis transmission. The criteria for diagnosis were a clinical picture characteristic of CL and ML in conjunction with parasite isolation or a positive delayed-type hypersensitivity response to Leishmania antigen, plus histological features of CL and ML. In all cases the immunological analysis was performed before therapy. Ten uninfected controls (UC) enrolled at the study as control group were healthy subjects leaving in a place where there is no L. braziliensis transmission. This research was conducted with approval of the Ethical Committee of the Maternidade Climério de Oliveira, and informed consent was obtained from each participant.
PBMC were obtained from heparinized venous blood layered over a Ficoll-Hypaque gradient (GE Healthacare, Uppsala-Sweden) then washed and resuspended in RPMI 1640 complete medium with 10% heat-inactivated human AB serum (Sigma, St Louis, MO) at a concentration of 3 × 106 cells/ml. These cells were added to 24-well plates and kept unstimulated or were stimulated with soluble Leishmania antigen (SLA) (5μg/ml) for 96 hours at 37°C, in 5% CO2. After this time, the supernates were collected and stored frozen until analyzed for cytokines. IL-17, IFN-γ and TNF-α was measured by ELISA (R&D Systems, MN).
A total of 16 samples from tegumentary leishmaniasis patients were evaluated. Ten patients presented with CL and six with ML. Tissue biopsies were incubated for 30 minutes in 30% sucrose at 4°C and then transferred into TissueTek media and kept at -70°C until processing. Individual 4-5um cryosections were placed in saline-precoated slides, fixed for 10 minutes with acetone, washed with phosphate buffered saline (PBS) for 15 minutes and submitted to either hematoxilin-eosin (HE) staining or to immunofluorescence using specific monoclonal antibodies. Sections were incubated with an antibody mixture (anti-CD4-FITC and anti-IL-17-PE), and acquired in a laser scanning confocal microscope (Zeiss), as previously done by us . Isotype controls were added to the immunofluorescence reactions, to confirm the lack of non-specific staining. Monoclonal antibodies were purchased from e-Biosciences (San Diego, CA).
Analyses were performed by counting the total number of cells in the fields and calculating the average of cells/section for each patient. The counts were performed blindly and the results were expressed as the average of cells/field for each parameter for each patient and then the values were averaged for each group. The results are representative of two experiments/patient.
Statistical analysis was performed using the Kruskal-Wallis Test with post-hoc testing to compare IL-17 data between UC, CL and ML patients (InStat). Statistical analysis of the confocal data was performed using the JMP statistical software from SAS. The comparisons of means between different treatments for a given parameter were done using non-parametric (one tailed, considering unequal variance of groups) t-Test. Results were considered statistically different when the analysis returned a p< 0.05.
The ability of PBMC from 30 CL and 15 ML patients to produce IL-17 is shown in Figure 1. IL-17 was detected in 70% of CL patients and in all of the ML patients. In cultures that didn't receive stimulus with SLA, the median of IL-17 levels were 0 pg/ml (0-102 pg/ml) and 0 pg/ml (0-59 pg/ml) for ML and CL patients, respectively. There was a tendency for lower levels of IL-17 in patients with short duration of illness, but there was no correlation between IL-17 and severity of the disease, evaluated by size of the lesion (data not shown). The concentration of IL-17 was higher in ML (140pg/ml, ranging from 16- 438 pg/ml) and in CL (50pg/ml, 0- 625pg/ml) than in UC (0 pg/ml, 0-41pg/ml, p<0.001). High concentration of TNF-α (1553pg/ml, 35-3103pg/ml) and IFN-γ (3733pg/ml, 49-19020 pg/ml) were detected in CL and in ML (1538pg/ml,438-3366pg/ml and 10200 pg/ml, 529-22660 pg/ml), respectively. There was a direct correlation between TNF-α and IL-17 (r = 0.5904 and p < 0.0001) and there was an inverse correlation between IFN-γ and IL-17 production (r = 0.4718 and p = 0.0005).
Since IL-17 is thought to be associated with severe inflammatory lesions, we next assessed whether IL-17+ cells were present in biopsies of lesions from CL and ML patients. Biopsies were analyzed for the number of IL-17+ cells, as well as the number of CD4+ cells, expressing this cytokine, using double-staining as described in Material and Methods. Our analysis showed a significant frequency of IL-17+ cells in CL and ML lesions. Approximately 40% of the cells within the inflammatory infiltrate expressed IL-17 in both CL and ML lesions. There was a tendency for a higher number of IL-17+ cells or CD4+IL-17+ in the lesions from ML than CL, with a p value of 0.15 (Figure 2A). Correlative analysis between the intensity of the inflammatory infiltrate (as measured by counting the total number of cells/field) and the number of IL-17+ cells showed a strong positive correlation in lesions from CL (r2 = 0.86, p = 0.0001) (Figure 2B).
T helper cells that produce IL-17 (Th17) have been shown to play a pivotal role in autoimmunity and chronic inflammatory diseases [4, 5]. While IL-17 participates in defense mechanisms against certain pathogens, very little is known about IL-17 production in parasitic diseases in humans. Here we show that IL-17 production by PBMCs is enhanced during the course of CL and ML infection, and that IL-17 is present within the lesions caused by L. braziliensis.
IL-17 was secreted in supernatants of PBMC cultures after stimulation with Leishmania antigen in the majority of the patients with CL and in all patients with ML. The concentrations of IL-17 in CL and ML were much lower than IFN-γ and TNF-α. As an inverse correlation between IFN-γ and IL-17 was documented, it is possible that IL-17 production could be more elevated in these patients. In CL and ML tissue the majority of IL-17 came from Th17, but up to 30% of the cells secreting IL-17 were negative for CD4, indicating that other cell types are secreting IL-17 in these patients. Others have shown that CD8 T cells, γ δ T cells, NK T cells and monocytes can also secrete IL-17  and in CL and ML a high percentage of cells secreting IFN-γ are non CD4+ or CD8+ T cells . Further analysis should be done to determine what other cells are secreting IL-17 in situ.
Several cytokines including TNF-α, IFN-γ and IL-10 play a role in the pathogenesis of CL and ML . We have previously shown an association between TNF-α and IFN-γ production and lesion size in CL . Alternatively, severity of leishmaniasis may be determined by response to antimony therapy. Both variables are dependent on duration of illness. In the present study patients with CL were evaluated between 30 to 90 days after disease presentation, when a classical ulcerated lesion was already established and a small number of ML were studied. Therefore we believe that we did not have sufficient power to evaluate if there was a correlation between IL-17 and severity of the disease in this study, yet an ongoing study is evaluating the association of IL-17 and these variables.
In intracellular infections IL-17 may participate in host defense mechanism as well as in tissue damage. In mice infected with T. gondii IL-17 produced by the innate immune response protects the animal, but was also shown to induce tissue damage  and IL-17 deficient mice have impaired granuloma formation and IFN-γ production upon Mycobacterium bovis bacilo Calmet-Guerin infection . Moreover, a correlation between the self healing of lesions and the frequency of CD4+IL-17+ cells was observed in mice infected with L. braziliensis . CL and ML are associated with an exaggerated type 1 immune response [6, 14], parasites are few in the lesions and drugs that inhibit TNF-α associated with antimony treatment, induce ulcer healing in patients with CL and ML refractory to antimony therapy alone . As TNF-α has been considered an important molecule in pathology associated with CL and ML the direct correlation between IL-17 and TNF-α gives support to the role of IL-17 in tissue damage in leishmaniasis.
This study shows that IL-17 is expressed in peripheral blood and in tissue of leishmaniasis patients and there was an association between cells expressing IL-17 and the intensity of the inflammatory infiltration. These data together with the observation of a correlation between IL-17 and TNF-α point towards the participation of IL-17 in the pathogenesis of leishmaniasis.
To Dr Luiz Henrique Guimaraes, Paulo Machado and Albert Schriefer for participation in clinical assistance of the patients.
Financial support: This study was supported by NIH grant # AI-30639, NIH/Fogarty International Center grant D43 TW007127 and National Institute of Studies in Tropical Diseases (INEDT-CNPq). EMC is a senior investigator of the CNPq.
Conflict of interest: The authors do not have commercial or other associations that might pose a conflict of interest.
Part of the information has been presented in 13th International Congress of Immunology, August 21-25, 2007 at Rio de Janeiro – Brazil as Round Table