Blockade of TLR2 and TLR4 Attenuates Inflammatory Response and Parasite Load in Cutaneous Leishmaniasis

Human cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is characterized by a pronounced inflammatory response associated with ulcer development. Monocytes/macrophages, the main cells harboring parasites, are largely responsible for parasite control. Toll-like receptor (TLR) signaling leads to the transcription of inflammatory mediators, such as IL-1β and TNF during innate immune response. TLR antagonists have been used in the treatment of inflammatory disease. The neutralization of these receptors may attenuate an exacerbated inflammatory response. We evaluated the ability of TLR2 and TLR4 antagonists to modulate host immune response in L. braziliensis-infected monocytes and cells from CL patient skin lesions. Following TLR2 and TLR4 neutralization, decreased numbers of infected cells and internalized parasites were detected in CL patient monocytes. In addition, reductions in oxidative burst, IL-1β, TNF and CXCL9 production were observed. TNF production by cells from CL lesions also decreased after TLR2 and TLR4 neutralization. The attenuation of host inflammatory response after neutralizing these receptors suggests the potential of TLR antagonists as immunomodulators in association with antimonial therapy in human cutaneous leishmaniasis.


INTRODUCTION
Human cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is characterized by an exacerbated cellular immune response and scarce numbers of parasites in lesions (1). While the presence of pro-inflammatory cytokines, such as IFN-g and TNF, are important for the control of parasite proliferation, total pathogen clearance is not achieved. Moreover, an exaggerated Th1 immune response has been associated with severe inflammation and disease pathology. Notably, levels of IL-10, a regulatory cytokine, remain low or absent (2)(3)(4)(5).
Following migration from peripheral blood to sites of inflammation, monocytes differentiate into macrophages that play an important role in antigen presentation and Leishmania killing (6,7).
The invasion and survival of Leishmania within host cells at early stages of infection involves interactions between molecules present on the surfaces of parasites and monocyte/macrophage receptors (8). Several studies have shown that different receptors mediate the internalization and phagocytosis of Leishmania spp. by macrophages (9). Recently, the participation of toll-like receptors (TLR) in protozoan recognition has been highlighted with regard to cytokine production, as well as in the generation of an effective inflammatory response (8). The binding of leishmania molecules to TLRs triggers the release of mediators, including cytokines, which initiate and regulate inflammatory responses necessary for controlling parasite proliferation and orchestrating the development of an adaptive immune response (10,11).
Many studies investigating the role of TLRs in leishmaniasis have been performed in experimental models, limiting our understanding of the roles played by these receptors in the context of human leishmaniasis. Macrophages from MyD88 -/ -TRIF -/ -L. panamensis-infected C57BL/6 mice, which are unable to activate TLR-dependent pathways, present a decreased ability to secrete TNF and increased parasite burden at early times of infection (12). In contrast, the absence of the receptor in TLR2deficient C57BL/6 mice infected with L. amazonensis decreased parasitic load and enhanced the recruitment of inflammatory cells to the site of infection at early infection stages (13). Viana et al. demonstrated higher TLR2 expression in human monocytes infected with L. braziliensis compared to Leishmania infantum, which was also found to positively correlate with TNF production (14). Our previous work has shown that monocytes from CL patients express ex vivo and after infection with L.braziliensis more TLR2 and TLR4 than monocytes obtained from healthy individuals; again, this was linked to TNF expression (15,16).
Although pentavalent antimony is the first line of treatment for CL, high rates of therapeutic failure have been documented (17,18). Some studies have shown that the administration of antimony in association with immunoregulatory drugs leads to more efficacious treatment outcomes than antimony alone (19)(20)(21). Natural and synthetic TLR antagonists, which reduce TLR signaling and effector functions, have been evaluated in preclinical and clinical models of inflammatory disease (22). The blockade of TLR2 by OPN301 (an IgG1 monoclonal anti-TLR2 antibody) in cells of patients with rheumatoid arthritis was found to decrease the production of TNF, IL-1b and IFN-y after stimulation with Pam3Cysk4 (a TLR2 agonist) (23). The present study endeavored to evaluate whether TLR2 and TLR4 neutralization leads to impaired monocyte infection, consequently modulating the inflammatory response observed in American tegumentary leishmaniasis (ATL) caused by L. braziliensis.

Patients
A total of 34 CL patients and 10 healthy subjects were included. All patients were examined at the Municipal Health Clinic of Corte de Pedra (Bahia, Brazil), located in a known region of L. braziliensis transmission. Diagnostic criteria included a clinical presentation characteristic of CL in conjunction with parasite isolation in culture, pathogen identification via histopathologic analysis, or L. braziliensis positivity by PCR. All patients were treated with i.v. pentavalent antimonial (Sb v ) (meglumine antimoniate; Sanofi-Aventis, Paris-France) daily at 20 mg/kg of body weight for 20 days. All experiments were performed prior to the administration of therapy. The CL group consisted of 20 males and 14 females with a median age of 45 years (range: . The control group was formed by 10 healthy subjects (HS) living in an urban area without exposure to L. braziliensis: 5 males and 5 females, median age 30 years (range: [22][23][24][25][26][27][28][29][30][31][32]. The present study received approval from the Institutional Review Board of the Professor Edgard Santos University Hospital Complex (HUPES-UFBA, protocol no. 693.111). Terms of informed consent were obtained from all study participants.

Parasite Cultures
L. braziliensis (MHOM/BR/2003/LTCP11245) isolates obtained from a skin lesion of a CL patient were initially cultivated in biphasic medium (NNN). Following isolation, parasites were cryopreserved in frozen nitrogen. None of the parasites selected for this study had been previously cultivated in liquid culture medium. After selection, parasites were expanded in Schneider's complete medium and identified as L. braziliensis by multilocus enzyme electrophoresis (24). All reagents and Schneider's medium were determined to be endotoxin-free by limulus amebocyte lysate (LAL) bacterial endotoxin testing (BioReliance, SIGMA-ALDRICH). We used L. braziliensis promastigotes in the stationary phase (14).

Soluble Leishmania Antigen Preparation
Soluble Leishmania antigen (SLA) was prepared from L. braziliensis (MHOM/BR/2001) isolated from a patient with CL as previously described (25). This antigen was tested for endotoxins by LAL and used at a concentration of 5 mg/mL.

Evaluation of L. braziliensis Infection and Parasite Viability Following TLR2 and TLR4 Neutralization
For the neutralization of TLR2 and TLR4, PBMC were incubated for 1 hour (37°C, 5% CO 2 ) in polystyrene tubes containing RPMI 1640 medium with 100uM each of anti-TLR2 and anti-TLR4 (Abcam). Following TLR2 and TLR4 neutralization, PBMC were infected with L. braziliensis stationary phase promastigotes (5:1) for 2 or 48 hours (37°C, 5% CO 2 ). After infection, the numbers of infected cells and intracellular parasites were determined through the microscopic evaluation of 100 monocytes, employing May Grunewald-Giemsa staining from cytocentrifuge preparations. Alternatively, after 72 hours of infection, monocytes were washed and RPMI medium was replaced with 0.5 mL of Schneider's medium (Sigma-Aldrich) supplemented with 10% fetal calf serum to quantify the number of viable parasites. Cells were then cultured for an additional 72 hours at 26°C. Finally, viable extracellular motile promastigotes were counted on a hemocytometer (26,27). Also, PBMC were infected at a ratio of 5:1 for 2 hours with fluorescent GFP-L.braziliensis (MHOM/BR/00/BA/866) grown in Schneider's culture medium supplemented with 20% fetal bovine serum, 100 U/mL penicillin and 100 mg/ml streptomycin, and then maintained at 24°C.
To evaluate TNF expression, the neutralization of TLR2 and TLR4 was performed as described above and cells were then stimulated with SLA (5 µg/mL) for 6 hours (37°C, 5% CO 2 ). All cells were washed with PBS (1,500 rpm, 5 min, 4°C), fixed in 4% paraformaldehyde and permeabilized with BD Perm/Wash ™ solution (BD Biosciences) for 15 min at 4°C in the absence of light. Intracellular staining was performed with anti-TNF (PerCP Cy-5.5) antibodies for 30 min, cells were washed and resuspended in 400 µl PBS for flow cytometry analysis on a BD FACS CANTO ™ II. A total of 200,000 events were acquired. Data analysis was performed using FlowJo software (Free Star Inc.).

Evaluation of TLR2 and TLR4 Expression in Cells From CL Patient Biopsies
Skin samples were obtained from CL patient lesions using a 4-mm punch. Biopsies were treated with collagenase for 90 mins (37°C, 5% CO 2 ), dissociated and cell suspensions were filtered using a 50 mm Medicon filter (BD pharmingen). To evaluate TLR2 and TLR4 expression, surface molecules were labeled using anti-CD14 (APC) (eBioscience, San Diedo, CA, USA), anti-TLR2 PE (clone TL2.1) and anti-TLR4 PE (clone HTA125) (IMGENEX, San Diego, CA, USA). Receptor expression was analyzed by flow cytometry, with 200,000 events acquired per sample.
In addition, biopsies from L. braziliensis patients were cultured in complete RPMI medium in the presence or absence of 100uM anti-TLR2 and 100uM anti-TLR4 for 72 hours (37°C, 5% CO 2 ). Biopsy culture supernatants were collected and stored at −70°C until the time of IL-10, IL-1b, TNF and CXCL9 quantification by ELISA (R&D Systems) in accordance with the manufacturer's instructions. Expression results are expressed in pg/ml.

Statistical Analysis
The Mann-Whitney test was employed for comparisons, while nonparametric Wilcoxon signed-rank testing was used to compare results obtained from cells derived from a single patient under different experimental conditions. All data are presented as medians and respective interquartile range (IQR). Statistical analyses were performed using GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego, CA, USA), with an alpha value of P<0.05 considered statistically significant.

TLR2 and TLR4 Expression Increase in Monocytes From CL Patients During L. braziliensis Infection
The expression of TLR2 and TLR4 receptors was evaluated in monocytes from CL patients and cells from HS after in vitro infection with L. braziliensis promastigotes ( Figure 1). First, we observed the frequency of TLR2 and TLR4 in the same cells from CL patients after L.braziliensis infection, and the expression of TLR4 was higher than TLR2 74(58-94) versus 90(87-98), p<0.05. The same was observed in HS cells, ( Figure 1B). Median TLR2 and TLR4 expression, represented by mean fluorescence intensity (MFI), was found to be higher in L. braziliensisinfected monocytes from CL patients compared to cells from HS ( Figures 1C, D). We observed a positive correlation between GFP MFI with TLR2 and TLR4. However, only TLR4 MFI has significant correlation with GFP MFI (r= 0.7683 and p<0.05) ( Figures 1E, F).

Leishmania Viability After TLR2 and TLR4 Neutralization
The viability of Leishmania promastigotes was estimated by assessing the proliferation of extracellular motile parasites in infected monocytes from CL patients cultivated in the presence or absence of anti-TLR2 and anti-TLR4 antibodies. We observed that the neutralization of both TLR2 and TLR4 resulted in fewer viable parasites: 8 x10 5 (2-10x10 5 ) in comparison to cells cultured in the absence of antibodies: 26x10 5 (19-36x10 5 ) (p<0.05) ( Figure 2E).

TLR2 and TLR4 Neutralization Decreases Oxidative Burst in Monocytes From CL Patients
It has been demonstrated that higher oxidative burst occurs in CL monocytes infected with L. braziliensis compared to HS monocytes; rather than controlling parasite growth, this phenomenon has been associated with pathology (15).
We evaluated the effects of anti-TLR2 and TLR4 antibodies on oxidative burst production by L. braziliensis-infected CL monocytes ( Figure 3A). In the presence of neutralizing antibodies, a significant decrease in the MFI of DHR was seen in cells from CL patients: 626 (104-1091) for the blockade of TLR2 and 496 (214-922) for TLR4, versus 1373 (478-1876) (p<0.01). The neutralization of both receptors further decreased the production of oxidative radicals in monocytes from CL patients to 484 (221-981) versus 1373 (478-1876) (p <0.001) ( Figure 3B). In cells from HS, the neutralization of TLR2 and TLR4 also reduced DHR expression, yet in the absence of statistical significance (data not shown).

TLR2 and TLR4 Neutralization Decreases TNF and IL-1b Production in Monocytes From CL Patients
An association between IFN-g and TNF production and lesion size has been demonstrated in L. braziliensis infection, and these proinflammatory cytokines have also been linked to more severe forms of disease (4,5,28). Furthermore, both inflammasome activation and IL-1b have been associated with disease severity in leishmaniasis (29). We observed lower levels of IL-1b production in L. braziliensis-infected monocytes following the simultaneous neutralization of TLR2 and TLR4: 34 pg/ml (4-55) versus 125 pg/ ml  in untreated cultures (p<0.05) ( Figure 4A). TNF production was also observed to decrease significantly in response to simultaneous TLR2 and TLR4 receptor neutralization: 581 pg/ml (229-858) versus 1204 pg/ml (496-2527) (p<0.01) ( Figure 4B). However, the neutralization of these receptors did not alter IL-10 production ( Figure 4C).

TLR2 and TLR4 Neutralization Decreases the Production of CXCL9 in Monocytes From CL Patients
A previous study documented that L. braziliensis-infected macrophages from CL patients produce more CXCL9 than macrophages obtained from individuals with subclinical infection or cells from HS (30). Accordingly, we investigated the effect of TLR neutralization on the production of CXCL9 and CXCL10, chemokines triggered by IFN-g. The production of CXCL9 was observed to decrease significantly after simultaneously neutralizing both receptors: 2,420 pg/ml (120-12,066) versus 11,693 pg/ml (2,522-17,000) (p<0.001) ( Figure 4D). In contrast, no differences in CXCL10 levels were observed in the presence of neutralizing antibodies ( Figure 4E).

Evaluation of TLR2 and TLR4 Expression After Stimulation With SLA
As the neutralization of TLR2 and TLR4 was observed to decrease the internalization and numbers of L. braziliensis-infected monocytes, cells were stimulated with SLA to investigate the modulation of inflammatory molecule production.

Effect of TLR2 and TLR4 Neutralization on Inflammatory Molecule Production in Cells From CL Skin Biopsies
The inflammatory infiltrate in CL lesions is known to be characterized by cells that produce inflammatory cytokines, such as TNF, IFN-g and IL1b (4,31). A recent study evaluating cells from CL patient skin biopsies reported increased IL-1b production in the supernatants of cultured cells compared to healthy skin biopsies (31). Based on these findings, we investigated whether the neutralization of TLR2 and TLR4 would interfere with the production of proinflammatory molecules (IL-1b, TNF, CXCL9) and IL-10 in biopsy cultures from CL patients.

DISCUSSION
Evidence has shown that an exacerbated inflammatory response is associated with the development of ulcers in cutaneous leishmaniasis caused by L. braziliensis. It follows that the identification of mechanisms capable of modulating this response may aid in the development of immunomodulatory therapies. The premise of our study was based on previous reports demonstrating that monocytes from CL patients express more TLR2 and TLR4 in association with elevated TNF production (16,32). The present results indicate that the neutralization of these receptors can indeed modulate the exacerbated inflammatory response documented in CL caused by L. braziliensis. At early times of infection, the ability of Leishmania to invade and survive within the host is dependent on interactions between molecules present in parasites and host cell receptors, which also influence the outcome of infection. Toll-like receptors, especially TLR2 and TLR4, have been implicated in the recognition of various Leishmania species (8). Different of human CL, it is well known that during active human visceral leishmaniasis, cellmediated immune responses are suppressed and consequently a decrease in IFN-g which is related to the production of regulatory cytokines such as IL-10. In these patients, increased TLR2 and TLR4 expression in lymphocytes and monocytes was associated with increased production of TNF-a, IL-10 and TGF-b and decreased production of IFN-g and NO (33). In diffuse cutaneous (DCL) leishmaniasis that is characterized by uncontrolled parasite dissemination and poor production of IFN-g patients showed reduced NK cell numbers that down-regulated TLR2, TLR1 and TLR6 expression as well as reduced cytokine production, as compared to CL patients (34). The in vitro neutralization of TLR2 and TLR4 led to decreased parasitic load and fewer infected monocytes derived from CL patients. Several studies have previously demonstrated low parasitic load and resistance to infection in TLR2-or TLR4deficient experimental animal models (13,35).
Our findings indicate that both receptors are important for parasite internalization, since neutralization decreased the frequency of infected cells and numbers of intracellular parasites after two hours of infection. These results were then confirmed in experiments employing GFP-L. braziliensis to infect monocytes from CL patients, as flow cytometry revealed a significant decrease in the frequency of infected cells after the simultaneous blockade of both receptors (Supplementary Figure 1).
In Leishmania infection, neutrophils, the first cell type to migrate to the site of infection, are responsible for parasite death (36). A study involving healthy L. amazonensis-infected human neutrophils revealed the expression of TLR2 and TLR4 by these cells. Receptor neutralization subsequently decreased the numbers of infected cells and intracellular parasites, suggesting that these receptors may participate in the internalization of leishmania by other innate immune cells (37).
Parasite proliferation inside phagocytic cells leads to cell lysis and the release of Leishmania (26). Herein, similarly to a reduction in the number of intracellular parasites, we also noted a decrease in the release of viable L. braziliensis promastigotes following the blockade of TLR2 and TLR4, which reinforces the notion that these receptors participate in the internalization of L. braziliensis by host monocytes. There are several reports in literature demonstrating an important role of TLR2 and TLR4 in the infection by Leishmania. However, these papers have not clarified the exact role of these receptors in the uptake of parasites, existing controversial depending on the cell type as well as the Leishmania species (37). Our data support the idea that both receptors seem to be important in the uptake of L.braziliensis, since the TLR2 and TLR4 blockade is the only condition able to decrease the number of internalized leishmania. Alternatively, these receptors may be included in a lipid raft that contains different phagocytic receptors already described for Leishmania (38,39). Our group previously demonstrated that monocytes from CL patients infected with L. braziliensis presented greater respiratory burst compared to cells from HS, which was observed to decrease after treatment leading to lesion cure (15). Here we found that the blockade of TLR2 and TLR4 decreased respiratory burst in infected CL monocytes. Contact between Leishmania lipophosphoglycans (LPG) and TLR4 stimulates the synthesis of NADPH oxidase, thus increasing the production of reactive oxygen species. However, the mechanisms underlying TLR-mediated stimulation of oxidative radical production have not been well elucidated. Srivastava et al. (40) demonstrated elevated TLR2 expression in mouse macrophages infected with L. major, which was associated with a higher oxidative response induced by the recognition of LPG by TLR2. Consequently, these authors noted the activation of MyD88 and increased iNOS expression (40).
Although the production of these oxidative radicals has been linked to parasite elimination in mice, the role of NO in L. braziliensis killing in humans remains unclear. In fact, in CL caused by this species of leishmania, NO production has been correlated with lesion size (15). Hence, it follows that the blockade of TLRs may be an important tool for the inhibition of oxidative radical production, which is more strongly associated with disease pathogenesis than parasite elimination. Although the decline in ROS levels could lead to an increase in the number of parasites, our hypothesis is that the block of TLR2 and 4 lead to a decrease in the uptake of Leishmania, decreasing the parasite burden and therefore, allowing a better response to control parasites and possibly in vivo, decreasing the inflammatory responses.
Several studies have previously demonstrated that the activation of TLR2 and TLR4, as well as the subsequent activation of transcription factors leading to the production of inflammatory molecules, are important in the control of infection by various Leishmania spp. (41,42). Among these molecules, TNF, which is also involved in the control of infection, plays an important role in tissue damage in the context of human disease caused by L. braziliensis. Thus, the present study investigated whether the blockade of TLR2 and TLR4 could interfere with the production of inflammatory molecules by CL patient monocytes. Our evaluation of the production of TNF and IL-1b revealed markedly reduced TNF production following the neutralization of both receptors. LPG, a molecule present on the surface of leishmania that stimulates the production of IL-12 and TNF through the activation of MyD88, constitutes an important ligand for TLR2 (43). Other studies have also shown that LPG and glycoinositol phospholipids, another type of molecule present on the surface of L. braziliensis, induce the production of TNF and NO through binding with TLR4 (44)(45)(46). Thus, since TLR2 and TLR4 are clearly involved in the production of TNF, the neutralization of these receptors expectedly led to decreased production of this cytokine.
IL-1b, a cytokine associated with various inflammatory diseases (31,47), has also been associated with the pathogenesis of cutaneous leishmaniasis (48)(49)(50). As the signaling of TLRs via MyD88, resulting in the activation of NF-ĸb, can also lead to the transcription of inflammatory gene components, mainly IL-1b (51,52), we further evaluated whether TLR blocking could modulate the production of this cytokine. Similar to what was observed with TNF, the neutralization of both receptors decreased the production of IL-1b. Our results are agreed with studies that demonstrate the synergy between the TLR2 and TLR4 for the induction of the immune response (53,54).
Cells from patients with CL caused by L. braziliensis produce either low levels of IL-10 or none at all. Although present in the lesions of these patients, this cytokine's regulatory action may be impaired by the absence of its receptors (3,4). A study conducted in CL patients with mucosal leishmaniasis demonstrated that the neutralization of IFN-g decreased TNF production in association with increased levels of IL-10, suggesting that the excessive production of IFN-g and TNF is associated with the absence or attenuation of a strong IL-10-mediated inflammatory response (28). Thus, we evaluated whether decreases in TNF could also affect the production of IL-10 by CL monocytes. We found no changes in the levels of this cytokine following TRL receptor blocking, which suggests that different pathways are involved in the production of TNF and IL-10 in human CL caused by L. braziliensis. Our results are divergent from those reported by Galdino et al. (55), who demonstrated that the blockade of TLR4 decreased IL-10 production (55). Importantly, in contrast to our experimental model, these authors used PBMCs from healthy individuals primed with IFN-g and subsequently infected them with amastigote forms of L. braziliensis, which could help explain this discrepancy. Alternatively, it has also been reported that CD4 + CD25 − FOXP3 − cells are the source of IL-10 production in CL patients (56).
In addition to the production of proinflammatory cytokines, Leishmania infection induces the expression of numerous genes related to the production of chemokines (35,57). Our results indicate that the expression of TLR2 and TLR4 is associated with CXCL9 production, but not CXCL10.
CXCL9 and CXCL10, chemokines important to the recruitment and activation of Th1-type cells, are notable for participation in the pathogenesis of several inflammatory diseases (58, 59). The activation of TLR2 and TLR4 can also activate the transcription of CXCL9 and CXCL10 via the phosphorylation of IRF3, an interferon regulatory factor (60). In human CL caused by L. braziliensis, macrophages from CL patients produce high levels of CXCL9 compared to cells obtained from individuals with subclinical infection or healthy subjects (30). High systemic production of CXCL9 has been noted in CL caused by L. braziliensis, which becomes even higher in disseminated forms of disease, constituting an emerging and more severe form of CL (61). Another study found the production of this chemokine by PBMCs from CL patients stimulated with SLA to be associated with therapeutic failure to pentavalent antimony (62). Thus, CXCL9 seems to contribute more to the pronounced inflammatory response observed in CL than CXCL10. While no studies in the literature have linked the expression of TLRs to chemokine production in Leishmania infection, some reports on inflammatory disease did detect an association. In autoimmune arthritis, for instance, the stimulation of TLR2 and TLR4 using agonists resulted in a substantial increase in CXCL9 production, which supports the role of these receptors in the expression of this chemokine (63,64).
Since a diminished inflammatory immune response could also occur due to weaker infection that consequently affects the proliferation of parasites in monocytes, we also evaluated the effects of TLR-neutralizing antibodies following stimulation with soluble leishmania antigen (SLA). SLA stimulation was initially observed to increase the expression of TLR2 and TLR4 in monocytes from CL patients compared to unstimulated cultures. Moreover, the neutralization of TLR2 and TLR4 decreased TNF expression in monocytes after stimulation with SLA. These results seem to indicate that, in our in vivo experimental model, even after inducing infection and the partial clearance of parasites, the presence of SLA is able to continue stimulating the immune response by increasing the expression TLR2 and TLR4, in addition to the production of inflammatory molecules (32).
Finally, as TLR2 and TLR4 have been documented in CL patient lesions (65,66), we investigated the role of TLR2 and TLR4 receptors in lesion biopsies obtained from CL patients. Some studies have been shown the TNF and IL-1b is highly expressed in lesions from CL patients (31,67). In this study biopsied cells were observed to express TLR2 and TLR4, while treatment with anti-TLR2 and anti-TLR4 decreased the levels of TNF, IL-1b and CXCL9 production by these cells.
The present findings provide convincing evidence that the blockade of TLR receptors can attenuate the inflammatory response observed in human cutaneous leishmaniasis caused by L. braziliensis. As several studies have demonstrated the use of this approach in the treatment of inflammatory diseases (68), our results present novel perspectives for the development of immunomodulatory therapies that may complement conventional treatment options for cutaneous leishmaniasis.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Institutional Review Board of the Professor Edgard Santos University Hospital Complex. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
PC and OB participated equally in the study design and in the writing of the manuscript. PC and AD participated equally in all of the experiments. WO participated in the human monocytes infection and processing of samples on the flow cytometer.
LG is a physician and participated in the diagnostic of the patients in the endemic area. CB participated in the study design and in the discussion of the results. EC 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 contributed to the article and approved the submitted version.

FUNDING
This work was supported by the National Institutes of Health (AI 136032 to EC). EC and OB are senior researchers at the Brazilian Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Cientıfco e Tecnoloǵico (CNPq).