Leishmaniasis is a neglected disease, for which current treatment presents numerous issues. Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The roles of the programmed death-1 (PD-1) receptor on lymphocytes and its ligand (PD-L1) on antigen-presenting cells have been well studied in tumor and other infection models; but little is known about their roles in non-healing cutaneous leishmaniasis. In this study, we observed that L. amazonensis induced PD-1 expression on both CD4+ and CD8+ T cells and PD-L1 on dendritic cells on BALB/c mice. We tested the therapeutic potential of anti-PD-1 and anti-PD-L1 monoclonal antibodies (MoAbs) against a non-healing L. amazonensis infection in BALB/c mice, and that anti-PD-1 and anti-PD-L1 treatment significantly increased IFN-γ-producing CD4+ and CD8+ T cells, respectively. Compared with infection controls, mice treated with anti-PD-1 and anti-PD-L1, but not anti-PD-L2, displayed bigger lesions with significantly lower parasite loads. Treatment did not affect anti-Leishmania antibody (IgM, IgG, IgG1 and IgG2a) or IL-10 production, but anti-PD-1 treatment reduced both IL-4 and TGF-β production. Together, our results highlight the therapeutic potential of an anti-PD-1-based treatment in promoting the reinvigoration of T cells for the control of parasite burden.

Neutrophils play an important role in the outcome of leishmaniasis, contributing either to exacerbating or controlling the progression of infection, a dual effect whose underlying mechanisms are not clear. We recently reported that CD4+ and CD8+ T cells, and dendritic cells of Leishmania amazonensis-infected mice present high expression of PD-1 and PD-L1, respectively. Given that the PD-1/PD-L1 interaction may promote cellular dysfunction, and that neutrophils could interact with T cells during infection, we investigated here the levels of PD-L1 in neutrophils exposed to Leishmania parasites. We found that both, promastigotes and amastigotes of L. amazonensis induced the expression of PD-L1 in the human and murine neutrophils that internalized these parasites in vitro. PD-L1-expressing neutrophils were also observed in the ear lesions and the draining lymph nodes of L. amazonensis-infected mice, assessed through cell cytometry and intravital microscopy. Moreover, expression of PD-L1 progressively increased in neutrophils from ear lesions as the disease evolved to the chronic phase. Co-culture of infected neutrophils with in vitro activated CD8+ T cells inhibits IFN-γ production by a mechanism dependent on PD-1 and PD-L1. Importantly, we demonstrated that in vitro infection of human neutrophils by L braziliensis induced PD-L1+ expression and also PD-L1+ neutrophils were detected in the lesions of patients with cutaneous leishmaniasis. Taken together, these findings suggest that the Leishmania parasite increases the expression of PD-L1 in neutrophils with suppressor capacity, which could favor the parasite survival through impairing the immune response.

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BackgroundWe have previously demonstrated that intranasal vaccination of highly susceptible BALB/c mice with whole Leishmania amazonensis antigens (LaAg) leads to protection against murine cutaneous leishmaniasis. Here, we evaluate the response of partially resistant C57BL/6 mice to vaccination as a more representative experimental model of human cutaneous leishmaniasis.MethodsC57BL/6 mice from different animal facilities were infected with L. amazonensis (Josefa strain) to establish the profile of infection. Intranasal vaccination was performed before the infection challenge with two doses of 10 μg of LaAg alone or associated with the adjuvant ADDAVAX® by instillation in the nostrils. The lesion progression was measured with a dial caliper and the parasite load by limited dilution assay in the acute and chronic phases of infection. Cytokines were quantified by ELISA in the homogenates of infected footpads.ResultsC57BL/6 mice from different animal facilities presented the same L. amazonensis infection profile, displaying a progressive acute phase followed by a controlled chronic phase. Parasites cultured in M199 and Schneider’s media were equally infective. Intranasal vaccination with LaAg led to milder acute and chronic phases of the disease. The mechanism of protection was associated with increased production of IFN-gamma in the infected tissue as measured in the acute phase. Association with the ADDAVAX® adjuvant did not improve the efficacy of intranasal LaAg vaccination. Rather, ADDAVAX® reduced vaccination efficacy.ConclusionThis study demonstrates that the efficacy of adjuvant-free intranasal vaccination with LaAg is extendable to the more resistant C57Bl/6 mouse model of infection with L. amazonensis, and is thus not exclusive to the susceptible BALB/c model. These results imply that mucosal immunomodulation by LaAg leads to peripheral protection irrespective of the genetic background of the host.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1822-9) contains supplementary material, which is available to authorized users.

Cutaneous leishmaniasis remains both a public health and a therapeutic challenge. To date, no ideal therapy for cutaneous leishmaniasis has been identified, and no universally accepted therapeutic regimen and approved vaccines are available. Due to the mesenchymal stromal cell (MSC) immunomodulatory capacity, they have been applied in a wide variety of disorders, including infectious, inflammatory, and allergic diseases. We evaluated the potential effects of bone marrow MSC therapy in a murine model of cutaneous leishmaniasis. In vitro, coculture of infected macrophages with MSC increased parasite load on macrophages in comparison with controls (macrophages without MSCs). In vivo, BALB/c mice were infected with 2 × 106 Leishmania amazonensis (Josefa strain) promastigotes in the footpad. 7 and 37 days after infection, animals were treated with 1 × 105 MSCs, either intralesional (i.l.), i.e., in the same site of infection, or intravenously (i.v.), through the external jugular vein. Control animals received the same volume (50 µL) of phosphate-buffered saline by i.l. or i.v. routes. The lesion progression was assessed by its thickness measured by pachymetry. Forty-two days after infection, animals were euthanized and parasite burden in the footpad and in the draining lymph nodes was quantified by the limiting dilution assay (LDA), and spleen cells were phenotyped by flow cytometry. No significant difference was observed in lesion progression, regardless of the MSC route of administration. However, animals treated with i.v. MSCs presented a significant increase in parasite load in comparison with controls. On the other hand, no harmful effect due to MSCs i.l. administered was observed. The spleen cellular profile analysis showed an increase of IL-10 producing T CD4+ and TCD8+ cells in the spleen only in mice treated with i.v. MSC. The excessive production of IL-10 could be associated with the disease-aggravating effects of MSC therapy when intravenously administered. As a conclusion, in the current murine model of L. amazonensis-induced cutaneous disease, MSCs did not control the damage of cutaneous disease and, depending on the administration route, it could result in deleterious effects.

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The leishmaniases are a group of diseases caused by Leishmania parasites, which have different clinical manifestations. Leishmania (Leishmania) amazonensis is endemic in South America and causes cutaneous leishmaniasis (CL), which can evolve into a diffuse form, characterized by an anergic immune response. Since the leishmaniases mainly affect poor populations, it is important to understand the involvement of immunonutrition, how the immune system is modulated by dietary nutrients and the effect this has on Leishmania infection. Vitamin D3 (VitD) is an immunonutrient obtained from diet or endogenously synthesized, which suppresses Th1 and Th17 responses by favoring T helper (Th) 2 and regulatory T cell (Treg) generation. Based on these findings, this study aims to evaluate dietary VitD influence on L. (L.) amazonensis experimental infection in C57BL/6 and BALB/c mice. Thus, C57BL/6 and BALB/c VitD deficient (VDD) mice were generated through dietary VitD restriction 45 days prior to infection. Both strains of VDD mice showed a more controlled lesion development compared to mice on a regular diet (Ctrl). There were no differences in serum levels of anti- Leishmania IgG1 and IgG2a, but there was a decrease in IgE levels in BALB/c VDD mice. Although CD4 + T cell number was not changed, the CD4 + IFN-y + T cell population was increased in both absolute number and percentage in C57BL/6 and BALB/c VDD mice compared to Ctrl mice. There was also no difference in IL-4 and IL-17 production, however, there was reduction of IL-10 production in VDD mice. Together, our data indicate that VitD contributes to murine cutaneous leishmaniasis susceptibility and that the Th1 cell population may be related to the resistance of VDD mice to L. (L.) amazonensis infection.

Leishmaniasis is a complex of neglected diseases caused by parasites of the genus Leishmania , such as Leishmania (Leishmania) amazonensis , the ethiologic agent of diffuse cutaneous leishmaniasis in Brazil. In this work, we investigated a new experimental model of infection for L. amazonensis : the Sv129 mouse. First, we subcutaneously infected Sv129 mice with 2 × 10 5 or 2 × 10 6 L. amazonensis parasites of the Josefa strain. A progressive lesion developed for both inoculation doses, showing that Sv129 mice are susceptible, independent of parasite dose. We next investigated the mechanisms associated with the pathogenesis of infection. We did not observe an increase of frequency of interferon-gamma (IFN- γ)-producing CD4 + and CD8 + T cells, a phenotype similar to that seen in BALB/c mice. There was an increased of frequency and number of IL-17-producing γδ (gamma-delta) T cells in infected Sv129 mice compared to naïve SV129 and an increased frequency of this population compared to infected BALB/c mice. In addition, Sv129 mice presented high levels of both IgG1 and IgG2a, suggesting a mixed Th1 and Th2 response with a skew toward IgG1 production based on IgG1/IgG2a ratio. Susceptibility of the Sv129 mice was further confirmed with the use of another strain of L. amazonensis , LTB0016. In this work, we characterized the Sv129 mice as a new model of susceptibility to Leishmania amazonensis infection, during infection there was controlled IFN-γ production by CD4 + or CD8 + T cells and induced IL-17 production by γδ T cells.