Natalia Tavares

CD8+ Granzyme B+–Mediated Tissue Injury vs. CD4+IFNγ+–Mediated Parasite Killing in Human Cutaneous Leishmaniasis

A protective or deleterious role of CD8+T cells in human cutaneous leishmaniasis (CL) has been debated. The present report explores the participation of CD8+T cells in disease pathogenesis as well as in parasite killing. CD8+T cells accumulated in CL lesions as suggested by a higher frequency of CD8+CD45RO+T cells and CD8+CLA+T cells compared with peripheral blood mononuclear cells. Upon Leishmania braziliensis restimulation, most of the CD8+T cells from the lesion expressed cytolytic markers, CD107a and granzyme B. Granzyme B expression in CL lesions positively correlated with lesion size and percentage of TUNEL-positive cells. We also observed a significantly higher percentage of TUNEL-positive cells and granzyme B expression in the biopsies of patients showing a more intense necrotic process. Furthermore, coculture of infected macrophages and CD8+T lymphocytes resulted in the release of granzyme B, and the use of granzyme B inhibitor, as well as z-VAD, Fas:Fc, or anti-IFN-γ, had no effect upon parasite killing. However, coculture of infected macrophages with CD4+T cells strongly increased parasite killing, which was completely reversed by anti-IFN-γ. Our results reveal a dichotomy in human CL: CD8+ granzyme B+T cells mediate tissue injury, whereas CD4+IFN-γ+T cells mediate parasite killing.

Lutzomyia longipalpis Saliva or Salivary Protein LJM19 Protects against Leishmania braziliensis and the Saliva of Its Vector, Lutzomyia intermedia

Background Leishmania transmission occurs in the presence of insect saliva. Immunity to Phlebotomus papatasi or Lutzomyia longipalpis saliva or salivary components confers protection against an infection by Leishmania in the presence of the homologous saliva. However, immunization with Lutzomyia intermedia saliva did not protect mice against Leishmania braziliensis plus Lu. intermedia saliva. In the present study, we have studied whether the immunization with Lu. longipalpis saliva or a DNA plasmid coding for LJM19 salivary protein would be protective against L. braziliensis infection in the presence of Lu. intermedia saliva, the natural vector for L. braziliensis. Methodology/Principal Findings Immunization with Lu. longipalpis saliva or with LJM19 DNA plasmid induced a Delayed-Type Hypersensitivity (DTH) response against Lu. longipalpis as well as against a Lu. intermedia saliva challenge. Immunized and unimmunized control hamsters were then intradermally infected in the ears with L. braziliensis in the presence of Lu. longipalpis or Lu. intermedia saliva. Animals immunized with Lu. longipalpis saliva exhibited smaller lesion sizes as well as reduced disease burdens both at lesion site and in the draining lymph nodes. These alterations were associated with a significant decrease in the expression levels of IL-10 and TGF-β. Animals immunized with LJM19 DNA plasmid presented similar findings in protection and immune response and additionally increased IFN-γ expression. Conclusions/Significance Immunization with Lu. longipalpis saliva or with a DNA plasmid coding LJM19 salivary protein induced protection in hamsters challenged with L. braziliensis plus Lu. intermedia saliva. These findings point out an important role of immune response against saliva components, suggesting the possibility to develop a vaccine using a single component of Lu. longipalpis saliva to generate protection against different species of Leishmania, even those transmitted by a different vector.

Leishmania amazonensis infection impairs differentiation and function of human dendritic cells

Dendritic cells (DCs) are of utmost importance in initiating an immune response and may also function as targets for pathogens. The presence of pathogens inside DCs is likely to impair their functions and thus, influence immune responses. In the present report, we evaluated the impact of the presence of Leishmania amazonensis during differentiation and maturation of human monocyte‐derived DCs. The presence of live L. amazonensis parasites during DC differentiation led to a significant decrease in CD80 (92%) and CD1a (56%) expression and an increase in CD86 (56%) cell surface expression. Phenotypic changes were accompanied by a lower secretion of IL‐6, observed after 6 days of DC differentiation in the presence of L. amazonensis. DCs differentiated in the presence of L. amazonensis were used as APC in an autologous coculture, and lower amounts of IFN‐γ were obtained compared with control DCs differentiated in the absence of parasites. The effect of heat‐killed parasites, but not of Leishmania antigen, during DC differentiation and maturation was similar to that observed with viable parasites. During maturation, the presence of live L. amazonensis parasites, but not of soluble Leishmania antigen, led to a decrease in IL‐6 and IL‐10 production. In this way, we observed that the parasite is able to abrogate full DC differentiation, causing a delay in the immune response and likely, favoring its establishment in human hosts.

Understanding the Mechanisms Controlling Leishmania amazonensis Infection In Vitro: The Role of LTB4 Derived From Human Neutrophils

Neutrophils are rapidly recruited to the site of Leishmania infection and play an active role in capturing and killing parasites. They are the main source of leukotriene B4 (LTB4), a potent proinflammatory lipid mediator. However, the role of LTB4 in neutrophil infection by Leishmania amazonensis is not clear. In this study, we show that L. amazonensis or its lipophosphoglycan can induce neutrophil activation, degranulation, and LTB4 production. Using pharmacological inhibitors of leukotriene synthesis, our findings reveal an LTB4-driven autocrine/paracrine regulatory effect. In particular, neutrophil-derived LTB4 controls L. amazonensis killing, degranulation, and reactive oxygen species production. In addition, L. amazonensis infection induces an early increase in Toll-like receptor 2 expression, which facilitates parasite internalization. Nuclear factor kappa B (NFkB) pathway activation represents a required upstream event for L. amazonensis–induced LTB4 synthesis. These leishmanicidal mechanisms mediated by neutrophil-derived LTB4 act through activation of its receptor, B leukotriene receptor 1 (BLT1).

DNA vaccination with KMP11 and Lutzomyia longipalpis salivary protein protects hamsters against visceral leishmaniasis

It was recently shown that immunization of hamsters with DNA plasmids coding LJM19, a sand fly salivary protein, partially protected against a challenge with Leishmania chagasi, whereas immunization with KMP11 DNA plasmid, a Leishmania antigen, induced protection against L. donovani infection. In the present study, we evaluated the protective effect of immunization with both LJM19 and KMP11 DNA plasmid together. Concerning the protection against an infection by L. chagasi, immunization with DNA plasmids coding LJM19 or KMP11, as well as with both plasmids combined, induced IFN-γ production in draining lymph nodes at 7, 14 and 21 days post-immunization. Immunized hamsters challenged with L. chagasi plus Salivary Gland Sonicate (SGS) from Lutzomyia longipalpis showed an enhancement of IFN-γ/IL-10 and IFN-γ/TGF-β in draining lymph nodes after 7 and 14 days of infection. Two and five months after challenge, immunized animals showed reduced parasite load in the liver and spleen, as well as increased IFN-γ/IL-10 and IFN-γ/TGF-β ratios in the spleen. Furthermore, immunized animals remained with a normal hematological profile even five months after the challenge, whereas L. chagasi in unimmunized hamsters lead to a significant anemia. The protection observed with LJM19 or KMP11 DNA plasmids used alone was very similar to the protection obtained by the combination of both plasmids.

Dendritic Cells and Leishmania Infection: Adding Layers of Complexity to a Complex Disease

Leishmaniasis is a group of neglected diseases whose clinical manifestations depend on factors from the host and the pathogen. It is an important public health problem worldwide caused by the protozoan parasite from the Leishmania genus. Cutaneous Leishmaniasis (CL) is the most frequent form of this disease transmitted by the bite of an infected sandfly into the host skin. The parasites can be uptook and/or recognized by macrophages, neutrophils, and/or dendritic cells (DCs). Initially, DCs were described to play a protective role in activating the immune response against Leishmania parasites. However, several reports showed a dichotomic role of DCs in modulating the host immune response to susceptibility or resistance in CL. In this review, we discuss (1) the interactions between DCs and parasites from different species of Leishmania and (2) the crosstalk of DCs and other cells during CL infection. The complexity of these interactions profoundly affects the adaptive immune response and, consequently, the disease outcome, especially from Leishmania species of the New World.

Degranulating Neutrophils Promote Leukotriene B4 Production by Infected Macrophages To Kill Leishmania amazonensis Parasites

Neutrophils mediate early responses against pathogens, and they become activated during endothelial transmigration toward the inflammatory site. In the current study, human neutrophils were activated in vitro with immobilized extracellular matrix proteins, such as fibronectin (FN), collagen, and laminin. Neutrophil activation by FN, but not other extracellular matrix proteins, induces the release of the granules’ contents, measured as matrix metalloproteinase 9 and neutrophil elastase activity in culture supernatant, as well as reactive oxygen species production. Upon contact with Leishmania amazonensis–infected macrophages, these FN-activated neutrophils reduce the parasite burden through a mechanism independent of cell contact. The release of granule proteases, such as myeloperoxidase, neutrophil elastase, and matrix metalloproteinase 9, activates macrophages through TLRs, leading to the production of inflammatory mediators, TNF-α and leukotriene B4 (LTB4), which are involved in parasite killing by infected macrophages. The pharmacological inhibition of degranulation reverted this effect, abolishing LTB4 and TNF production. Together, these results suggest that FN-driven degranulation of neutrophils induces the production of LTB4 and TNF by infected macrophages, leading to the control of Leishmania infection.

Heme Drives Oxidative Stress-Associated Cell Death in Human Neutrophils Infected with Leishmania infantum

Free heme is an inflammatory molecule capable of inducing migration and activation of neutrophils. Here, we examine the heme-driven oxidative stress-associated cell death mechanisms in human neutrophils infected with Leishmania infantum, an etiologic agent of visceral leishmaniasis (VL). We first performed exploratory analyses in a population of well characterized treatment-naïve VL patients as well as uninfected controls, who were part of previously reported studies. We noted a positive correlation between serum concentrations of heme with heme oxygenase-1 (HO-1) and lactate deydrogenase, as well as, a negative correlation between heme values and peripheral blood neutrophils counts. Moreover, in vitro infection with L. infantum in the presence of heme enhanced parasite burden in neutrophils, while increasing the production of reactive oxygen species and release of neutrophilic enzymes. Additional experiments demonstrated that treatment of infected neutrophils with ferrous iron (Fe+2), a key component of the heme molecule, resulted in increased parasite survival without affecting neutrophil activation status. Furthermore, stimulation of infected neutrophils with heme triggered substantial increases in HO-1 mRNA expression as well as in superoxide dismutase-1 enzymatic activity. Heme, but not Fe+2, induced oxidative stress-associated cell death. These findings indicate that heme promotes intracellular L. infantum survival via activation of neutrophil function and oxidative stress. This study opens new perspectives for the understanding of immunopathogenic mechanisms involving neutrophils in VL.

Integrated Analysis Reveals That miR-193b, miR-671, and TREM-1 Correlate With a Good Response to Treatment of Human Localized Cutaneous Leishmaniasis Caused by Leishmania braziliensis

Localized cutaneous leishmaniasis (LCL) is a chronic disease characterized by ulcerated skin lesion(s) and uncontrolled inflammation. The mechanisms underlying the pathogenesis of LCL are not completely understood, and little is known about posttranscriptional regulation during LCL. MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression and can be implicated in the pathogenesis of LCL. We investigated the involvement of miRNAs and their targets genes in human LCL using publicly available transcriptome data sets followed by ex vivo validation. Initial analysis highlighted that miRNA expression is altered during LCL, as patients clustered separately from controls. Joint analysis identified eight high confidence miRNAs that had altered expression (−1.5 ≤ fold change ≥ 1.5; p < 0.05) between cutaneous ulcers and uninfected skin. We found that the expression of miR-193b and miR-671 are greatly associated with their target genes, CD40 and TNFR, indicating the important role of these miRNAs in the expression of genes related to the inflammatory response observed in LCL. In addition, network analysis revealed that miR-193b, miR-671, and TREM1 correlate only in patients who show faster wound healing (up to 59 days) and not in patients who require longer cure times (more than 60 days). Given that these miRNAs are associated with control of inflammation and healing time, our findings reveal that they might influence the pathogenesis and prognosis of LCL.

Corrigendum to “Dendritic Cells and Leishmania Infection: Adding Layers of Complexity to a Complex Disease”

[This corrects the article DOI: 10.1155/2016/3967436.].