Claire da Silva Santos

Human mucosal leishmaniasis: neutrophils infiltrate areas of tissue damage that express high levels of Th17-related cytokines.

Mucosal leishmaniasis (ML) is characterised by severe tissue destruction. Herein, we evaluated the involvement of the IL‐17‐type response in the inflammatory infiltrate of biopsy specimens from 17 ML patients. IL‐17 and IL‐17‐inducing cytokines (IL‐1β, IL‐23, IL‐6 and TGF‐β) were detected by immunohistochemistry in ML patients. IL‐17+ cells exhibited CD4+, CD8+ or CD14+ phenotypes, and numerous IL‐17+ cells co‐expressed the CC chemokine receptor 6 (CCR6). Neutrophils, a hallmark of Th17‐mediated inflammation, were regularly detected in necrotic and perinecrotic areas and stained positive for neutrophil elastase, myeloperoxidase and MMP‐9. Taken together, these observations demonstrate the existence of Th17 cells in ML lesions associated with neutrophils in areas of tissue injury and suggest that IL‐17 is involved in ML pathogenesis.

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.

Saliva from Lutzomyia longipalpis Induces CC Chemokine Ligand 2/Monocyte Chemoattractant Protein-1 Expression and Macrophage Recruitment

Saliva of bloodfeeding arthropods has been incriminated in facilitating the establishment of parasite in their host. We report on the leukocyte chemoattractive effect of salivary gland homogenate (SGH) from Lutzomyia longipalpis on saliva-induced inflammation in an air pouch model. SGH (0.5 pair/animal) was inoculated in the air pouch formed in the back of BALB/c or C57BL/6 mice. L. longipalpis SGH induced a significant influx of macrophages in BALB/c but not in C57BL/6 mice. SGH-induced cell recruitment reached a peak at 12 h after inoculation and was higher than that induced by the LPS control. This differential cell recruitment in BALB/c mice was directly correlated to an increase in CCL2/MCP-1 expression in the air pouch lining tissue. In fact, treatment with bindarit, an inhibitor of CCL2/MCP-1 synthesis, and also with a specific anti-MCP-1 mAb resulted in drastic reduction of macrophage recruitment and inhibition of CCL2/MCP-1 expression in the lining tissue. CCL2/MCP-1 production was also seen in vitro when J774 murine macrophages were exposed to L. longipalpis SGH. The SGH effect was abrogated by preincubation with serum containing anti-SGH IgG Abs as well as in mice previously sensitized with L. longipalpis bites. Interestingly, the combination of SGH with Leishmania chagasi induced an increased recruitment of neutrophils and macrophages when compared with L. chagasi alone. Taken together these results suggest that SGH not only induces the recruitment of a greater number of macrophages by enhancing CCL2/MCP-1 production but also synergizes with L. chagasi to recruit more inflammatory cells to the site of inoculation.

The Role of CD4 and CD8 T Cells in Human Cutaneous Leishmaniasis

Leishmaniasis, caused by infection with parasites of the Leishmania genus, affects millions of individuals worldwide. This disease displays distinct clinical manifestations ranging from self-healing skin lesions to severe tissue damage. The control of Leishmania infection is dependent on cellular immune mechanisms, and evidence has shown that CD4 and CD8 T lymphocytes play different roles in the outcome of leishmaniasis. Although the presence of CD4 T cells is important for controlling parasite growth, the results in the literature suggest that the inflammatory response elicited by these cells could contribute to the pathogenesis of lesions. However, recent studies on CD8 T lymphocytes show that these cells are mainly involved in tissue damage through cytotoxic mechanisms. In this review, we focus on the recent advances in the study of the human adaptive immunological response in the pathogenesis of tegumentary leishmaniasis.

IFN-γ expression is up-regulated by peripheral blood mononuclear cells (PBMC) from non-exposed dogs upon Leishmania chagasi promastigote stimulation in vitro.

Abstract While the response to Leishmania spp. is well characterized in mice and humans, much less is known concerning the canine immune response, particularly soon after exposure to the parasite. Early events are considered to be a determinant of infection outcome. To investigate the dogs early immune response to L. chagasi, an in vitro priming system (PIV) using dog naïve PBMC was established. Until now, dog PIV immune response to L. chagasi has not been assessed. We co-cultivated PBMC primarily stimulated with L. chagasi in vitro with autologous infected macrophages and found that IFN-γ mRNA is up-regulated in these cells compared to control unstimulated cells. IL-4 and IL-10 mRNA expression by L. chagasi-stimulated PBMC was similar to control unstimulated PBMC when incubated with infected macrophages. Surprisingly, correlation studies showed that a lower IFN-γ/IL-4 expression ratio correlated with a lower percentage of infection. We propose that the direct correlation between IFN-γ/IL-4 ratio and parasite load is dependent on the higher correlation of both IFN-γ and IL-4 expression with lower parasite infection. This PIV system was shown to be useful in evaluating the dog immune response to L. chagasi, and results indicate that a balance between IFN-γ and IL-4 is associated with control of parasite infection in vitro.

Proteome Profiling of Human Cutaneous Leishmaniasis Lesion

In this study, we used proteomics and biological network analysis to evaluate the potential biological processes and components present in the identified proteins of biopsies from cutaneous leishmaniasis (CL) patients infected by Leishmania braziliensis in comparison with normal skin. We identified 59 proteins differently expressed in samples from infected and normal skin. Biological network analysis employing identified proteins showed the presence of networks that may be involved in the cell death mediated by cytotoxic T lymphocytes. After immunohistochemical analyses, the expression of caspase-9, caspase-3, and granzyme B was validated in the tissue and positively correlated with the lesion size in CL patients. In conclusion, this work identified differentially expressed proteins in the inflammatory site of CL, revealed enhanced expression of caspase-9, and highlighted mechanisms associated with the progression of tissue damage observed in lesions.

Differential Expression of the Eicosanoid Pathway in Patients With Localized or Mucosal Cutaneous Leishmaniasis

Unfettered inflammation is thought to play critical role in the development of different clinical forms of tegumentary leishmaniasis. Eicosanoids are potent mediators of inflammation and tightly associated with modulation of immune responses. In this cross-sectional exploratory study, we addressed whether targets from the eicosanoid biosynthetic pathway, assessed by multiplexed expression assays in lesion biopsy and plasma specimens, could highlight a distinct biosignature in patients with mucocutaneous leishmaniasis (MCL) or localized cutaneous leishmaniasis (LCL). Differences in immunopathogenesis between MCL and LCL may result from an imbalance between prostaglandins and leukotrienes, which may serve as targets for future host-directed therapies.

Lutzomyia longipalpis Saliva Drives Interleukin-17-Induced Neutrophil Recruitment Favoring Leishmania infantum Infection

During bloodfeeding, the presence of sand fly saliva in the hemorrhagic pool where Leishmania is also inoculated modulates the development of host immune mechanisms creating a favorable environment for disease progression. To date, information obtained through experimental models suggests that sand fly saliva induces cellular recruitment and modulates production of eicosanoids. However, the effect of sand fly saliva in the different steps of the inflammatory response triggered by Leishmania remains undefined. Here we further investigate if interaction of Lutzomyia longipalpis salivary gland sonicate (SGS) with different host cells present during the initial inflammatory events regulate Leishmania infantum infectivity. Initially, we observed that incubation of human peripheral blood mononuclear cells (PBMC) with Lu. longipalpis SGS in the presence of L. infantum significantly increased IL-10 but did not alter expression of IFN-γ and TNF-α by CD4+ T cells induced by the parasite alone. Interestingly, incubation of PBMC with Lu. longipalpis SGS alone or in the presence of L. infantum resulted in increased IL-17 production. The presence of IL-17 is related to neutrophil recruitment and plays an important role at the site of infection. Here, we also observed increased migration of neutrophil using an in vitro chemotactic assay following incubation with supernatants from PBMC stimulated with L. infantum and Lu. longipalpis SGS. Neutrophil migration was abrogated following neutralization of IL-17 with specific antibodies. Moreover, culture of human neutrophils with L. infantum in the presence of Lu. longipalpis SGS promoted neutrophil apoptosis resulting in increased parasite viability. Neutrophils operate as the first line of defense in the early stages of infection and later interact with different cells, such as macrophages. The crosstalk between neutrophils and macrophages is critical to determine the type of specific immune response that will develop. Here, we observed that co-culture of human macrophages with autologous neutrophils previously infected in the presence of Lu. longipalpis SGS resulted in a higher infection rate, accompanied by increased production of TGF-β and PGE2. Our results provide new insight into the contribution of Lu. longipalpis SGS to L. infantum-induced regulation of important inflammatory events, creating a favorable environment for parasite survival inside different host cells.

Corrections to: “CD8+ Granzyme B+–Mediated Tissue Injury versus 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.