Evaluation of the Ability of Miltefosine Associated with Topical GM-CSF in Modulating the Immune Response of Patients with Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL) due to L. braziliensis is associated with an exaggerated inflammatory response and tissue damage. Miltefosine is more effective than pentavalent antimony (Sbv) in the treatment of CL, and here, we evaluate the ability of Sbv, miltefosine, and GM-CSF administered intravenously, orally, or topically, respectively, to modify the immune response. Patients were treated with miltefosine plus GM-CSF, miltefosine plus placebo, or Sbv. Mononuclear cells were stimulated with soluble Leishmania antigen (SLA) on day 0 and day 15 of therapy, and cytokine levels were determined in supernatants by ELISA. The lymphocyte proliferation and oxidative burst were evaluated by flow cytometry, and the degree of infection and Leishmania killing by optical microscopy. Proliferation of CD4+ T cells were enhanced in patients using miltefosine and in CD8+ T cells when GM-CSF was associated. Enhancement in the oxidative burst occurred in the miltefosine plus GM-CSF group on day 15 of therapy. Moreover, the number of L. braziliensis in infected monocytes on day 15 as well as the percentage of infected cells was lower after 48- and 72-hour culture in cells from patients treated with miltefosine plus GM-CSF. In addition to the ability of miltefosine to kill Leishmania, the changes in the immune response caused by miltefosine and GM-CSF may increase the cure rate of CL patients using these drugs.

Granzyme B Produced by Natural Killer Cells Enhances Inflammatory Response and Contributes to the Immunopathology of Cutaneous Leishmaniasis.

BACKGROUND: Skin lesions from patients infected with Leishmania braziliensis has been associated with inflammation induced by cytotoxic CD8+ T cells. In addition, CD8+ T cell-mediated cytotoxicity has not been linked to parasite killing. Meanwhile, the cytotoxic role played by natural killer (NK) cells in cutaneous leishmaniasis (CL) remains poorly understood. METHODS: In this study, we observed higher frequencies of NK cells in the peripheral blood of CL patients compared with healthy subjects, and that NK cells expressed more interferon-γ, tumor necrosis factor (TNF), granzyme B, and perforin than CD8+ T cells. RESULTS: We also found that most of the cytotoxic activity in CL lesions was triggered by NK cells, and that the high levels of granzyme B produced in CL lesions was associated with larger lesion size. Furthermore, an in vitro blockade of granzyme B was observed to decrease TNF production. CONCCLUSIONS: Our data, taken together, suggest an important role by NK cells in inducing inflammation in CL, thereby contributing to disease immunopathology.

Early Cutaneous Leishmaniasis Patients Infected With Leishmania braziliensis Express Increased Inflammatory Responses After Antimony Therapy.

Background: Early cutaneous leishmaniasis (ECL) is characterized by a nonulcerated papular lesion and illness duration less than 30 days. Approximately 4 weeks later, the cutaneous leishmaniasis (CL) ulcers appear. We were surprised to find that failure after antimony therapy (Sb5) is higher in ECL than CL. We hypothesize that the inflammatory response in ECL patients may increase during Sb5 therapy, which leads to treatment failure. Methods: A cohort of 44 ECL patients infected by Leishmania braziliensis was established to evaluate the response to Sb5 and to compare immunologic responses in ECL patients with CL and healthy subjects. Results: A hierarchical clustering based on cytokine levels showed a weak positive correlation between proinflammatory cytokine levels and those patients that failed Sb5 treatment. Although Sb5 therapy decreased interferon-γ and tumor necrosis factor levels in CL patients, we were surprised to find that an increase in these cytokines was observed in ECL patients. Moreover, interleukin (IL)-10 was less able to down-modulate immune responses in ECL. Conclusions: The enhanced production of proinflammatory cytokines, due in part to the decreased ability of IL-10 to down-modulate immune response during therapy in ECL, promotes the development and persistence of leishmania ulcer despite antimony therapy.

Cytotoxic activity in cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a chronic disease caused by species of the protozoan Leishmania and characterised by the presence of ulcerated skin lesions. Both parasite and host factors affect the clinical presentation of the disease. The development of skin ulcers in CL is associated with an inflammatory response mediated by cells that control parasite growth but also contribute to pathogenesis. CD8+ T cells contribute to deleterious inflammatory responses in patients with CL through cytotoxic mechanisms. In addition, natural killer cells also limit Leishmania infections by production of interferon-γ and cytotoxicity. In this review, we focus on studies of cytotoxicity in CL and its contribution to the pathogenesis of this disease.

Cytotoxic activity in cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is a chronic disease caused by species of the protozoan Leishmania and characterised by the presence of ulcerated skin lesions. Both parasite and host factors affect the clinical presentation of the disease. The development of skin ulcers in CL is associated with an inflammatory response mediated by cells that control parasite growth but also contribute to pathogenesis. CD8+ T cells contribute to deleterious inflammatory responses in patients with CL through cytotoxic mechanisms. In addition, natural killer cells also limit Leishmania infections by production of interferon-γ and cytotoxicity. In this review, we focus on studies of cytotoxicity in CL and its contribution to the pathogenesis of this disease.

A Potential Role for Mononuclear Phagocytes in Cutaneous Ulcer Development in Human Immunodeficiency Virus-Leishmania braziliensis Coinfection.

Skin ulcer development in cutaneous leishmaniasis due to Leishmania braziliensis infection is associated with a mononuclear cell infiltrate and high levels of tumor necrosis factor (TNF). Herein, we show that despite the absence of Leishmania-driven TNF, a cutaneous leishmaniasis patient with acquired immunodeficiency syndrome developed a skin ulcer. The presence of mononuclear phagocytes and high levels of TNF, chemokine (C-C motif) ligand 2 (CCL2), and metalloproteinase-9 in tissue are identified as potential contributors to immunopathology observed in L. braziliensis-infected patients.

Intermediate monocytes contribute to pathologic immune response in Leishmania braziliensis infections.

Ulcer development in patients with cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is associated with high levels of tumor necrosis factor (TNF). We found that early after infection, before ulcer development, the frequency of CD16(+) (both intermediate [CD14(+)CD16(+)] and nonclassical [CD14(dim)CD16(+)]) monocytes was increased in the peripheral blood of patients with L. braziliensis, compared with uninfected controls. These results suggest that CD16(+) monocytes might promote disease. Also, we found that intermediate monocytes expressed CCR2 and that increased levels of CCL2 protein were present in lesions from patients, suggesting that intermediate monocytes are more likely than nonclassical monocytes to migrate to the lesion site. Finally, we found that the intermediate monocytes produced TNF. Our results show that intermediate monocytes are increased in frequency soon after infection; express CCR2, which would promote their migration into the lesions; and, owing to their production of TNF, can enhance the inflammatory response.

Matrix metalloproteinase 9 production by monocytes is enhanced by TNF and participates in the pathology of human cutaneous Leishmaniasis.

INTRODUCTION: Cutaneous leishmaniasis (CL) due to L.braziliensis infection is characterized by a strong inflammatory response with high levels of TNF and ulcer development. Less attention has been given to the role of mononuclear phagocytes to this process. Monocytes constitute a heterogeneous population subdivided into classical, intermediate and non-classical, and are known to migrate to inflammatory sites and secrete inflammatory mediators. TNF participates in the induction of matrix metalloproteinases (MMPs). MMP-9 is an enzyme that degrades basal membrane and its activity is controlled by the tissue inhibitor of metalloproteinase. METHODS: Mononuclear cells were obtained from ex-vivo labeling sub-populations of monocytes and MMP-9, and the frequency was determined by flow cytometry. Culture was performed during 72 hours, stimulating the cells with SLA, levels of MMP-9 and TIMP-1 in the supernatants were determined by ELISA. RESULTS: We observed that cells from CL lesions secrete high amounts of MMP-9 when compared to healthy subjects. Although MMP-9 was produced by monocytes, non-classical ones were the main source of this enzyme. We also observed that TNF produced in high level during CL contributes to MMP-9 production. CONCLUSIONS: These observations emphasize the role of monocytes, TNF and MMP-9 in the pathogenesis of L. braziliensis infection.