Different inoculum of Leishmania braziliensis concentrations influence immunopathogenesis and clinical evolution in the ear dermis hamster model of cutaneous leishmaniasis.

The golden hamster (Mesocricetus auratus) is commonly used as a promising model for Leishmania braziliensis infection developing skin-ulcerated lesions. However, different protocols using high concentration of parasites inoculated in the footpad result in severe clinical disease. Here, we further investigate the outcome of the site of infection and concentration of L. braziliensis parasites inoculated on the immunopathogenesis and clinical evolution. Initially, hamsters were infected in the ear dermis or hind footpad with a concentration of 1 × 105 parasites. Animals infected in the ear dermis developed a disease, with an increased parasite load that more closely resembled human cutaneous leishmaniasis lesions comparing to the group infected in the footpad. Next, we evaluated if different parasite concentrations (104 , 105 and 106 ) inoculated in the ear dermis would impact the course and clinical aspects of infection. Hamsters infected with 104 and 105 parasites developed mild lesions compared to the group infected with 106 that presented severe and persistent lesions. The parasite load varied between the different parasite concentrations. The inflammatory response was more intense when infection was initiated with 106 parasites accompanied by an increased initial expression of IL-4, IL-10 and arginase in the lymph node followed by expression of both pro-and anti-inflammatory cytokines comparing to groups infected with 104 and 105 parasites. In conclusion, the number of parasites inoculated, and the initial site of infection could influence the inflammatory response, and clinical presentation. Our results suggest that the ear dermis infection model induces a chronic disease that relates to immunopathological aspects of CL natural infection.

CXCL10 immunomodulatory effect against infection caused by an antimony refractory isolate of Leishmania braziliensis in mice.

Leishmania braziliensis is the main causative agent of American tegumentary leishmaniasis in Brazil. Current treatment includes different drugs that have important side effects and identification of cases of parasite resistance to treatment support the search for new therapeutic strategies. Recent findings have indicated that CXCL10, a chemokine that recruits and activates Th1 cells, NK cells, macrophages, dendritic cells and B lymphocytes, is a potential alternative to treat Leishmania infection. Here, we tested CXCL10 immunotherapy against experimental infection caused by an antimony-resistant isolate of Leishmania braziliensis. Following infection, mice were treated with CXCL10 for 7 days after onset of lesions. We demonstrate that mice treated with CXCL10 controlled lesion progression and parasite burden more efficiently comparing to controls. An increased IFN-γ, IL-10, TGF-ß and low IL-4 production combined with a distinct inflammatory infiltrate composed by activated macrophages, lymphocytes and granulomas was observed in the CXCL10-treated group comparing to controls. However, CXCL10 and Glucantime combined therapy did not improve CXCL10-induced protective effect. Our findings reinforce the potential of CXCL10 immunotherapy as an alternative treatment against infection caused by L. braziliensis resistant to conventional chemotherapy.

Astronium fraxinifolium Schott Exerts Leishmanicidal Activity by Providing a Classically Polarized Profile in Infected Macrophages.

BACKGROUND: Leishmania braziliensis is prevalent in Latin American countries, including Brazil. It causes cutaneous and mucocutaneous leishmaniasis, leading to high morbidity, and has a low cure rate. Treatment is based on pentavalent antimonials; nonetheless, there are problems related to high toxicity, high cost, and parasitic resistance. Discovery of new leishmanicidal drugs without these limitations and that stimulate the cellular immune response is necessary. PURPOSE: The present work evaluates whether Astronium fraxinifolium Schott exerts leishmanicidal activity against L. braziliensis by providing a classically polarized profile in infected macrophages. METHODS: For the evaluation of the A. fraxinifolium Schott leishmanicidal activity, amastigote cell death was demonstrated in infected RAW 267.4 macrophages treated with an ethanolic extract from the plant sapwood (EEAF). For the evaluation of the EEAF capacity in providing a classically polarized profile in infected macrophages, the following analyses were done: detection of LAMP-1 protein by the baculovirus technology, measurement of superoxide anion by the NBT testing, quantification of TNF-α, IL-12p40, IL-10, IL-4, and TGF-ß by sandwich-type enzyme immune assays, and iNOS and COX-2 expression by RT-PCR technique. RESULTS: The EEAF significantly reduced amastigote counts inside the cells. Vacuoles were visualized in infected and treated cells before and after May-Grünwald-Giemsa staining. A strong LAMP-1 protein fluorescence revealed phagosome maturation in infected cells treated with the EEAF. No production of superoxide was visualized in infected cells treated with the plant material. Nonetheless, high levels of TNF-α, IL-12p40, and IL-10 were found in cell supernatants, but reduced levels of TGF-ß and no IL-4 production. We identified augmented mRNA expression for COX-2, but no expression of iNOS mRNA. CONCLUSION: Our results demonstrated that A. fraxinifolium induced a classically polarized profile in infected macrophages but also provided a less harmful environment by stimulating the production of certain anti-inflammatory mediators, such as IL-10.