Resumo

Paracoccidioidomycosis (PCM) is a mycoses endemic in Latin American. PCM is primarily caused by Paracoccidioides brasiliensis as well as the recently described closely related species P. lutzii. Current treatment approaches require protracted administration of systemic antibiotics and relapses frequently occur despite months of initial therapy. Hence, there is a need for innovative approaches to treatment. In the present study we analyzed the impact of two monoclonal antibodies generated against Hsp60 from Histoplasma capsulatum on the outcome experimental murine PCM. Yeast cells of P. lutzii (Pb01) were incubated with or without monoclonal antibody against Hsp60 from H. capsulatum - 4E12 (IgG2a) or 7B6 (IgG2b) - and these cells were placed in contact with macrophage J774.16 cells in ratio 5:1 (macrophage:fungal cell). An irrelevant IgG was used as an additional control. The number of phagocytosed yeast was determined by light microscopy and was defined the phagocytic index. BALB/c mice (6 to 8 weeks female) were each anesthetized intraperitoneally with solution of ketamina/xylazine and inoculated intratracheally with 1 x 10⁶ yeast cells of Pb01. After 15 days, mice were euthanized and the fungal burden in lungs, liver and spleen was determined by CFU counts in BHI medium. In addition, we performed histology of the lungs by Grocott Silver and analyze cytokines in this organ using ELISA kits (IL-4, IL-10, IL-12 and IFN-γ). All procedures were approved by the Animal Committee of USP. Statistical comparisons were made by analysis of variance (one-way ANOVA) followed by a Turkey-Kramer posttest in GraphPad Prism version 5.00 for Windows. We observed an increase of T_H1 cytokines accompanied by a reduction in fungal burden and pulmonary injury and a decrease of T_H2 cytokines. The protection mediated by MAb 4E12 was more efficient in the reduction of fungal burden and was associated with the enhanced phagocytosis of P. lutzii yeast cells by J774.16 macrophages. Although, historically the protection against PCM has been attributed to a vigorous cellular immune response, we show that there are protective anti-hsp60 antibodies that exhibit an important role in disease control. These results provide new prospects for immunotherapy of paracoccidioidomycosis and other mycoses.