Avaliação do papel das células T CD8+ e análise proteômica nas Biópsias de pacientes com Leishmaniose Cutânea Localizada infectados por L. braziliensis

CD8+ T cells are essential in the defense against virus, but little is known of their participation in the host defense against parasites, such as Leishmania. In the present study, we investigated the participation of CD8+ T cells in the inflammatory process and parasite killing, as well as proteome profiles in the biopsies from localized cutaneous leishmaniasis patients (LCL) infected by L. brazliensis. We found a higher percentage of CD8+ T cells in lesions of LCL patients, as suggested by the higher frequency of CD8+CD45RO+T cells and CD8+CLA +T cells compared to PBMC. Upon L. braziliensis-restimulation, most of CD8+T cells from the lesion expressed cytolytic markers, CDI07a and Granzyme B. Furthermore, co-culture of infected macrophages and CD8+T lymphocytes resuIted in release of granzyme B and the use of granzyme B inhibitor, as well as z-V AD, Fas:Fc or anti-IFN-y had no effect upon parasite killing. On the other hand, co-culture of infected macrophages with CD4+T cells strongly increased parasite killing, which was completely reversed by anti-IFN-y, pointing out the decisive role of CD4+IFN-y +T cells in parasite killing. Besides that, a total of 150 differentially expressed proteins were observed in the lesions of LCL patients and normal skin. Fifty-nine proteins were identified. Among them, 13 were up or down regulated in LCL lesions compared to normal skin, 27 proteins were unique in the lesions of LCL patients and 18 were unique in the normal skin samples. These proteins were associated with biological regulation, including apoptosis, cell cycle and immune response. To explore interactions between the identified proteins and proteins and genes that may be affected by them, networks and subnetworks were generated. After immunohistochemistry analyses, the presence of casp~~e 9, caspase 3 and granzyme B were validated in the lesions site. Granzyme B expression in the lesions of LCL patients positively correlated with caspase 9 expression and the percentage of TUNEL-positive cells. We also observed a significant higher percentage of TUNEL-positive cells and granzyme B expression in the biopsies of patients showing a more intense necrotic processo Besides that, the presence of granzime B, caspase 9 and caspas e 3 were positively correlated with the lesion size. In this study we can conclude that CD8+ Granzyme B+T cells are involved in the pathogenesis of L. braziliensis due to their cytotoxic potential to induce apoptotic mechanism in the inflammatory site that favors the progression oftissue damage observed in LCL patients.