Regulação da resposta imune por linfócitos T reguladores Foxp3+ de sangue e de pele de pacientes com hanseníase

Abstract

Leprosy is an infectious disease caused by the obligate intracellular pathogen Mycobacterium leprae. The disease has a broad spectrum of clinical forms with a strong Th1 cellular response in tuberculoid leprosy, whereas in lepromatous leprosy this response is defective. T regulatory cells (Treg) are cells that express the lineage-specific transcription factor Foxp3 and can suppress the effector responses. These cells have essential participation in the homeostasis, controlling the intensity of inflamatory responses and then limiting tissue damage. A better understanding of the negative modulation of the immune response and the involvement of Tregs in this modulation can give us important informations about the leprosy pathogenesis and evolution. Thus, we evaluated M. leprae-specific Tregs of the blood and the memory phenotype of these cells in skin lesions of leprosy patients. The analysis of Treg in the skin lesions is indispensable since central events of the immune system regulation in leprosy occur in the tissues. Flow cytometry was used to identify Treg CD4+ and T conventional CD4+ (Tconv) frequencies in response to mycobacterial antigens. We observed an inverse relationship between Tregs CD4+ and Tconv responsive to ML1419c, a recombinant protein of M. leprae. In multibacillary leprosy patients higher frequencies of Tregs and lower of Tconv were observed when compared to paucibacillary patients, indicating a possible suppressive action of Tregs on Tconv. We used a mechanical and enzimatic dissociation technique to obtain cells in suspension from a skin lesion biopsy for phenotype caracterization. We observed that the Tregs in the lesion presented an effector memory phenotype and were in greater numbers in the lesion than in the blood. Immunofluorescence microscopy analysis allowed us to detect CD4+ and CD8+ Treg in the evaluated samples, however the majority of the Tregs was CD4+ in these lesion infiltrates. Therefore, our evaluation of a leprosy skin lesion are consistent with the presence of Tregs almost exclusively of the effector memory phenotype in critical sites of immune regulation in leprosy. A future goal of our study is to detect the stable suppressive function of Treg and the preliminary steps required for this evaluation were completed. Isolation of Treg from peripheral blood mononuclear cells for in vitro suppresion assays and evaluation of the methylation status of TSDR region in the foxp3 gene were accomplished. Our results show that the methodology used here is applicable for our goals in the investigation of Treg participation in the downregulation of the immune response in leprosy.