Papel do óxido nítrico no desenvolvimento de lesões cardíacas na fase aguda e crônica da infecção experimental pelo Trypanosoma cruzi.

Abstract

Chagas disease affects approximately 20 million people in Latin America. Some of these subjects could develop multiple organ commitment, manly heart. Generation of genetically modified animals provided important insights to immune system studies, by better clarifying the role played by many molecules in defense responses against pathogens and also in tissue damage. Among these molecules, nitric oxide presents a great trypanomicid action associated with high risk of tissue damage, mainly due to its ability of inducing cell death and its negative inotropic effect. In this study, the aim was evaluate the contribution of nitric oxide, IFN-y and MCP-1 in the development of inflammatory responses and cardiac fibrosis during both acute and chronic phases of Trypanosoma cruzi experimental infection. Forty-five C57BL/6 mice were used in this study, being 27 from wild type strain, 3 of which used as uninfected control group, and 18 iNOS genetically deficient (Knock out) mice, infected intra peritonealy with 3,000 trypomastigote forms of Y or Colombiana strains of T. cruzi, in groups consisted of 6 animals each. Four groups were euthanized at day 15th (acute phase) and 3 groups at day 120th post-infection (chronic phase). Fragments of the heart were processed and stained with hematoxylin-eosin for inflammatory infiltrate analysis, with picrosirius for fibrosis evaluation and immunohistochemistry for in situ detection of T. cruzi amastigotes. Spleen cells were cultured in presence of parasite antigens and supernatant used for IFN-y and MCP-1 tritation by ELISA. Our study showed that the number of T. cruzi amastigotes of Colombian strain was significantly higher in heart tissues of C57BL/6KO animals than in wild type littermates. Also, mice infected with Colombian strain presented significantly more amastigotes in heart tissues than animals infected with Y strain. Tissue inflammation analysis revealed that during acute phase of infection, C57BL/6KO mice infected with Colombian strain presented higher inflammatory infiltrate in comparison to Y strain infection. No difference was observed when KO mice were compared to wild type mice infected with each parasite strains. Interestingly, C57BL/6 wild type mice chronically infected with Colombian strain showed increased inflammatory infiltrate when compared to matched group infected with Y strain. Although C57BL/6KO mice presented a more extensive fibrotic area in the conjunctive tissue when infected by Colombian strain, there were no differences between wild type and KO mice infected with each strains. In this study, iNOS-deficient animals presented higher inflammation probably because of their intense tissular parasitism. Lack of any difference in fibrosis analysis between wild type and iNOS-deficient mice, suggests that nitric oxide may not be directly involved in fibrosis generation during Chagas disease acute phase. When IFN- y production was evaluated, there was no difference between studied groups. As C57BL/6KO mice presented higher parasite loads than wild type animals, the similar IFN- production observed in both groups would confirm the essential role played by nitric oxide in infection control, being corroborated by the observation that many iNOS-deficient animals died before chronic phase. Moreover, MCP-1 basal levels were significantly stronger in Colombian strain-infected C57BL/6KO mice during acute phase than in other groups. Conversely, during chronic phase, C57BL/6 wild type mice infected with Y strain produced the higher MCP-1 levels observed in basal and T. cruzi stimulated cell cultures. Thus, nitric oxide deficient mice showed the highest tissue parasitism and inflammation, without significant difference in fibrosis, but with the most elevated levels of MCP-1 production. We conclude that nitric oxide and MCP-1 are key molecules involved in cardiac lesions development during acute and chronic phases of experimental Chagas disease.