Strategy to assess the overall cytokine profile of circulating leukocytes and its association with distinct clinical forms of human Chagas disease

Herein we have employed an alternative strategy to assess the cytokine patterns of circulating leukocytes and correlate dominant cytokine profiles with indeterminate-IND and cardiac-CARD clinical forms of Chagas disease. We have first calculated median percentages of cytokine-positive leukocytes of our study sample to establish, for each cytokine-positive cell population, the cut-off edge that would segregate 'low' and 'high' cytokine producers to build colour diagrams and draw a panoramic cytokine chart. Using this approach we demonstrated that most IND individuals presented a dominant regulatory cytokine profile, whereas CARD individuals displayed a dominant inflammatory cytokine pattern. In addition, radar chart analysis confirmed the dichotomic cytokine balance between IND and CARD groups and further allowed the identification of the relative contribution of each cell population for the global cytokine pattern. Data analysis demonstrated that CD4(+) T cells were the major cell population defining the regulatory profile in IND, whereas monocytes and CD4(+) T cells determined the inflammatory cytokine pattern in CARD individuals. Interestingly, in vitro stimulation with trypomastigote Trypanosoma cruzi antigen was able to invert the cytokine balances in IND and CARD groups. Upon antigenic stimulation, changes in the frequencies of IL-10-producing CD4(+) T cells and monocytes drove IND individuals towards an inflammatory pattern and CARD towards a regulatory cytokine profile. A similar inversion could be found after in vivo treatment of IND and CARD individuals with benzonidazole. Altogether, these findings shed some light into the complex cytokine network underlying the immunopathogenesis of Chagas disease and provide putative immunological biomarkers of disease severity and therapeutic response.