6. Figure Legends
Figure 1. Parasitemia curve of mice infected withTrypanosoma cruzi that received prior ingestion of EtOH. Mice (n=6/group) were infected intraperitoneally with 5×10³ with trypomastigotes (Y strain) bloodstream twenty four hours after the last intragastric ethanol administration (ETOH-I) or control group (C-I). Parasitemia was monitored daily from day 2 to day 13 post-infection. Results were expressed as trypomastigotes/0.1 mL blood. Pre-patent period (PPP): Period between inoculation and the first day of parasite detection in the animal’s peripheral blood; Patent period (PP): Period between the first day of positive fresh examination and complete negativity of parasitemia; Maximum peak of parasitemia (MPP): This corresponds to the maximum parasitemia; Day of maximum peak of parasitemia (DMPP): This corresponds to the day of maximum parasitemia occurrence. * Indicates statistically significant difference between groups.
Figure 2. Evaluation of cytokine production (IL-4, IL-10, IFN-g) by unstimulated splenic lymphocytes from mice infected withT. cruzi that received ethanol previously. For fifteen consecutive days, C57BL/6 female mice (n = 6) were given 50% (v/v) ethanol (EtOH) or filtered water by gavage. After 24 hours of last administration, the animals were infected with T. cruzi and fourteen days post-infection, spleens were removed and prepared for immunophenotyping, by flow cytometry, of (A) T CD4+ Lymphocytes, (B) T CD8+ Lymphocytes and (C) B Lymphocytes, performed acquiring 30,000 events/sample on a FACScalibur cytometer. Data were analyzed using FlowJo software v10.0.1. * Indicates statistically significant difference (p <0.05) between groups submitted to the same intervention (EtOH or control). Connecting lines indicate statistical difference between groups of different intervention.
Figure 3. Evaluation of cytokine production (IL-4, IL-10, IFN-g, TNF-a) by unstimulated innate immune splenic cells of mice infected with T. cruzi that received ethanol previously. For fifteen consecutive days, C57BL/6 female mice (n = 6) were given 50% (v/v) ethanol (EtOH) or filtered water by gavage. After 24 hours of last administration, the animals were infected with T. cruzi and fourteen days post-infection, spleens were removed and prepared for immunophenotyping by flow cytometry of (A) NK cells, (B) Macrophages and (C) Dendritic cells, performed acquiring 30,000 events/sample on a FACScalibur cytometer. Data were analyzed using FlowJo software v10.0.1. * Indicates statistically significant difference (p <0.05) between groups submitted to the same intervention (EtOH or control). Connecting lines indicate statistical difference between groups of different intervention.
Figure 4. Evaluation of cytokine production (IL-4, IL-10, IFN-g) by stimulated splenic lymphocytes of mice infected with T. cruzi that received ethanol previously. For fifteen consecutive days, C57BL/6 female mice (n = 6) were given 50% (v/v) ethanol (EtOH) or filtered water by gavage. After 24 hours of last administration, the animals were infected with T. cruzi and fourteen days post-infection, spleens were removed and prepared, cultured and stimulated with soluble epimastigote antigen during 12 hours. After that, preparation for immunophenotyping was performed in a flow cytometry for analysis of (A) T CD4+ Lymphocytes, (B) T CD8+ Lymphocytes and (C) B Lymphocytes with the acquisition of 30,000 events/sample on a FACScalibur cytometer. Data were analyzed using FlowJo software v10.0.1.* Indicates statistically significant difference (p or control). Connecting lines indicate statistical difference between groups of different intervention.
Figure 5. Evaluation of cytokine production (IL-4, IL-10, IFN-g, TNF-a) by stimulated innate immune splenic cells of mice infected with T. cruzi that received ethanol previously. For fifteen consecutive days, C57BL/6 female mice (n = 6) were given 50% (v/v) ethanol (EtOH) or filtered water by gavage. After 24 hours of last administration, the animals were infected with T. cruzi and fourteen days post-infection, spleens were removed and prepared, cultured and stimulated with soluble epimastigote antigen during 12 hours. After that, preparation for immunophenotyping was performed in a flow cytometry for analysis of (A) NK cells, (B) Macrophages and (C) Dendritic cells with the acquisition of 30,000 events/sample on a FACScalibur cytometer. Data were analyzed using FlowJo software v10.0.1.* Indicates statistically significant difference (p or control). Connecting lines indicate statistical difference between groups of different intervention.
Figure 6. Determination of the stimulation index (SI) of lymphocyte cultures and innate immune cells from the spleen of mice infected with T. cruzi that previously received ethanol. The SI was calculated as the ratio between the immunophenotyping values obtained in cultures stimulated with Trypanosoma cruzi antigens and the values obtained in unstimulated cultures (control). This parameter quantifies the magnitude of the specific cytokine production induced by exposure to T. cruzi antigens in (A) T CD4+ Lymphocytes, (B) T CD8+ Lymphocytes, (C) B Lymphocytes, (D) NK cells, (E) Macrophages, and (F) Dendritic cells.
Figure 7. Illustrative image showing the summary of the main results obtained in the study. Top of the figure: In ethanol-treatedT. cruzi infected-mice, CD4+ T and B splenic lymphocytes, in unstimulated culture, showed reduced production of the cytokine IL-4, while CD8+ T lymphocytes demonstrated an increase in this cytokine. Macrophages showed reduction in the production of IL-10 and TNF-a. Bottom of the figure: In ethanol-treated T. cruzi infected-mice, CD4+ and CD8+ T lymphocytes, in stimulated culture, demonstrated an increase in IL-4 production, while B lymphocytes showed reduction. Macrophages and NK cells showed reduction in the production of IL-10, TNF-a and IL-4, IL-10 and TNF-a, respectively.