Aspirin Modulates Innate Inflammatory Response and Inhibits the Entry of Trypanosoma cruzi in Mouse Peritoneal Macrophages
Figure 4
Effect of ASA on macrophage activity depends on production. (a) Aspirin treatment stimulated iNOs expression in T. cruzi-infected macrophages. Immunocytochemistry for iNOS was performed on coverslip-adherent cells using the labeled streptavidin biotin method with a LSAB KIT (DAKO Japan, Kyoto, Japan) without microwave accentuation. (1) Intracellular iNOS protein cannot be detected by immunocytochemistry in uninfected (control) macrophages, (2) T. cruzi-infected cell, (3) ASA (0.625 mM) was an effective inducer of iNOS expression in peritoneal macrophages, and (4) the addition of PGE2 (3.52 ng mL−1) to culture media increased iNOS mRNA expression in macrophages infected. Macrophages were treated for 30 minutes separately with 0.625 μM ASA. After treatment, macrophages were washed and incubated with aminoguanidine (AG, 1.0 mM) (b) or L-NAME (c) at 1.0 mM for 1 h at 37°C. After treatment, macrophages were washed again and interacted with 5 : 1 trypomastigotes for 2 hours at 37°C, after which they are washed, fixed with Bouin’s fixative, and stained with Giemsa. Quantification was carried out under a light microscope where the number of intracellular parasites was counted in a total of at least 500 cells. Results are the mean ± standard error for triplicate determinations and are representative of two independent experiments. (d) Effect of aspirin upon trypomastigote release in aspirin-treated T. cruzi-infected macrophages. Cells were infected with T. cruzi trypomastigotes and treated daily with ASA at 0.625 mM; after 4 days of treatment, trypomastigotes release to supernatants was found and was measured until day 7 after infection. Results are the mean ± standard error for triplicate determinations and are representative of two independent experiments. for a comparison with infected cells cultured in medium alone. for a comparison with infected cells cultured treated with ASA.
