Estudo da cardiopatia chagásica em camundongos que apresentam disfunção colinérgica

Abstract

Recently was described the antiinflammatory role of acetylcholine (ACh). Similar to its role as a neurotransmitter, ACh is a mediator of the immune system, acting negatively in inflammatory processes. In order to evaluate the role of ACh in cardiac inflammation, VAChT KDHOM mice, which have approximately 65 % reduction in the expression of the vesicular acetylcholine transporter (VAChT) and the consequent reduction in the ACh release, were infected with 50 trypomastigotes of Colombian Trypanosoma cruzi strain. Although there have been significant differences in parasitemia, cardiac parasitism was higher (~ 50 %) in VAChT KDHOM mice at 35 days post infection (dpi), similar to that of WT mice at 40 and 55dpi. This increase in cardiac parasite can be explained, at least in part, by a reduction in iNOS expression in the heart of VAChT KDHOM mice. Heart histology data revealed higher inflammatory infiltrate in hearts of VAChT KDHOM mice at 35 dpi when compared to WT hearts. To better understand the increased cardiac inflammatory infiltrate, measurements of inflammatory cytokines were made in hearts of WT and VAChT KDHOM mice at 20, 35, 40 , and 55 dpi , and the results were expressed in ng/ml. We didn't observe differences in the basal production of cytokines between control groups. The levels of TNF- α (WT=0.625 ± 0.16 ; VAChT KDHOM= 2.00 ± 0.26 ) , IL-6 (WT =1.598 ± 0.32 ; VAChT KDHOM= 3.20 ± 0.14 ), IL-12 ( WT = 2.05 ± 0.73 ; VAChT KDHOM= 3.69 ± 0.37) and IFN-γ (WT= 0.682 ± 0.13 ; VAChT KDHOM= 2.273 ± 0.35 ) remained high in the hearts of VAChT KDHOM mice in 55dpi , while they decreased in the hearts of infected WT mice. In contrast, levels of IL-10 production decreased after 40 dpi in the hearts of VAChT KDHOM mice (40dpi=1.399±0.293; 55dpi=0.872±0.07). The production of TGF-β, even with the drop between 40 and 55 dpi, continued higher in hearts of VAChT KDHOM mice compared to WT (WT =1.61 ± 0.37 ; VAChT KDHOM = 0.67 ± 5.12). The expression of INF-γ producing CD4+ and CD8+ T cells in spleen of controls and infected mice from WT and VAChT KDHOM was investigated by flow cytometry (FACS). The CD4+ T cells expression in the spleen of WT and VAChT KDHOM mice was similar during T. cruzi infection. However, the infection increased the CD8+ T cells expression in the spleen of VAChT KDHOM mice at 55 dpi when compared to WT mice (WT = 2.13; VAChT KDHOM= 3.89). After the observation that the basal expression of mRNA for the FOXP-3 encoding gene was five times greater in the heart of VAChT KDHOM mice when compared to WT, and that this profile was observed during the infection, T reg lymphocytes were counted, using FACS, in both genotypes. The results showed that both in the thymus (WT=1.09; VAChT KDHOM=3.38) and in the spleen (WT=2.18; VAChT KDHOM=68.5) a higher population of Treg cells was found in VAChT KDHOM mice at 35 dpi. To prove that the reduction of ACh release was the primary factor for the observed phenotypes in VAChT KDHOM mice, these mice were infected with T. cruzi and treated for 55 days with pyridostigmine (PYR), a cholinesterase inhibitor. FACS results showed that PYR treatment reversed the expression of INF-γ producing CD8+ cells in the spleen of VAChT KDHOM mice to levels similar to that found in WT infected mice. Furthermore, the expression of regulatory lymphocytes in the spleen was further increased infected VAChT KDHOM mice treated with PYR, suggesting that this mechanism may counterregulate T lymphocytes IFN- γ producers. The assessment of cardiac function revealed that infection by T. cruzi caused a less evident remodeling in VAChT KDHOM mice than in WT mice, which may be due to increased Treg lymphocytes expression in VAChT KDHOM mice. The data indicate that ACh modulates the cardiac inflammatory process in infection by T. cruzi and also suggest a crucial role of ACh for the control of inflammation in the heart.