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Silvia Guatimosimhttp://lattes.cnpq.br/7958786029463633Fabiana Simão MachadoCarla Maximo PradoHelio Cesar SalgadoLuciana de Oliveira Andradehttp://lattes.cnpq.br/5958955185157038Aline Alves Lara Gomes2024-03-13T13:59:50Z2024-03-13T13:59:50Z2014-02-21http://hdl.handle.net/1843/65755Recentemente foi descrito o papel antiinflamatório da acetilcolina (ACh). Similar ao seu papel de neurotransmissor, a ACh é um mediador do Sistema Imune, agindo negativamente em processos inflamatórios. Com intuito de avaliar o papel da ACh no processo inflamatório cardíaco, camundongos VAChT KDHOM, que apresentam aproximadamente 65% de redução na expressão do transportador vesicular de ACh (VAChT) e consequente redução na liberação de ACh, foram infectados com 50 formas tripomastigotas da cepa Colombiana de Trypanosoma cruzi. Apesar de não ter sido observada diferença significativa na parasitemia, o parasitismo cardíaco foi maior (~50%) nos camundongos VAChT KDHOM aos 35 dias pós infecção (dpi), sendo similar ao dos camundongos WT aos 40 e 55dpi. Esse aumento no parasitismo cardíaco pode se explicado, pelo menos em parte, pela redução na expressão de iNOS no coração dos camundongos VAChT KDHOM. Cortes histológicos cardíacos revelaram maior infiltrado inflamatório em corações de camundongos VAChT KDHOM aos 35 dpi quando comparado aos corações de camundongos WT. Para melhor entender o aumento do infiltrado inflamatório cardíaco foram feitas dosagens de citocinas inflamatórias no coração dos camundongos WT e VAChT KDHOM aos 20, 35, 40 e 55 dpi, os resultados foram expressos em ng/mL. Não houve diferença na produção basal de citocinas entre os camundongos WT e VAChT KDHOM controles. Os níveis de 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) e IFN-γ (WT=0.682±0.13; VAChT KDHOM =2.273±0.35) mantiveram-se elevados nos corações dos camundongos VAChT KDHOM aos 55dpi, enquanto que os mesmos decairam nos corações dos camundongos WT infectados. Contrariamente, os níveis de produção da IL-10 decairam após 40 dpi nos corações de camundongos VAChT KDHOM (40dpi=1.399±0.293; 55dpi=0.872±0.07). Já a produção de TGF-β, mesmo com a queda entre 40 e 55 dpi, continuou maior nos corações dos camundongos VAChT KDHOM quando comparada aos WT aos 55dpi (WT=1.61±0.37; VAChT KDHOM=5.12±0.67). A expressão de linfócitos T CD4+ e CD8+ produtores de INF-γ em baço de camundongos WT e VAChT KDHOM, controles e infectados, foi investigada através de citometria de fluxo (FACS). A expressão de células T CD4+ em baço de camundongos WT e VAChT KDHOM foi semelhante durante a infecção por T.cruzi. Porém, a infecção aumentou a expressão de linfócitos T CD8+ no baço de camundongos VAChT KDHOM aos 55 dpi quando comparado aos camundongos WT (WT=2.13; VAChT KDHOM=3.89). Após ser identificada que a expressão basal de RNAm para o gene que codifica FOXP-3 foi cinco vezes maior no coração dos camundongos VAChT KDHOM quando comparados aos WT e que esse comportamento foi observado durante a infecção, contagem de linfócitos T reg foi avaliada através de FACS nos dois genótipos. Os resultados encontrados mostraram que tanto no timo (WT=1.09 ; VAChT KDHOM=3.38) quanto no baço (WT=2.18; VAChT KDHOM=68.5) foi encontrada maior expressão de células Treg nos camundongos VAChT KDHOM aos 35 dpi. Para comprovar que a redução na liberação de ACh era o fator primordial para os fenótipos observados em camundongos VAChT KDHOM, esses camundongos foram infectados com T. cruzi e tratados durante 55 dias com piridostigmina (PIR), um inibidor de colinesterase. Os resultados de FACS mostraram que o tratamento com PIR reverteu a porcentagem de expressão de células T CD8+ produtoras de IFN-γ em baçode camundongos VAChT KDHOM para níveis semelhantes aos encontrados em camundongos WT infectados. Além disso, a expressão de linfócitos T regulatórios aumentou ainda mais no baço de camundongos VAChT KDHOM infectados e tratados com PIR, sugerindo que seja esse o mecanismo contrarregulatório dos linfócitos T produtores de IFN-γ. A avaliação da função cardíaca revelou que infecção com T. cruzi causou um remodelamento menos evidente em camundongos VAChT KDHOM do que em camundongos WT, o que pode ser devido a maior expressão de linfócitos Treg em camundongos VAChT KDHOM. Os dados obtidos indicam que a ACh regula o processo inflamatório cardíaco em infecção por T. cruzi e também sugerem um papel crucial da ACh para a contenção da inflamação no coração.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.porUniversidade Federal de Minas GeraisPrograma de Pós-Graduação em Ciências Biológicas - Fisiologia e FarmacologiaUFMGBrasilICB - INSTITUTO DE CIÊNCIAS BIOLOGICAShttp://creativecommons.org/licenses/by-nc-nd/3.0/pt/info:eu-repo/semantics/openAccessFarmacologiaAcetilcolinaInflamaçãoTrypanosoma cruziAcetilcolinaInflamaçãoTregTrypanosoma cruziEstudo da cardiopatia chagásica em camundongos que apresentam disfunção colinérgicainfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisreponame:Repositório Institucional da UFMGinstname:Universidade Federal de Minas Gerais (UFMG)instacron:UFMGORIGINALTese_biblioteca_Aline Alves Lara Gomes.pdfTese_biblioteca_Aline Alves Lara Gomes.pdfapplication/pdf2013462https://repositorio.ufmg.br/bitstream/1843/65755/1/Tese_biblioteca_Aline%20Alves%20Lara%20Gomes.pdfa742d5357473c57a9e6402d2f0435bfdMD51CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.ufmg.br/bitstream/1843/65755/2/license_rdfcfd6801dba008cb6adbd9838b81582abMD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82118https://repositorio.ufmg.br/bitstream/1843/65755/3/license.txtcda590c95a0b51b4d15f60c9642ca272MD531843/657552024-03-13 10:59:51.209oai:repositorio.ufmg.br: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ório InstitucionalPUBhttps://repositorio.ufmg.br/oaiopendoar:2024-03-13T13:59:51Repositório Institucional da UFMG - Universidade Federal de Minas Gerais (UFMG)false
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