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Previous issue date: 2016-12-13 / Introdu??o: Os c?es s?o considerados os principais reservat?rios dom?sticos de Leishmania infantum no Brasil e apresentam um amplo espectro de manifesta??es cl?nicas, variando de perda de peso, anemia grave, hepatoesplenomegalia e les?es d?rmicas disseminadas. O objetivo deste estudo foi avaliar o perfil de express?o g?nica em c?lulas do ba?o e do sangue perif?rico de c?es naturalmente infectados por L. infantum e investigar as altera??es histopatol?gicas no ba?o, f?gado, intestino e pele, visando identificar poss?veis vias imunorreguladoras envolvidas na patog?nese da LVC. Metodologia: Vinte e um c?es oriundos do Centro de Controle de Zoonoses de Natal-RN foram estudados e um subgrupo destes animais (n=8) foi utilizado para estudos de express?o g?nica global. A carga de Leishmania nos diversos tecidos, foi estimada por qPCR e cultura para Leishmania foi realizada. O RNA total do ba?o e do sangue perif?rico foi extra?do e as bibliotecas de cDNA foram obtidas e sequenciadas em plataforma Illumina. As sequ?ncias obtidas foram filtradas e alinhadas contra o genoma de Canis familiaris pelo software Bowtie. A express?o g?nica diferencial foi analisada usando o protocolo do Cufflinks, a anota??o funcional e as caracter?sticas moleculares dos genes foram obtidas no banco de dados Gene Ontology e KEGG atrav?s do pacote STRINGdb. Fragmentos de f?gado, duodeno, pele e ba?o foram coletados, processados e corados com hematoxilina/eosina e imunofluoresc?ncia para an?lises histopatol?gicas. Resultados: As cargas parasit?rias estimadas para ba?o e f?gado apresentam correla??o, por?m n?o h? correla??o entre a carga parasit?ria destes tecidos com aquela presente no sangue. Aproximadamente 756 genes se mostraram super expressos no ba?o de animais sintom?ticos, como CTLA4, CCL2 e IL27; al?m destes, cerca de 1.190 genes apresentaram express?o reprimida neste mesmo grupo, como BMP6, C1QB e C1QC. As vias de ativa??o de c?lulas NK, receptores dos tipos Toll-like e NOD-like expressas no ba?o de animais sintom?ticos estavam com express?o elevada, enquanto as vias de s?ntese de ?ncoras de GPI e ativa??o do imunoproteossoma estavam reprimidas. A progress?o da LVC resulta em desorganiza??o da citoarquitetura do ba?o, com perda parcial da delimita??o entre polpa branca e vermelha. ? observado infiltrado inflamat?rio no f?gado, duodeno e pele. Validando o achado do transcriptoma, foi observada por imunofluoresc?ncia a presen?a de CTLA4, CCR7 e NLRP3 no ba?o de animais sintom?ticos. Conclus?es: A desregula??o de mecanismos tipicamente associados ? imunidade inata pode estar diretamente envolvida na patogenia da LVC. Al?m disso, uma alta efici?ncia no processamento e apresenta??o de ant?genos parecem ser respons?veis, pela resist?ncia ao desenvolvimento dos sinais cl?nicos e integridade tissular durante a infec??o por L. infantum em c?es. / Background: Dog is the main domestic reservoir of Leishmania infantum in Brazil. Those animals present a wide spectrum of clinical manifestations, varying from weight loss, anemia to hepatosplenomegaly and skin lesions. The goal of this study was to evaluate the gene expression profile in the spleen and peripheral blood cells from naturally Leishmania-infected dogs and to study the histology of the spleen, liver, gut and skin, in order to determine possible molecular pathways and histopathological process underlying the mechanisms related to disease progression and resistance. Methodology: 21 dogs was studied and a sub group (n=8) was selected for transcriptomic studies. In addition, spleen, liver, gut and blood parasitism were estimated by qPCR and parasite was isolated from the spleen by culture. Total RNA was extracted and cDNA libraries were constructed and sequenced in an Illumina platform. The reads obtained were filtered and aligned against Canis familiaris genome using Bowtie. Differential gene expression was analyzed using the Cufflinks workflow, functional annotation and molecular features for each gene was obtained from Gene Ontology and KEGG database using STRINGdb. A fragment of liver, gut, skin and another spleen fragment were collected, processed and stained by HE and immunofluorescence for histopathological analysis. Results: Parasite load estimated in the spleen and liver were strongly associated, however no correlation was observed between them and circulating parasite estimation. Approximately, 756 genes were upregulated in the spleens of symptomatic dogs, as CTLA4, CCL2 and IL27, furthermore, about 1,190 genes were downregulated in the same group of animals, as BMP6, C1QB and C1QC. We found that NK cells activation, toll-like and NOD-like receptors pathways are highly expressed in the spleen of symptomatic dogs, while GPI-anchors synthesis and immunoproteasome activation related genes are downregulated in these animals. Additionally, we observed that in symptomatic animals, the cytoarchitecture of spleen is altered, with partial loss of the delimitation between white and red pulp. Furthermore, an intense inflammatory infiltrate in the liver, gut and skin of these animals. Confirming the RNA-Seq findings, we observed by immunofluorescence CTLA4, CCR7 and NLRP3 are overexpressed the spleen of symptomatic dogs. Conclusions: These findings suggest that misregulation of innate immunity is involved in CVL and an efficient antigen presentation maybe decisive for resistance to clinical signs development and tissue integrity during L. infantum infection in dogs.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/23361 |
| Date | 13 December 2016 |
| Creators | Nascimento, Paulo Ricardo Porfirio do |
| Contributors | 15603016434, http://lattes.cnpq.br/7120263570947836, Brodskyn, Cl?udia Ida, 23380489549, http://lattes.cnpq.br/8510726976369443, Santos, Elizeu Antunes dos, 41305655400, http://lattes.cnpq.br/6762251930590306, Pasquali, Matheus Augusto de Bittencourt, 97496065072, http://lattes.cnpq.br/2819075769518114, Souza, Sandro Jos? de, 70990409953, http://lattes.cnpq.br/8479967495464590, Jer?nimo, Selma Maria Bezerra |
| Publisher | PROGRAMA DE P?S-GRADUA??O EM BIOQU?MICA, UFRN, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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