Estudios de los mecanismos de inmunomodulación por Fasciola hepáticaApoptosis y linfocitos T reguladores

Résumé

Fasciolosis caused by Fasciola hepatica is an economically important disease of ruminants in temperate climates. F. hepatica has developed a variety of mechanisms to modulate or suppress the host response making it ineffective, which allows the parasite to survive in the host for years. The parasite host immune response modulation is a serious obstacle for developing protective vaccines for ruminants. The knowledge of the immunomodulation mechanisms carried out by F. hepatica to avoid the host immune response is a key to improve the vaccines development. In helminth infections, eosinophils play important roles both in developing tissue pathology and in the host effector response. Apoptosis of effector cells such as eosinophils and macrophages may play a role in the host immune evasion/suppression induced by helminth infection. Studies in the rat model have demonstrated that secreted excretory products of F. hepatica (FhESP) are able to induce apoptosis in eosinophils and peritoneal macrophages -in vitro- and in the inflammatory infiltrate - in vivo-, suggesting that apoptosis of effector cells may play a role in the host immune evasion/suppression induced by F. hepatica infection. The use of activated caspasa 3 antibodies is an easy, reliable and sensitive method to study the presence of apoptosis in tissue sections. The aim of this study, included in the First Work of this Thesis was to identify and quantify apoptosis of eosinophils in the hepatic inflammatory infiltrates of sheep challenged with F. hepatica during the migratory and biliary stages of infection. The aim of the Second Work was to evaluate the presence and number of inflammatory peritoneal leucocytes undergoing apoptosis in peritoneal fluid from sheep experimentally infected with F. hepatica during the peritoneal migratory stages in the host. Twenty five- merino sheep were used. They were divided into five groups (n=5): Group 1 was used as an uninfected control group (UC) and groups 2–5 were orally infected with one dose of 200 F. hepatica metacercariae and sheep from these groups were sacrificed by an intravenous injection of thiobarbital at 1, 3, 9, and 18 dpi, respectively. Apoptosis was detected using three different methods: 1) immunocytochemistry (ICC) with a polyclonal antibody anti-active caspase-3; 2) Flow cytometry assay using the Annexin V-FITC/propidium iodide (PI) kit; and 3) transmission electron microscopy (TEM). The aim of the Third Work of this Thesis was to evaluate the presence of Foxp3+ Tregs in liver and hepatic lymph nodes (HLN) from experimentally infected sheep and goats during acute and chronic stages of infection.