Mice (= 7C8/group) received an intraperitoneal injection of 15 g of monoclonal rat IgG2anti-Siglec-F antibody the day before and after infection with and every 3 days until sacrifice. juvenile flukes penetrate the hosts intestine wall and reach the liver between 4 and 6 days. Eventually, the flukes reach the bile ducts, where they become sexually mature (3). causes chronic infections due to sophisticated immune modulation strategies that make possible its long-period survival in the host. Indeed, several studies have shown that the parasite induces regulatory dendritic cells (4, 5), alternative activated macrophages (6), and a type 2 modified immune response characterized by an important regulatory T cell (Treg) component (4, 7). Eosinophils, granulocytes belonging to the innate immune system, participate mainly in the defense against Losartan multicellular parasites and in several Th2-driven immune disorders, such as asthma, atopic dermatitis, and eosinophilic esophagitis (8). The classical functions of eosinophils include mainly degranulation triggered by antibodies in a mechanism known as antibody-dependent cell cytotoxicity (ADCC) (8, 9). However, in the last years, novel immunomodulatory functions have been reported such as the regulation of glucose metabolism in the adipose tissue (10), enhancement of plasma cells survival (11), and suppression of Th1 immune responses (12, 13). Eosinophils play a pivotal role in fighting against some helminth infections (9), reflected by their dramatic increase in response to IL-5 produced by CD4+ type 2 helper T cells (Th2) (14). Although their capacity for killing helminths through ADCC has been Losartan well demonstrated (15, 16), their role remains controversial. While in some cases eosinophils appear to be beneficial for the host (17) in others their presence is redundant (18) or even of apparent benefit for the parasite (19). In infection, however, the role of Losartan eosinophils remains unknown. In this study we characterized eosinophils during experimental infection with in mice and demonstrate that they contribute to limit liver damage induced by the parasite by reducing the production of IL-10 by CD4+ T cells. In addition, our results indicate that eosinophils play a role in specific humoral immunity by inducing the production of more effective antibodies in triggering eosinophil degranulation. Thus, the present study contributes to the elucidation of immunomodulatory mechanisms mediated by eosinophils during infection and collaborates with the understanding of their role in helminth infections. Materials and Methods Ethics Statement Mouse experiments were carried out in accordance with strict guidelines from the National Committee on Animal Research (Comisin Nacional de Experimentacin Animal, CNEA, http://www.cnea.gub.uy/, National Law 18.611, Uruguay) according to the international statements on animal use in biomedical research from the Pan American Health Organization (PAHO) and World Health Organization (WHO). The protocol was approved by the Uruguayan Committee on Animal Research. Cattles livers were collected during the routine work of a local abattoir (Frigorfico Carrasco) in Montevideo (Uruguay). Mice Six- to eight-week-old female BALB/c mice were purchased from DILAVE Laboratories (Uruguay). Animals were kept in the animal house (URBE, Facultad de Medicina, UdelaR, Uruguay) with water and food supplied were obtained from the bile ducts of bovine livers, washed in phosphate-buffered saline (PBS) pH 7.4, then mechanically disrupted and sonicated. After centrifugation at 40,000 for 60 min, supernatants were collected and dialyzed against PBS. The obtained lysate (FhTE) was quantified and stored at -80C. The endotoxin Rabbit polyclonal to EpCAM levels were determined by using the Limulus Amebocyte Lysate kit Pyrochrome (Associates of Cape Cod). Adult worms were collected during the routine work of a local abattoir (Frigorfico Carrasco and Sarubbi) in Montevideo (Uruguay). Protocols were approved by the Uruguayan Committee on Animal Research (CHEA, Protocol Number: 070153-000820-17). Infections BALB/c mice were orally infected with 10 metacercariae (Montevideo, Uruguay) per animal. After 8, 15, or 21 days post-infection (d.p.i.) mice were bled and peritoneal exudate cells (PECs), spleens, and livers were removed. noninfected animals were used as controls. At least 4 animals were used per group. Peritoneal exudate cells were harvested by washing the peritoneal cavity with 10 ml of cold PBS. For the isolation of hepatic leukocytes, a previously described protocol was followed (18). Briefly, livers were mechanically dissociated and the cell suspension was left on ice for 15-20 min. The supernatant was then recovered in a new tube and centrifuged at 120 for 7 min at 4C. The pellet was resuspended in 40% Percoll gradient and centrifuged at 600 for 20 min at 20C, after which the hepatic leukocytes were obtained. Finally, red cells.
