| Haemonchus contortus, also known as red stomach worm or barber’s pole worm, is very common parasite and one of the most pathogenic nematodes of ruminants. Adult worms are attached to abomasal mucosa and feed on the blood. This parasite is responsible for anemia, bottle jaw, and death of infected sheep and goats.Galectins are a family of S-type lectins, found in vertebrates and invertebrates, that are featured by various roles in apoptosis, chemoattraction, cell adhesion, cell proliferation, cytokine secretion and immune responses. Growing evidence from recent studies indicates that host galectins can function as pattern recognition receptors (PRRs) that crosslink glycans on the surfaces of viruses, bacteria and helminths, which, like many other receptor-ligand systems, can trigger a cascade of transmembrane signaling events in different biological processes such as cell-cell interaction, cell-pathogen interaction as well as activation and homeostasis of immune cells.In our previous research, we reported that two isoforms of galectins, Hco-gal-m (Acc. No. AY253330) and Hco-gal-f (Acc. No. AY253331), derived, respectively, frommale (m) and female (f) H. contortus, varied by one amino acid in the C-terminal CRD. Both galectins could hemagglutinate human, dog, rabbit, chicken and mouse erythrocytes, but not the erythrocytes of goat, one natural host of H. contortus. They also had the ability to inhibit the mRNA transcription of goat cytokines and induce apoptosis of goat PBMC in a dose-dependent manner. Vaccination of goats with rHco-gal-m/f significantly reduced the fecal egg output, as well as worm burdens, and increased levels of IgG in the vaccinated groups compared to the negative controls post challenge with H. contortus.All these findings suggested that galectins of parasitic origin actively participated in the interactions between mammalian hosts and infective pathogens and might contribute to parasitic invasion or modulation of the host immune response. However, the mechanisms underlying the immune modulation and evasion induced by parasitic galectins remain unclear. So, the research work was sequentially carried as follows:1. Developmental expression and localization of the galectins in Haemonchus contortusIn this study, the timing and site of galectin expression in Haemonchus contortus were analyzed. To evaluate the developmental expression of the galectin (Hco-gal-m/f), real-time PCR was performed with specific primers and cDNA from different life forms of H. contortus. The results showed that Hco-gal-m/f was expressed in all life cycle stages without eggs as well as L3, and maximally expressed in adult worms. This stage-restricted expression was further confirmed by western blot using the antibody eluted from recombinant Hco-gal-m/f (rHco-gal-m/f). Immunohistochemical staining with anti-rHco-gal-m/f serum as the primary antibody was further conducted to examine the localization of Hco-gal-m/f in adult worms. It showed that Hco-gal-m/f was expressed in the intestinal surface of the adult worm’s gut.2. Transcriptional and proteomic analysis reveal recombinant galectins of Haemonchus contortus down-regulated functions of goat PBMC and modulation of several signaling cascades in vitroIn this research, a combined proteomic and transcriptomic analysis was performed to understand the mechanisms underlying the immunomodulation induced by recombinant galectins of Haemonchus contortus (rHco-gal-m/f) on goat peripheral blood mononuclear cells (PBMC). We demonstrated that rHco-gal-m/f could be distinguished by antisera from goats experimentally infected with H. contortus and bound to the surface of goat PBMC. Following rHco-gal-m/f exposure,16 differentially expressed proteins were identified, which function in biological processes such as stimulus response, biological regulation and localization. According to Gene Ontology Annotation,15 proteins (93.8%) had binding activity and 9 proteins (56.3%) had catalytic activity. A series of transcriptomic analyses were performed subsequently to assess the expression change of certain pathway members. The integrated results of proteomic and transcriptomic analysis suggested that the activation of VEGF pathway, free radical producing pathway, NFκB pathway and ubiquitin-proteasome pathway was inhibited following exposure to rHco-gal-m/f, while the TLR pathway and CASPASE pathway were activated. Cytokine production and T cell differentiation were also influenced. Cell migration assays were performed and the results were in accordance with the change of the proteins and genes. The protein and gene profiles determined here identified several mechanisms underlying the rHco-gal-m/f-induced immunomodulation of goat PBMC.3. Proteomics characterization of goat peripheral blood mononuclear cells primed with mitogens and the inhibitory mechanism of Haemonchus contortus derived galectins on cytokine productionIn this study, proteomic study combined transcriptomic analysis were performed to understand the mechanisms underlying the activation of goat peripheral blood mononuclear cells (PBMC) primed with mitogen and the inhibitory mechanism of galectins from Haemonchus contortus (rHco-gal-m/f) on cytokine production. When goat PBMC were activated with ConA, totally 22 differentially expressed protein were identified, which mostly involved in the biological processes including matrix organization, actin filament capping, regulation of translational initiation and regulation of cell proliferation. A series of transcriptomic analysis were performed subsequently to assess the expression change of certain pathway members associated with cell activation and cytokine production. The result suggested that the exposure of ConA promoted the activation of cytokine-cytokine receptor, MAPKs, STATs as well as CDKs in goat PBMC. However, following rHco-gal-m/f engagement, the activation of these signal pathways was reversed. Our study provided some marker proteins which might be used to indicate the activation of goat PBMCs and to evaluate the involvement of inflammatory activated goat PBMCs in certain diseases. Furthermore, the gene profiles provided a valuable basis for a better understanding of the mechanisms underlying the inhibitory activity of rHco-gal-m/f on cytokine production in goat PBMC.4. Galectin Hco-gal-m from Haemonchus Contortus modulates goat monocytes and T cells function in different patternsMonocytes and T cells are two major subpopulations of peripheral blood mononuclear cells (PBMC) and play an essential role in the innate and adaptive immune systems. Different members of galectin family show multiple and distinct regulatory effects on different cell types. In this study, flow cytometric analyses revealed that rHco-gal-m could bind to both monocytes and T cells. The engagement of rHco-gal-m decreased the production of IL-6, IL-10 and TNF-a in T cells, however, it significantly increased the secretion of IL-10 in monocytes. After rHco-gal-m exposure, the expression of MHC-II on monocytes and that of CD25 on T cells were restricted. Consequently, T cell proliferations were potently inhibited by rHco-gal-m. Finally, we demonstrated that rHco-gal-m induced apoptosis in T cells, but not significantly in monocytes. These results indicated that rHco-gal-m modulated goat monocytes and T cells function in different patterns. |
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