Arginine kinase from Haemonchus contortus decreased the proliferation and increased the apoptosis of goat PBMCs in vitro.
ABSTRACT: Arginine kinase (AK), an important member of phosphagen kinase family has been extensively studied in various vertebrates and invertebrates. Immunologically, AKs are important constituents of different body parts, involved in various biological and cellular functions, and considered as immune-modulator and effector for pro-inflammatory cytokines. However, immunoregulatory changes of host cells triggered by AK protein of Haemonchus contortus, a parasitic nematode of ruminants, are still unknown. The current study was focused on cloning and characterisation of Hc-AK, and its regulatory effects on cytokines level, cell migration, cell proliferation, nitric oxide production and apoptosis of goat peripheral blood mononuclear cells (PBMCs) were observed.The full-length sequence of the Hc-AK gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sub-cloned into the prokaryotic expression vector pET-32a. The biochemical characteristics of recombinant protein Hc-AK, which was purified by affinity chromatography, were performed based on the enzymatic assay. Binding of rHc-AK with PBMCs was confirmed by immunofluorescence assay (IFA). Immunohistochemical analysis was used to detect localisation of Hc-AK within adult worms sections. The immunoregulatory effects of rHc-AK on cytokine secretions, cell proliferation, cell migration, nitric oxide production and apoptosis were determined by co-incubation of rHc-AK with goat PBMCs.The full-length ORF (1080 bp) of the Hc-AK gene was successfully cloned, and His-tagged AK protein was expressed in the Escherichia coli strain BL21. The recombinant protein of Hc-AK (rHc-AK) was about 58.5 kDa together with the fused vector protein of 18 kDa. The biochemical assay showed that the protein encoded by the Hc-ak exhibited enzymatic activity. Western blot analysis confirmed that the rHc-AK was recognised by the sera from rat (rat-antiHc-AK). The IFA results showed that rHc-AK could bind on the surface of goat PBMCs. Immunohistochemically, Hc-AK was localised at the inner and outer membrane as well as in the gut region of adult worms. The binding of rHc-AK to host cells increased the levels of IL-4, IL-10, IL-17, IFN-?, nitric oxide (NO) production and cell apoptosis of goat PBMCs, whereas, TGF-?1 levels, cell proliferation and PBMCs migration were significantly decreased in a dose dependent manner.Our findings suggested that rHc-AK is an important excretory and secretory (ES) protein involved in host immune responses and exhibit distinct immunomodulatory properties during interaction with goat PBMCs.
Project description:BACKGROUND:RNA interference (RNAi) is an important tool to determine the role of genes. RNAi has been widely used to downregulate target molecules, resulting in the reduction of mRNA for protein expression. Matrix metalloprotease 12A (MMP-12) is known to have important roles during embryonic development, organ morphogenesis and pathological processes in animals. However, MMP-12 from Haemonchus contortus has not been characterized. METHODS:Haemonchus contortus MMP-12 gene was cloned and recombinant protein of MMP-12 (rHc-MMP-12) was expressed. Binding activities of rHc-MMP-12 to goat peripheral blood mononuclear cells (PBMCs) were assessed by immunofluorescence assay (IFA) and the immuno-regulatory effects of rHc-MMP-12 on cell proliferation and nitric oxide production were observed by co-incubation of rHc-MMP-12 with goat PBMCs. Furthermore, a soaking method was used to knockdown the expression of Hc-MMP12 gene using three siRNA, targeting different regions of the gene and infectivity of effective siRNA on the development of H. contortus was evaluated in goat. RESULTS:rHc-MMP-12 was successfully expressed in an expression vector as well as the tissues of the cuticle of adult H. contortus worms and a successful binding with PBMCs surface were observed. Increased cellular proliferation and nitric oxide production by goat PBMCs was observed in a dose-dependent manner. Quantitative real time PCR (qRT-PCR) results confirmed the successful silencing of Hc-MMP-12 gene in siRNA of 1, 2 and 3 treated third-stage larvae (L3) of H. contortus in vitro. The most efficient qRT-PCR-identified siRNA template was siRNA-2, with a 69% suppression rate compared to the control groups. Moreover, in an in vivo study, silencing of the Hc-MMP-12 gene by siRNA-2 reduced the number of eggs (54.02%), hatchability (16.84%) and worm burden (51.47%) as compared to snRNA-treated control group. In addition, a shorter length of worms in siRNA-2-treated group was observed as compared to control groups. CONCLUSIONS:Our results indicate that siRNA-mediated silencing of Hc-MMP-12 gene in H. contortus significantly reduce the egg counts, larval hatchability, and adult worm counts and sizes. The findings of the present study demonstrate important roles of Hc-MMP-12 in the development of H. contortus.
Project description:During host-parasite interactions, binding of excretory/secretory proteins (ESPs) on the host immune cells is considered the fundamental phase for regulation of immune responses. In this study, gene encoding Haemonchus contortus tropomyosin (Hc-TpMy), was successfully cloned and expressed, and the recombinant protein after host cell surface attachment was evaluated for immune functional analysis with goat peripheral blood mononuclear cells (PBMCs) in vitro. The isopropyl-?-D-thiogalactopyranoside (IPTG)-induced recombinant protein was successfully recognized by the sera of rat experimentally infected with rHc-TpMy. The immunofluorescence assay detected attachment of rHc-TpMy on the surface of host PBMCs. Furthermore, immunoregulatory roles of rHc-TpMy on cytokines expression, PBMC proliferation, migration, nitric oxide (NO) production, apoptosis and monocytes phagocytosis were observed. The results showed that expression of IL-4 and IFN-? cytokines, cell proliferation, NO production and PBMC migration were significantly suppressed by goat PBMCs after co-incubation with rHc-TpMy protein. However, the productions of IL-10, IL-17 and TGF-?1 cytokines, PBMCs apoptosis and monocytes phagocytosis were elevated at dose dependent manner. Our findings indicated that rHc-TpMy is an important ES binding protein exhibit distinct immuno-suppressive roles on goat PBMCs which might be a potential molecular target to control haemonchosis in future.
Project description:Galectins are glycan-binding proteins that are widely expressed and distributed in mammalian tissues as well as cells of innate and adaptive immune responses. CD4+ T-helper cells differentiate into effector subsets in response to cytokines. T helper 9 cells are one of the recently described subsets of effector T cells that are relatively new and less studied. In this study, galectin domain containing protein from Haemonchus contortus (Hc-GDC) was cloned, expressed in pET32a, and immunoblotting was performed. Localization of recombinant (r)Hc-GDC on outer and inner surface of H. contortus worm and binding with goat Peripheral Blood Mononuclear cells (PBMCs) were performed using immunofluorescence assay. Moreover, effects of rHc-GDC on proliferation, apoptosis, cell migration, and the nitric oxide production in goat PBMCs were evaluated. Furthermore, modulatory effects of rHc-GDC on production of Th1, Th2, and Th9 cells were evaluated by flowcytometry and on interferon gamma, interleukin (IL)-4 and IL-9 were evaluated by quantitative real-time polymerase chain reaction. The results demonstrated that rHc-GDC was successfully cloned, expressed in expression vector as well as in the gut surface of adult H. contortus worm and successful binding with PBMCs surface were observed. Immunoblotting results revealed that rHc-GDC is an important active protein of H. contortus excretory and secretory products. Moreover, the interaction of rHc-GDC with host cells increased the production of Th2, Th9 cells, IL4, IL-9, PBMC proliferation, nitric oxide, and cell migration. No effects of rHc-GDC were observed on PMBC apoptosis, production of Th1 cells, and secretions of IFN- and IL-10 cytokines. These findings indicate that recombinant GDC protein from H. contortus modulates the immune functions of goat PBMCs and has the potential to enhance protective immunity by inducing T helper-9-derived IL-9 in vitro.
Project description:Small size excretory/secretory (ES) antigens of the Haemonchus contortus parasite have intense interest among researchers for understanding the molecular basis of helminths immune regulation in term of control strategies. Immunomodulatory roles of H. contortus ES-15 kDa (HcES-15) on host immune cells during host-parasite interactions are unknown. In this study, the HcES-15 gene was cloned and expression of recombinant protein (rHcES-15) was induced by isopropyl-ß-D-thiogalactopyranoside (IPTG). Binding activity of rHcES-15 to goat peripheral blood mononuclear cells (PBMCs) was confirmed by immunofluorescence assay (IFA) and immunohistochemical analysis showed that H. contortus 15 kDa protein localized in the outer and inner structure of the adult worm, clearly indicated as the parasite's ES antigen. The immunoregulatory role on cytokines production, cell proliferation, cell migration, nitric oxide (NO) production, apoptosis, and phagocytosis were observed by co-incubation of rHcES-15 with goat PBMCs. The results showed that cytokines IL-4, IL-10, IL-17, the production of nitric oxide (NO), PBMCs apoptosis, and monocytes phagocytosis were all elevated after cells incubated with rHcES-15 at differential protein concentrations. We also found that IFN-?, TGF-?1, cells proliferation and migration were significantly suppressed with the interaction of rHcES-15 protein. Our findings indicated that low molecular ES antigens of H. contortus possessed discrete immunoregulatory roles, which will help to understand the mechanisms involved in immune evasion by the parasite during host-parasite interactions.
Project description:<h4>Background</h4>Hepatocellular carcinoma-associated antigen 59 (HCA59), which is one of the most important excretory/secretory products of Haemonchus contortus (HcESPs), is known to have antigenic functions. However, its immunomodulatory effects on host cells are poorly understood.<h4>Methods</h4>Here, we cloned the HCA59 gene and expressed the recombinant protein of HCA59 (rHCA59). Binding activities of rHCA59 to goat peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) were checked by immunofluorescence assay (IFA) and the immunoregulatory effects of rHCA59 on cytokine secretions, cell migration, cell proliferation, nitric oxide production, and changes in expression of genes in related pathways were observed by co-incubation of rHCA59 with goat PBMCs and DCs. Monocyte phagocytosis and characterization of goat blood DC subsets were detected by flow cytometry.<h4>Results</h4>The IFA results revealed that rHCA59 could bind to PBMCs and DCs. Treatment of PBMCs with rHCA59 significantly increased cellular proliferation and NO production in a dose-dependent manner, while cell migration was vigorously blocked. Treatment with rHCA59 significantly suppressed monocytes phagocytosis. The quantity of surface marker CD80 on DCs increased significantly after rHCA59 treatment. In addition, the expression of genes included in the WNT pathway was related to the differentiation and maturation of DCs, and the production of IL-10 and IL-17 produced by PBMCs was altered.<h4>Conclusions</h4>Our findings illustrated that rHCA59 could enhance host immune responses by regulating the functions of goat PBMCs and DCs, which would benefit our understanding of HCA59 from parasitic nematodes contributing to the mechanism of parasitic immune evasion.
Project description:Trehalose phosphate synthase (TPS), a key enzyme in trehalose synthesis, is not present in mammals but critical to the viability of a wide range of lower organisms. However, almost nothing is known about the function of Hc-TPS (GT1-TPS structural domain protein from <i>Haemonchus contortus</i>). In this study, Hc-TPS gene was cloned and the recombinant protein (rHc-TPS) was expressed and purified. The quantitative real-time PCR (qPCR) results showed that Hc-TPS was transcribed at different stages of <i>H. contortus</i>, with higher levels of transcription at the molting and embryo stages. Immunofluorescence analysis showed that Hc-TPS was widely distributed in adults, but the expression was mainly localized on the mucosal surface of the intestine as well as in the embryos of female worms. The impacts of rHc-TPS on peripheral blood mononuclear cell (PBMC) proliferation, nitric oxide (NO) generation, transcriptional expression of cytokines, and related pathways were examined by co-incubating rHc-TPS with goat PBMCs. The results showed that rHc-TPS significantly inhibited PBMC proliferation and NO secretion in a dose-dependent manner. We also found that rHc-TPS activated the interleukin (IL)-10/signal transducer and activator of transcription 3/suppressor of cytokine signaling 3 (IL-10/STAT3/SOCS3) axis and significantly promoted SOCS3 expression, while inhibiting interferon-gamma (INF-γ), IL-4, IL-9, and IL-2 pathways. Our findings may contribute to understanding the immune evasion mechanism for the parasite during host-parasite interactions and also help to provide ideas for discovering new drug targets.
Project description:A 24 kDa protein is one of the important components in Haemonchus contortus (barber pole worm) excretory/secretory products (HcESPs), which was shown to have important antigenic function. However, little is known about the immunomodulatory effects of this proteinon host cell. In the present study gene encoding 24kDa excretory/secretory protein (HcES-24) was cloned. The recombinant protein of HcES-24 (rHcES-24) was expressed in a histidine-tagged fusion protein soluble form in Escherichia coli. Binding activity of rHcES-24 to goat PBMCs was confirmed by immunofluorescence assay (IFA) and its immunomudulatory effect on cytokine secretion, cell proliferation, cell migration and nitric oxide production were observed by co-incubation of rHcES-24. IFA results revealed that rHcES-24 could bind to the PBMCs. The interaction of rHcES-24 increased the production of IL4, IL10, IL17 and cell migration in dose dependent manner. However, rHcES-24 treatment significantly suppressed the production of IFN?, proliferation of the PBMC and Nitric oxide (NO) production. Our findings showed that the rHcES-24 played important regulatory effects on the goat PBMCs.
Project description:ADP-ribosylation factors (ARFs) are members of the Ras-related small GTPase family involved in the vesicular trafficking regulation. Immunomodulatory effects of these proteinson host cell arenot being addressed yet. H. contortus small GTPase ADP-ribosylation 1 gene (HcARF1) was cloned and recombinant protein of HcARF1 (rHcARF1) was successfully expressed in Escherichia coli. Binding activity of rHcARF1 to goat PBMCs was confirmed by immunofluorescence assay (IFA) and its immunomudulatory effects on cytokine secretion, cell proliferation, cell migration and nitric oxide production (NO) were observed by co-incubation of rHcARF1. IFA results revealed that rHcARF1 could bind to the PBMCs. The interaction of rHcARF1 modulated the cytokine production, the production of IL-4, IL-10 and IL-17 was increased in a dose dependent manner, however, the IFN-? production was significantly decreased. Cell migration and NO production were significantly increased by rHcARF1, whereas, rHcARF1 treatment significantly suppressed the proliferation of the PBMC in a dose dependent manner. Our findings showed that the rHcARF1 play important roles on the goat PBMCs.
Project description:BACKGROUND:The molecular structure of Fasciola gigantica 14-3-3 protein has been characterized. However, the involvement of this protein in parasite pathogenesis remains elusive and its effect on the functions of innate immune cells is unknown. We report on the cloning and expression of a recombinant F. gigantica 14-3-3 epsilon protein (rFg14-3-3e), and testing its effects on specific functions of goat peripheral blood mononuclear cells (PBMCs). METHODS:rFg14-3-3e protein was expressed in Pichia pastoris. Western blot and immunofluorescence assay (IFA) were used to examine the reactivity of rFg14-3-3e protein to anti-F. gigantica and anti-rFg14-3-3e antibodies, respectively. Various assays were used to investigate the stimulatory effects of the purified rFg14-3-3e protein on specific functions of goat PBMCs, including cytokine secretion, proliferation, migration, nitric oxide (NO) production, phagocytosis, and apoptotic capabilities. Potential protein interactors of rFg14-3-3e were identified by querying the databases Intact, String, BioPlex and BioGrid. A Total Energy analysis of each of the identified interaction was performed. Gene Ontology (GO) enrichment analysis was conducted using Funcassociate 3.0. RESULTS:Sequence analysis revealed that rFg14-3-3e protein had 100% identity to 14-3-3 protein from Fasciola hepatica. Western blot analysis showed that rFg14-3-3e protein is recognized by sera from goats experimentally infected with F. gigantica and immunofluorescence staining using rat anti-rFg14-3-3e antibodies demonstrated the specific binding of rFg14-3-3e protein to the surface of goat PBMCs. rFg14-3-3e protein stimulated goat PBMCs to produce interleukin-10 (IL-10) and transforming growth factor beta (TGF-?), corresponding with low levels of IL-4 and interferon gamma (IFN-?). Also, this recombinant protein promoted the release of NO and cell apoptosis, and inhibited the proliferation and migration of goat PBMCs and suppressed monocyte phagocytosis. Homology modelling revealed 65% identity between rFg14-3-3e and human 14-3-3 protein YWHAE. GO enrichment analysis of the interacting proteins identified terms related to apoptosis, protein binding, locomotion, hippo signalling and leukocyte and lymphocyte differentiation, supporting the experimental findings. CONCLUSIONS:Our data suggest that rFg14-3-3e protein can influence various cellular and immunological functions of goat PBMCs in vitro and may be involved in mediating F. gigantica pathogenesis. Because of its involvement in F. gigantica recognition by innate immune cells, rFg14-3-3e protein may have applications for development of diagnostics and therapeutic interventions.
Project description:Hco-gal-m and -f were two isoforms of galectin cloned from male and female Haemonchus contortus, respectively, and it was demonstrated that recombinant Hco-gal-m and -f could act as immune suppressors. However, little is known about the receptors or binding partners of these galectins in the host. The research of the molecular mechanisms that govern the interactions between these galectins and host molecules will fill a gap in our understanding how parasite galectins interact with host cells.A yeast two-hybrid system was used to identify the binding partners of Hco-gal-m and -f in this research. The interaction between rHco-gal-m and candidate binding protein was validated by co-immunoprecipitation. The localization of transmembrane protein 63A (TMEM63A) in peripheral blood mononuclear cells (PBMCs) was detected by immunofluorescence. The distribution of TMEM63A in T cells, B cells and monocytes in PBMCs was detected by flow cytometry. The immunomodulatory effects of Hco-gal-m and TMEM63A on cell proliferation, migration, apoptosis, nitric oxide production and cytokine secretion were observed by co-incubation of rHco-gal-m and TMEM63A-siRNA with goat PBMCs and monocytes.We found that TMEM63A, a functionally unknown protein, from goat PBMCs could bind to Hco-gal-m and -f. Immunofluorescence showed that TMEM63A was localized to the cell membrane. Flow cytometric analysis revealed that TMEM63A was expressed in the majority of goat PBMCs. After using RNA interference to knockdown expression of TMEM63A, the PBMC proliferation and migration were significantly increased, while the influence of rHco-gal-m on monocyte phagocytosis, PBMC nitric oxide production and migration were potently blocked. In addition, the production of IL-10, IFN-? and TGF-? induced by rHco-gal-m were also altered.Our results show that TMEM63A is a binding partner of Hco-gal-m/f, and involved in the immune responses of host PBMCs induced by Hco-gal-m for the first time.