The Serine/Threonine-Protein Phosphatase 1 From Haemonchus contortus Is Actively Involved in Suppressive Regulatory Roles on Immune Functions of Goat Peripheral Blood Mononuclear Cells

Serine/threonine-protein phosphatases (STPs), as integral constituents of parasitic excretory/secretory proteins, are assumed to be released during the host–parasite interactions. However, knowledge about these phosphatases and their immunoregulatory and immune protective efficiencies with host peripheral blood mononuclear cells (PBMCs) is scant. In this study, an open reading frame of STP from Haemonchus contortus designated as HcSTP-1 was amplified and cloned using reverse-transcription-polymerase chain reaction (RT-PCR) method. The 951-bp nucleotides sequence was encoded to a protein of 316 amino acid residues, conserved in characteristics motifs GDXHG, GDYVDRG, GNHE, HGG, RG, and H. The HcSTP-1 protein was detected at approximately 35 kDa as recombinant protein fused in an expression vector system and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunohistochemically, HcSTP-1 was found to be localized in both male and female adult worm sections. Using immunofluorescence assay, the binding activity of rHcSTP-1 was confirmed on surface of goat PBMCs, which resulted in expression of multiple cytokines and various immunoregulatory activities in vitro. The RT-PCR results showed that mRNA level of interleukin-2, TGF-β1, IFN-γ, and IL-17 (with 10 µg/ml) was upregulated and IL-10 was decreased. However, IL-6 showed no change after PBMCs incubated with rHcSTP-1 protein. Further functional analysis showed that migratory activity of cells, intracellular nitrite production (NO), and apoptotic efficiency of PBMCs were elevated at significant level, whereas the proliferation of goat PBMCs and monocytes-associated major histocompatibility complex (MHC)-I and MHC-II expressions were decreased significantly at concentration-dependent fashion. Our results showed that the HcSTP-1 protein engaged in vital suppressive regulatory roles on host immune cells, which might represent a potential molecular target for controlling H. contortus infection in future

Characterization of Haemonchus contortus Excretory/Secretory Antigen (ES-15) and Its Modulatory Functions on Goat Immune Cells In Vitro

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-γ, TGF-β1, 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

Effects of Recombinant Toxoplasma gondii Citrate Synthase I on the Cellular Functions of Murine Macrophages In vitro

Toxoplasmosis, which is one of the most widespread zoonoses worldwide, has a high incidence and infection can result in severe disease in humans and livestock. Citrate synthase (CS) is a component of nearly all living cells that plays a vital role in the citric acid cycle, which is the central metabolic pathway of aerobic organisms. In the present study, the citrate synthase I gene of Toxoplasma gondii (T. gondii) (TgCSI) was cloned and characterized. The TgCSI gene had an open reading frame of 1665 bp nucleotides encoding a 555 amino acid protein with a molecular weight of 60 kDa. Using western blotting assay, the recombinant protein was successfully recognized by the sera of rats experimentally infected with T. gondii, while the native protein in the T. gondii tachyzoites was detected in sera from rats immunized with the recombinant protein of TgCSI. Binding of the protein to murine macrophages was confirmed by immuno fluorescence assay. Following incubation of macrophages with rTgCSI, the rTgCSI protein was found to have a dual function, with low concentrations (5–10 μg/mL) enhancing phagocytosis and high levels (80 μg/mL) inhibiting phagocytosis. Investigation of murine macrophage apoptosis illustrated that 5 μg/mL rTgCSI protein can significantly induce early apoptosis and late stage apoptosis (*p < 0.05), while 10 μg/mL rTgCSI protein significantly induced early apoptosis, but had no effect on late stage of apoptosis (**p < 0.01), and 80 μg/mL rTgCSI protein inhibited late stage apoptosis of macrophages (*p < 0.05). Cytokine detection revealed that the secretion of interleukin-10, interleukin-1β, transforming growth factor-β1 and tumor necrosis factor-α of macrophages increased after the cells were incubated with all concentration of rTgCSI, with the exception that 5 μg/mL rTgCSI had no effect on the secretion of interleukin-10 and interleukin-1β. However, secretion of NO and cell proliferation of the macrophages were substantially reduced. Taken together, these results suggested that TgCSI can affect the immune functions of murine macrophages by binding to the cells in vitro

Hepatocellular carcinoma-associated antigen 59 of Haemonchus contortus modulates the functions of PBMCs and the differentiation and maturation of monocyte-derived dendritic cells of goats in vitro

Abstract Background 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. Methods 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. Results 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. Conclusions 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

Immunomodulatory dynamics of excretory and secretory products on Th9 immune response during Haemonchus contortus infection in goat.

CD4+ T cells play critical roles in mediating adaptive immunity to a variety of pathogens. Recently, new subset of CD4+T named as T helper 9 cells that express the prototypical interleukin-9 (IL-9) cytokine have been recognized in human and mice models during different parasitic infections. Haemonchus contortus is a gastrointestinal nematode of small ruminants which cause high mortality in young animals. During infection, Excretory and Secretary Products (ESPs) are released in the host body. No other study has reported yet on immunomodulatory dynamics of H. contortus ESPs on Th9 immune response in vitro or in vivo. In this study, immunomodulatory effects of ESPs (5, 10, 20, 40, 80; μg/mL) incubated with goat PBMCs on Th9 cells, IL-9 immune response and TGF-β/Smad signaling regulator were evaluated in vitro. Moreover, for in vivo study, goats were infected with different doses (P-800, P-2400, and P-8000) of H. contortus infective larva (L3) and immunomodulatory effects on Th9 cells, IL-9 immune response and TGF-β/Smad signaling regulator were evaluated at 7, 10, 14, 18, 21, 28 Days Post Infection (DPI). Flow cytometry was performed to evaluate the effects on Th9 cells and quantitative real time polymerase chain reaction was performed to evaluate the IL-9 cytokine transcription level. Additionally, fecal egg counting was also performed in parallel to confirm the infection. All goats were dewormed at 29 DPI and all experiments were also performed at 35 DPI, one week post deworming. The finding indicated that 10, 20, 40, 80 μg/mL concentration of ESPs incubated with goat PBMCs showed significant increase in the production of Th9 cells, signature cytokine IL-9 and expression of TGF-β/Smad signaling regulator as compared to control group in vitro.All infected groups showed significant increase in production of Th9 cells and IL-9 cytokine and expression of TGF-β/Smad key genes at 18, 21, and 28 DPI as compared to control group. Likewise, at 14 DPI, P-2400 and P-8000 groups showed significant increase in production of Th9 cells, IL-9 cytokine and expression of TGF-β/Smad key genes. While at 10 DPI, production of Th9 cells and IL-9 was significantly increased in P-2400 & P-8000 groups, and at 7 DPI only P-8000 showed significantly increase in IL-9 production. No immunomodulatory effects were observed at 0 and 3 DPI. Additionally, significant gradually up-regulated key genes expression of TGF-β/Smad signaling regulator in all infected groups confirmed the above results. After deworming, production of Th9 cells, associated immune response and expression of signaling regulator in each group were significantly decreased. Based on this study, it is concluded that Th9 immune response was induced during H. contortus infection in goat by up-regulation of TGF-β/Smad signaling key genes

Nanoparticles (PLGA and Chitosan)-Entrapped ADP-Ribosylation Factor 1 of Haemonchus contortus Enhances the Immune Responses in ICR Mice

ADP-ribosylation factor 1 (HcARF1) is one of the Haemonchus contortus (H. contortus) excretory/secretory proteins involved in modulating the immune response of goat peripheral blood mononuclear cells (PBMC). Here, we evaluated the immunogenic potential of recombinant HcARF1 (rHcARF1) against H. contortus infection in Institute of Cancer Research (ICR) mice. Briefly, rHcARF1 was entrapped in poly (D, L-lactide-co-glycolide) (PLGA) and chitosan (CS) nanoparticles (NP) and injected into mice as a vaccine. Fifty-six ICR mice were assigned randomly into seven groups, with eight animals in each group, and they were vaccinated subcutaneously. At the end of the experiment (14th day), the blood and the spleen were collected from euthanized mice to detect lymphocyte proliferation, cytokine analysis, and the production of antigen-specific antibodies. Scanning electron microscope was used to determine the size, morphology, and zeta potential of nanoparticles. Flow cytometry was performed, which presented the increase percentages of CD4+ T cells (CD3e+CD4+), CD8+ T cells (CD3e+CD8+) and dendritic cells (CD11c+CD83+, CD11c+CD86+) in mice vaccinated with rHcARF1+PLGA NP. Immunoassay analysis show raised humoral (Immunoglobulin (Ig)G1, IgG2a, IgM) and cell-mediated immune response (Interleukin (IL)-4, IL-12, and IL-17, and Interferon (IFN)-γ) induced by rHcARF1+PLGA NP. Experimental groups that were treated with the antigen-loaded NP yield higher lymphocyte proliferation than the control groups. Based on these results, we could propose that the rHcARF1 encapsulated in NP could stimulate a strong immune response in mice rather than administering alone against the infection of H. contortus

Table_1_The Serine/Threonine-Protein Phosphatase 1 From Haemonchus contortus Is Actively Involved in Suppressive Regulatory Roles on Immune Functions of Goat Peripheral Blood Mononuclear Cells.PDF

<p>Serine/threonine-protein phosphatases (STPs), as integral constituents of parasitic excretory/secretory proteins, are assumed to be released during the host–parasite interactions. However, knowledge about these phosphatases and their immunoregulatory and immune protective efficiencies with host peripheral blood mononuclear cells (PBMCs) is scant. In this study, an open reading frame of STP from Haemonchus contortus designated as HcSTP-1 was amplified and cloned using reverse-transcription-polymerase chain reaction (RT-PCR) method. The 951-bp nucleotides sequence was encoded to a protein of 316 amino acid residues, conserved in characteristics motifs GDXHG, GDYVDRG, GNHE, HGG, RG, and H. The HcSTP-1 protein was detected at approximately 35 kDa as recombinant protein fused in an expression vector system and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunohistochemically, HcSTP-1 was found to be localized in both male and female adult worm sections. Using immunofluorescence assay, the binding activity of rHcSTP-1 was confirmed on surface of goat PBMCs, which resulted in expression of multiple cytokines and various immunoregulatory activities in vitro. The RT-PCR results showed that mRNA level of interleukin-2, TGF-β1, IFN-γ, and IL-17 (with 10 µg/ml) was upregulated and IL-10 was decreased. However, IL-6 showed no change after PBMCs incubated with rHcSTP-1 protein. Further functional analysis showed that migratory activity of cells, intracellular nitrite production (NO), and apoptotic efficiency of PBMCs were elevated at significant level, whereas the proliferation of goat PBMCs and monocytes-associated major histocompatibility complex (MHC)-I and MHC-II expressions were decreased significantly at concentration-dependent fashion. Our results showed that the HcSTP-1 protein engaged in vital suppressive regulatory roles on host immune cells, which might represent a potential molecular target for controlling H. contortus infection in future.</p

Data_Sheet_1_The Serine/Threonine-Protein Phosphatase 1 From Haemonchus contortus Is Actively Involved in Suppressive Regulatory Roles on Immune Functions of Goat Peripheral Blood Mononuclear Cells.zip

<p>Serine/threonine-protein phosphatases (STPs), as integral constituents of parasitic excretory/secretory proteins, are assumed to be released during the host–parasite interactions. However, knowledge about these phosphatases and their immunoregulatory and immune protective efficiencies with host peripheral blood mononuclear cells (PBMCs) is scant. In this study, an open reading frame of STP from Haemonchus contortus designated as HcSTP-1 was amplified and cloned using reverse-transcription-polymerase chain reaction (RT-PCR) method. The 951-bp nucleotides sequence was encoded to a protein of 316 amino acid residues, conserved in characteristics motifs GDXHG, GDYVDRG, GNHE, HGG, RG, and H. The HcSTP-1 protein was detected at approximately 35 kDa as recombinant protein fused in an expression vector system and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunohistochemically, HcSTP-1 was found to be localized in both male and female adult worm sections. Using immunofluorescence assay, the binding activity of rHcSTP-1 was confirmed on surface of goat PBMCs, which resulted in expression of multiple cytokines and various immunoregulatory activities in vitro. The RT-PCR results showed that mRNA level of interleukin-2, TGF-β1, IFN-γ, and IL-17 (with 10 µg/ml) was upregulated and IL-10 was decreased. However, IL-6 showed no change after PBMCs incubated with rHcSTP-1 protein. Further functional analysis showed that migratory activity of cells, intracellular nitrite production (NO), and apoptotic efficiency of PBMCs were elevated at significant level, whereas the proliferation of goat PBMCs and monocytes-associated major histocompatibility complex (MHC)-I and MHC-II expressions were decreased significantly at concentration-dependent fashion. Our results showed that the HcSTP-1 protein engaged in vital suppressive regulatory roles on host immune cells, which might represent a potential molecular target for controlling H. contortus infection in future.</p

Image_1_The Serine/Threonine-Protein Phosphatase 1 From Haemonchus contortus Is Actively Involved in Suppressive Regulatory Roles on Immune Functions of Goat Peripheral Blood Mononuclear Cells.PDF

<p>Serine/threonine-protein phosphatases (STPs), as integral constituents of parasitic excretory/secretory proteins, are assumed to be released during the host–parasite interactions. However, knowledge about these phosphatases and their immunoregulatory and immune protective efficiencies with host peripheral blood mononuclear cells (PBMCs) is scant. In this study, an open reading frame of STP from Haemonchus contortus designated as HcSTP-1 was amplified and cloned using reverse-transcription-polymerase chain reaction (RT-PCR) method. The 951-bp nucleotides sequence was encoded to a protein of 316 amino acid residues, conserved in characteristics motifs GDXHG, GDYVDRG, GNHE, HGG, RG, and H. The HcSTP-1 protein was detected at approximately 35 kDa as recombinant protein fused in an expression vector system and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunohistochemically, HcSTP-1 was found to be localized in both male and female adult worm sections. Using immunofluorescence assay, the binding activity of rHcSTP-1 was confirmed on surface of goat PBMCs, which resulted in expression of multiple cytokines and various immunoregulatory activities in vitro. The RT-PCR results showed that mRNA level of interleukin-2, TGF-β1, IFN-γ, and IL-17 (with 10 µg/ml) was upregulated and IL-10 was decreased. However, IL-6 showed no change after PBMCs incubated with rHcSTP-1 protein. Further functional analysis showed that migratory activity of cells, intracellular nitrite production (NO), and apoptotic efficiency of PBMCs were elevated at significant level, whereas the proliferation of goat PBMCs and monocytes-associated major histocompatibility complex (MHC)-I and MHC-II expressions were decreased significantly at concentration-dependent fashion. Our results showed that the HcSTP-1 protein engaged in vital suppressive regulatory roles on host immune cells, which might represent a potential molecular target for controlling H. contortus infection in future.</p