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Toxocara canis mucins among other excretory-secretory antigens induce in vitro secretion of cytokines by mouse splenocytes.

Długosz E, Wasyl K, Klockiewicz M, Wiśniewski M - Parasitol. Res. (2015)

Bottom Line: Cell stimulation with whole TES products was more effective and resulted in secretion of IL-4, IL-5, IL-6, IL-10, and TGF-β and downregulation of TNF-α production.However, splenocyte stimulation with both TES and ConA upregulated the production of IFN-γ and IL-17.This shows that TES antigens have strong immunomodulatory properties and are able to induce a broad range of effects on murine immune cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Parasitology, Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Ciszewskiego 8, 02-786, Warsaw, Poland, ewa_dlugosz@sggw.pl.

ABSTRACT
The effect of Toxocara larval antigens on cytokine secretion by mouse splenocytes was studied in vitro. Recombinant mucins were produced in Pichia pastoris yeast, and Toxocara excretory-secretory (TES) antigens were collected from in vitro culture of L2 larvae. Tc-MUC-2, Tc-MUC-3, Tc-MUC-4, and Tc-MUC-5 were expressed as glycoproteins and were specifically recognized by Toxocara canis-infected dog serum antibodies. Mouse splenocytes stimulated with recombinant mucins produced IL-5, IL-6, and TGF-β. Cell stimulation with whole TES products was more effective and resulted in secretion of IL-4, IL-5, IL-6, IL-10, and TGF-β and downregulation of TNF-α production. IFN-γ and IL-17 secretion was noted only after ConA treatment. Cells originating from infected animals produced significantly smaller amounts of these two cytokines compared to control cells, which suggests that Th1 and Th17 response in infected mice is strongly inhibited. However, splenocyte stimulation with both TES and ConA upregulated the production of IFN-γ and IL-17. This shows that TES antigens have strong immunomodulatory properties and are able to induce a broad range of effects on murine immune cells.

No MeSH data available.


SDS-PAGE (a, b) and Western blotting (c, d) analysis of Toxocara recombinant mucins produced in Pichia pastoris. 5 μg of Tc-MUC-2 (lane 1), Tc-MUC-3 (lane 2), Tc-MUC-4 (lane 3), and Tc-MUC-5 (lane 4) were separated on 12.5 % polyacrylamide gels and stained with Coomassie Blue (a), Glycoprotein Staining Kit (b), transferred on nitrocellulose membranes, and detected with monoclonal anti-polyhistidine antibodies (c) or T. canis-infected dog serum IgG antibodies (d) (blotting analysis with one of three infected sera is shown)
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Fig1: SDS-PAGE (a, b) and Western blotting (c, d) analysis of Toxocara recombinant mucins produced in Pichia pastoris. 5 μg of Tc-MUC-2 (lane 1), Tc-MUC-3 (lane 2), Tc-MUC-4 (lane 3), and Tc-MUC-5 (lane 4) were separated on 12.5 % polyacrylamide gels and stained with Coomassie Blue (a), Glycoprotein Staining Kit (b), transferred on nitrocellulose membranes, and detected with monoclonal anti-polyhistidine antibodies (c) or T. canis-infected dog serum IgG antibodies (d) (blotting analysis with one of three infected sera is shown)

Mentions: SDS-PAGE analysis of purified recombinant mucins showed single bands of 70 kDa for Tc-MUC-2 and Tc-MUC-4, 100 kDa for Tc-MUC-3, and more than 120 kDa for Tc-MUC-5 (Fig. 1a). Glycoprotein staining showed intensive glycosylation of all expressed mucins (Fig. 1b). The presence of glycan moieties on recombinant mucins was also confirmed by lectin-binding assay which showed that concanavalin A (ConA) binds to all four glycoproteins (data not shown). Recombinant mucins were specifically recognized by anti-HIS-tag (Fig. 1c) and serum antibodies of T. canis-infected dogs (Fig. 1d). Control uninfected dog serum did not react with investigated antigens (data not shown). Additionally, different recombinant proteins produced in P pastoris X33 strain were also tested as controls. Two Fasciola hepatica and one Hypoderma diana recombinant antigens were used. Antibodies from T. canis-infected or control dogs did not recognize these proteins (data not shown).Fig. 1


Toxocara canis mucins among other excretory-secretory antigens induce in vitro secretion of cytokines by mouse splenocytes.

Długosz E, Wasyl K, Klockiewicz M, Wiśniewski M - Parasitol. Res. (2015)

SDS-PAGE (a, b) and Western blotting (c, d) analysis of Toxocara recombinant mucins produced in Pichia pastoris. 5 μg of Tc-MUC-2 (lane 1), Tc-MUC-3 (lane 2), Tc-MUC-4 (lane 3), and Tc-MUC-5 (lane 4) were separated on 12.5 % polyacrylamide gels and stained with Coomassie Blue (a), Glycoprotein Staining Kit (b), transferred on nitrocellulose membranes, and detected with monoclonal anti-polyhistidine antibodies (c) or T. canis-infected dog serum IgG antibodies (d) (blotting analysis with one of three infected sera is shown)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4537704&req=5

Fig1: SDS-PAGE (a, b) and Western blotting (c, d) analysis of Toxocara recombinant mucins produced in Pichia pastoris. 5 μg of Tc-MUC-2 (lane 1), Tc-MUC-3 (lane 2), Tc-MUC-4 (lane 3), and Tc-MUC-5 (lane 4) were separated on 12.5 % polyacrylamide gels and stained with Coomassie Blue (a), Glycoprotein Staining Kit (b), transferred on nitrocellulose membranes, and detected with monoclonal anti-polyhistidine antibodies (c) or T. canis-infected dog serum IgG antibodies (d) (blotting analysis with one of three infected sera is shown)
Mentions: SDS-PAGE analysis of purified recombinant mucins showed single bands of 70 kDa for Tc-MUC-2 and Tc-MUC-4, 100 kDa for Tc-MUC-3, and more than 120 kDa for Tc-MUC-5 (Fig. 1a). Glycoprotein staining showed intensive glycosylation of all expressed mucins (Fig. 1b). The presence of glycan moieties on recombinant mucins was also confirmed by lectin-binding assay which showed that concanavalin A (ConA) binds to all four glycoproteins (data not shown). Recombinant mucins were specifically recognized by anti-HIS-tag (Fig. 1c) and serum antibodies of T. canis-infected dogs (Fig. 1d). Control uninfected dog serum did not react with investigated antigens (data not shown). Additionally, different recombinant proteins produced in P pastoris X33 strain were also tested as controls. Two Fasciola hepatica and one Hypoderma diana recombinant antigens were used. Antibodies from T. canis-infected or control dogs did not recognize these proteins (data not shown).Fig. 1

Bottom Line: Cell stimulation with whole TES products was more effective and resulted in secretion of IL-4, IL-5, IL-6, IL-10, and TGF-β and downregulation of TNF-α production.However, splenocyte stimulation with both TES and ConA upregulated the production of IFN-γ and IL-17.This shows that TES antigens have strong immunomodulatory properties and are able to induce a broad range of effects on murine immune cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Parasitology, Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Ciszewskiego 8, 02-786, Warsaw, Poland, ewa_dlugosz@sggw.pl.

ABSTRACT
The effect of Toxocara larval antigens on cytokine secretion by mouse splenocytes was studied in vitro. Recombinant mucins were produced in Pichia pastoris yeast, and Toxocara excretory-secretory (TES) antigens were collected from in vitro culture of L2 larvae. Tc-MUC-2, Tc-MUC-3, Tc-MUC-4, and Tc-MUC-5 were expressed as glycoproteins and were specifically recognized by Toxocara canis-infected dog serum antibodies. Mouse splenocytes stimulated with recombinant mucins produced IL-5, IL-6, and TGF-β. Cell stimulation with whole TES products was more effective and resulted in secretion of IL-4, IL-5, IL-6, IL-10, and TGF-β and downregulation of TNF-α production. IFN-γ and IL-17 secretion was noted only after ConA treatment. Cells originating from infected animals produced significantly smaller amounts of these two cytokines compared to control cells, which suggests that Th1 and Th17 response in infected mice is strongly inhibited. However, splenocyte stimulation with both TES and ConA upregulated the production of IFN-γ and IL-17. This shows that TES antigens have strong immunomodulatory properties and are able to induce a broad range of effects on murine immune cells.

No MeSH data available.