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Fibroblasts express immune relevant genes and are important sentinel cells during tissue damage in rainbow trout (Oncorhynchus mykiss).

Ingerslev HC, Ossum CG, Lindenstrøm T, Nielsen ME - PLoS ONE (2010)

Bottom Line: Supernatant stimuli significantly increased the expression of IL-1beta, TLR-3 and TLR-9, whereas the debris stimuli only increased expression of IL-1beta.A weaker, but significant response was also seen for TLR-9 and TLR-22.Rainbow trout fibroblasts were found to be highly immune competent with a significant ability to express cytokines and immune receptors.

View Article: PubMed Central - PubMed

Affiliation: Section for Aquatic Protein Biochemistry, Division for Seafood Research, DTU Food, National Food Institute, Lyngby, Denmark. hci@aqua.dtu.dk

ABSTRACT
Fibroblasts have shown to be an immune competent cell type in mammals. However, little is known about the immunological functions of this cell-type in lower vertebrates. A rainbow trout hypodermal fibroblast cell-line (RTHDF) was shown to be responsive to PAMPs and DAMPs after stimulation with LPS from E. coli, supernatant and debris from sonicated RTHDF cells. LPS was overall the strongest inducer of IL-1beta, IL-8, IL-10, TLR-3 and TLR-9. IL-1beta and IL-8 were already highly up regulated after 1 hour of LPS stimulation. Supernatant stimuli significantly increased the expression of IL-1beta, TLR-3 and TLR-9, whereas the debris stimuli only increased expression of IL-1beta. Consequently, an in vivo experiment was further set up. By mechanically damaging the muscle tissue of rainbow trout, it was shown that fibroblasts in the muscle tissue of rainbow trout contribute to electing a highly local inflammatory response following tissue injury. The damaged muscle tissue showed a strong increase in the expression of the immune genes IL-1beta, IL-8 and TGF-beta already 4 hours post injury at the site of injury while the expression in non-damaged muscle tissue was not influenced. A weaker, but significant response was also seen for TLR-9 and TLR-22. Rainbow trout fibroblasts were found to be highly immune competent with a significant ability to express cytokines and immune receptors. Thus fish fibroblasts are believed to contribute significantly to local inflammatory reactions in concert with the traditional immune cells.

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Quantitative real-time PCR for mechanically damaged fish.Expression is shown for the genes (A) IL-1β, (B) IL-8, (C) TGF-β, (D) TLR-3, (E) TLR-9 and (F) TLR-22. Black bars represent expression at the site of injury relative to control fish and white bars indicate expression at the non-damaged internal control site relative to control fish. The data are normalised relative to the expression of ribosomal protein S20 and analysed using the ΔΔCt method. Data are shown as −ΔΔCt values and fold expression. Bars represent mean values of −ΔΔCt + SD values from five individuals. * Depicts statistical significance between injured fish and control fish (P<0.05); Δ denotes statistical significant difference between site of injury and internal control site (P<0.05). A −ΔΔCt value of 0 means no regulation relative to control fish.
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pone-0009304-g004: Quantitative real-time PCR for mechanically damaged fish.Expression is shown for the genes (A) IL-1β, (B) IL-8, (C) TGF-β, (D) TLR-3, (E) TLR-9 and (F) TLR-22. Black bars represent expression at the site of injury relative to control fish and white bars indicate expression at the non-damaged internal control site relative to control fish. The data are normalised relative to the expression of ribosomal protein S20 and analysed using the ΔΔCt method. Data are shown as −ΔΔCt values and fold expression. Bars represent mean values of −ΔΔCt + SD values from five individuals. * Depicts statistical significance between injured fish and control fish (P<0.05); Δ denotes statistical significant difference between site of injury and internal control site (P<0.05). A −ΔΔCt value of 0 means no regulation relative to control fish.

Mentions: The device used for mechanical tissue damage is shown in figure 3 A–B. The following effects of mechanical injury of the muscle tissue on the expression of the genes IL-1β, IL-8, TGF-β and TLR-3, 5 m, 9 and 22 at the time points 4, 8 and 24 hours post injury (p.i.) is shown in figure 4 A-F. No significant changes were seen for IL-10 (P>0.05) (not illustrated). The study showed a strong induction of immune related genes, especially of the cytokines IL-1β, IL-8 and TGF-β in the injured muscle tissue at all samplings. The average up-regulation in the injured fish for the three sampling points was between 5.2 to 21.8 folds for IL-1β; 20.0 to 59.3 folds for IL-8 and 4.9 to 13.9 folds for TGF- β. For all three genes the mean expression level was increasing from 4 hours to 24 hours post injury and the expression at the site of injury was significantly higher at all sampling points compared to the non-injured samples from the same fish (P<0.05).


Fibroblasts express immune relevant genes and are important sentinel cells during tissue damage in rainbow trout (Oncorhynchus mykiss).

Ingerslev HC, Ossum CG, Lindenstrøm T, Nielsen ME - PLoS ONE (2010)

Quantitative real-time PCR for mechanically damaged fish.Expression is shown for the genes (A) IL-1β, (B) IL-8, (C) TGF-β, (D) TLR-3, (E) TLR-9 and (F) TLR-22. Black bars represent expression at the site of injury relative to control fish and white bars indicate expression at the non-damaged internal control site relative to control fish. The data are normalised relative to the expression of ribosomal protein S20 and analysed using the ΔΔCt method. Data are shown as −ΔΔCt values and fold expression. Bars represent mean values of −ΔΔCt + SD values from five individuals. * Depicts statistical significance between injured fish and control fish (P<0.05); Δ denotes statistical significant difference between site of injury and internal control site (P<0.05). A −ΔΔCt value of 0 means no regulation relative to control fish.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2823790&req=5

pone-0009304-g004: Quantitative real-time PCR for mechanically damaged fish.Expression is shown for the genes (A) IL-1β, (B) IL-8, (C) TGF-β, (D) TLR-3, (E) TLR-9 and (F) TLR-22. Black bars represent expression at the site of injury relative to control fish and white bars indicate expression at the non-damaged internal control site relative to control fish. The data are normalised relative to the expression of ribosomal protein S20 and analysed using the ΔΔCt method. Data are shown as −ΔΔCt values and fold expression. Bars represent mean values of −ΔΔCt + SD values from five individuals. * Depicts statistical significance between injured fish and control fish (P<0.05); Δ denotes statistical significant difference between site of injury and internal control site (P<0.05). A −ΔΔCt value of 0 means no regulation relative to control fish.
Mentions: The device used for mechanical tissue damage is shown in figure 3 A–B. The following effects of mechanical injury of the muscle tissue on the expression of the genes IL-1β, IL-8, TGF-β and TLR-3, 5 m, 9 and 22 at the time points 4, 8 and 24 hours post injury (p.i.) is shown in figure 4 A-F. No significant changes were seen for IL-10 (P>0.05) (not illustrated). The study showed a strong induction of immune related genes, especially of the cytokines IL-1β, IL-8 and TGF-β in the injured muscle tissue at all samplings. The average up-regulation in the injured fish for the three sampling points was between 5.2 to 21.8 folds for IL-1β; 20.0 to 59.3 folds for IL-8 and 4.9 to 13.9 folds for TGF- β. For all three genes the mean expression level was increasing from 4 hours to 24 hours post injury and the expression at the site of injury was significantly higher at all sampling points compared to the non-injured samples from the same fish (P<0.05).

Bottom Line: Supernatant stimuli significantly increased the expression of IL-1beta, TLR-3 and TLR-9, whereas the debris stimuli only increased expression of IL-1beta.A weaker, but significant response was also seen for TLR-9 and TLR-22.Rainbow trout fibroblasts were found to be highly immune competent with a significant ability to express cytokines and immune receptors.

View Article: PubMed Central - PubMed

Affiliation: Section for Aquatic Protein Biochemistry, Division for Seafood Research, DTU Food, National Food Institute, Lyngby, Denmark. hci@aqua.dtu.dk

ABSTRACT
Fibroblasts have shown to be an immune competent cell type in mammals. However, little is known about the immunological functions of this cell-type in lower vertebrates. A rainbow trout hypodermal fibroblast cell-line (RTHDF) was shown to be responsive to PAMPs and DAMPs after stimulation with LPS from E. coli, supernatant and debris from sonicated RTHDF cells. LPS was overall the strongest inducer of IL-1beta, IL-8, IL-10, TLR-3 and TLR-9. IL-1beta and IL-8 were already highly up regulated after 1 hour of LPS stimulation. Supernatant stimuli significantly increased the expression of IL-1beta, TLR-3 and TLR-9, whereas the debris stimuli only increased expression of IL-1beta. Consequently, an in vivo experiment was further set up. By mechanically damaging the muscle tissue of rainbow trout, it was shown that fibroblasts in the muscle tissue of rainbow trout contribute to electing a highly local inflammatory response following tissue injury. The damaged muscle tissue showed a strong increase in the expression of the immune genes IL-1beta, IL-8 and TGF-beta already 4 hours post injury at the site of injury while the expression in non-damaged muscle tissue was not influenced. A weaker, but significant response was also seen for TLR-9 and TLR-22. Rainbow trout fibroblasts were found to be highly immune competent with a significant ability to express cytokines and immune receptors. Thus fish fibroblasts are believed to contribute significantly to local inflammatory reactions in concert with the traditional immune cells.

Show MeSH
Related in: MedlinePlus