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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 the RTHDF cells.Expression is shown for the genes (A) IL-1β, (B) IL-8, (C) IL-10, (D) TLR-3 and (E) TLR-9. Black bars represent expression in E. coli LPS stimulated cells relative to control cells; white bars indicate expression in cells stimulated with debris from sonicated RTHDF cells relative to control cells and striped bars denotes expression in cells stimulated with supernatant from sonicated RTHDF cells relative to control cells. The data are normalised relative to the expression of elongation factor-1α and analysed using the ΔΔCt method. Data are shown as −ΔΔCt values and fold expression. Bars represent mean values of −ΔΔCt + SD values from three cell replicates. * Depicts statistical significance between stimulated cells and control cells (*P<0.05; **P<0.01; ***P<0.001). A −ΔΔCt value of 0 means no regulation relative to control cells.
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pone-0009304-g002: Quantitative real-time PCR for the RTHDF cells.Expression is shown for the genes (A) IL-1β, (B) IL-8, (C) IL-10, (D) TLR-3 and (E) TLR-9. Black bars represent expression in E. coli LPS stimulated cells relative to control cells; white bars indicate expression in cells stimulated with debris from sonicated RTHDF cells relative to control cells and striped bars denotes expression in cells stimulated with supernatant from sonicated RTHDF cells relative to control cells. The data are normalised relative to the expression of elongation factor-1α and analysed using the ΔΔCt method. Data are shown as −ΔΔCt values and fold expression. Bars represent mean values of −ΔΔCt + SD values from three cell replicates. * Depicts statistical significance between stimulated cells and control cells (*P<0.05; **P<0.01; ***P<0.001). A −ΔΔCt value of 0 means no regulation relative to control cells.

Mentions: Figure 2 A-E shows the effect of E. coli LPS (0111:B4), debris or supernatant from sonicated RTHDF cells on the expression of IL-1β, IL-8, IL-10, TLR-3 and TLR-9 in the RTHDF cells after 1, 4 and 24 hours of stimulation. No influence on the expression of TGF-β and TLR5m was seen and TLR-22 was not expressed in the RTHDF cells after 40 cycles of PCR (data not shown). Overall the variation in expression between cell replicates was much lower compared to individual fish. The highest effect on all genes was seen for IL-1β and IL-8 after stimulation with LPS. The expression increased significantly after 1 hour of stimulation for both genes peaked after 4 hours to 42.5 and 22 folds, respectively, followed by a decrease in expression to 9.4 and 9.9 folds after 24 hours, respectively (P<0.05). The pattern for IL-10 was different and the response was slower than for IL-1β and IL-8. IL-10 was only significantly expressed at 24 hours after stimulation by 2.7 folds relative to non-stimulated cells (P<0.05). Stimulation by debris revealed significant impact on only IL-1β, but the response was weak and only significantly elevated at 4 hours post stimulation by 2.2 folds (P<0.05). Supernatant from the sonicated RTHDF cells increased the expression of both IL-1β and IL-10, but weakly compared to the effects of LPS. The IL-1β expression increased to 1.9 folds after 4 hours of stimulation, while for IL-10 it was 2 folds after 24 hours relative to non-stimulated cells (P<0.05). TLR-3 and TLR-9 showed similar expression patterns. Both LPS and supernatant increased the expression significantly after 24 hours; LPS to approximately 4 fold for both genes and supernatant to 1.6 and 2.5 folds for TLR-3 and TLR-9, respectively (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 the RTHDF cells.Expression is shown for the genes (A) IL-1β, (B) IL-8, (C) IL-10, (D) TLR-3 and (E) TLR-9. Black bars represent expression in E. coli LPS stimulated cells relative to control cells; white bars indicate expression in cells stimulated with debris from sonicated RTHDF cells relative to control cells and striped bars denotes expression in cells stimulated with supernatant from sonicated RTHDF cells relative to control cells. The data are normalised relative to the expression of elongation factor-1α and analysed using the ΔΔCt method. Data are shown as −ΔΔCt values and fold expression. Bars represent mean values of −ΔΔCt + SD values from three cell replicates. * Depicts statistical significance between stimulated cells and control cells (*P<0.05; **P<0.01; ***P<0.001). A −ΔΔCt value of 0 means no regulation relative to control cells.
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Related In: Results  -  Collection

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pone-0009304-g002: Quantitative real-time PCR for the RTHDF cells.Expression is shown for the genes (A) IL-1β, (B) IL-8, (C) IL-10, (D) TLR-3 and (E) TLR-9. Black bars represent expression in E. coli LPS stimulated cells relative to control cells; white bars indicate expression in cells stimulated with debris from sonicated RTHDF cells relative to control cells and striped bars denotes expression in cells stimulated with supernatant from sonicated RTHDF cells relative to control cells. The data are normalised relative to the expression of elongation factor-1α and analysed using the ΔΔCt method. Data are shown as −ΔΔCt values and fold expression. Bars represent mean values of −ΔΔCt + SD values from three cell replicates. * Depicts statistical significance between stimulated cells and control cells (*P<0.05; **P<0.01; ***P<0.001). A −ΔΔCt value of 0 means no regulation relative to control cells.
Mentions: Figure 2 A-E shows the effect of E. coli LPS (0111:B4), debris or supernatant from sonicated RTHDF cells on the expression of IL-1β, IL-8, IL-10, TLR-3 and TLR-9 in the RTHDF cells after 1, 4 and 24 hours of stimulation. No influence on the expression of TGF-β and TLR5m was seen and TLR-22 was not expressed in the RTHDF cells after 40 cycles of PCR (data not shown). Overall the variation in expression between cell replicates was much lower compared to individual fish. The highest effect on all genes was seen for IL-1β and IL-8 after stimulation with LPS. The expression increased significantly after 1 hour of stimulation for both genes peaked after 4 hours to 42.5 and 22 folds, respectively, followed by a decrease in expression to 9.4 and 9.9 folds after 24 hours, respectively (P<0.05). The pattern for IL-10 was different and the response was slower than for IL-1β and IL-8. IL-10 was only significantly expressed at 24 hours after stimulation by 2.7 folds relative to non-stimulated cells (P<0.05). Stimulation by debris revealed significant impact on only IL-1β, but the response was weak and only significantly elevated at 4 hours post stimulation by 2.2 folds (P<0.05). Supernatant from the sonicated RTHDF cells increased the expression of both IL-1β and IL-10, but weakly compared to the effects of LPS. The IL-1β expression increased to 1.9 folds after 4 hours of stimulation, while for IL-10 it was 2 folds after 24 hours relative to non-stimulated cells (P<0.05). TLR-3 and TLR-9 showed similar expression patterns. Both LPS and supernatant increased the expression significantly after 24 hours; LPS to approximately 4 fold for both genes and supernatant to 1.6 and 2.5 folds for TLR-3 and TLR-9, respectively (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