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

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.

Figure 1. Constitutive expression of the examined genes.

The C _t-values for RPS20, ELF-1α, IL-1β, IL-8, IL-10, TGF-β, TLR-3, TLR-5m, TLR-9 and TLR-22 are shown for muscle tissue (black bars) and for RTHDF cells (white bars). The data are presented as mean expression of the control fish from all samples points and mean expression of control RTHDF cells from all sample points. The C _t value is defined as the threshold cycle number of PCR at which the sample fluorescent signal passes a fixed threshold above the baseline.

Figure 2. 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 ΔΔC _t method. Data are shown as −ΔΔC _t values and fold expression. Bars represent mean values of −ΔΔC _t + 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 −ΔΔC _t value of 0 means no regulation relative to control cells.

Figure 3. Damage procedures and sampling from mechanically injured rainbow trout.

The fish were injured on the left side behind the dorsal fin using the damage instrument. Muscle tissue samples were taken in the injured area while the internal control samples were taken from the same place relative to the dorsal fin on the right side of the fish (A). The vertical position of the site of injury/sampling site is shown in (B). Sampling of muscle tissue from non-injured control fish was performed in the same area as shown for injured fish (not shown).

Figure 4. 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 ΔΔC _t method. Data are shown as −ΔΔC _t values and fold expression. Bars represent mean values of −ΔΔC _t + 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 −ΔΔC _t value of 0 means no regulation relative to control fish.