PAMPs and DAMPs stimulate the expression of pro-inflammatory cytokines in vitro in a fibroblast cell-line from rainbow trout (Oncorhynchus mykiss)

The recognition of PAMPs by immune cells relies on conserved PRRs such as TLRs, NLRs and RLRs leading to activation of NFκB signalling pathways. These receptors are activated upon stimulation by different ligands such as bacterial or viral components. The binding of ligands to the receptors activates downstream signalling pathways, which subsequently leads to expression of pro-inflammatory cytokines and chemokines. DAMPs released from necrotic cells may also bind to and activate similar downstream signalling events. In telosts was found that mechanical damage of the muscle tissue using sterile needles induced a very rapid expression of the pro-inflammatory cytokines IL-1β, IL-8 and IL-10 as measured by real-time PCR. The results imply that cells located in the muscular tissue in addition to recruited cells are involved in the observed increased cytokine / chemokine expression. It is believed that this expression to a large extend is mediated by fibroblasts in the musculature. To investigate this, a fibroblast cell-line (RTHDF1) from the rainbow trout was stimulated with either LPS from E. coli, cell debris or supernatant from sonicated fibroblasts. Whereas LPS stimulation resulted in a significant up-regulation of the expression of IL-1β, IL-8 and IL-10 and stimulation with supernatant from sonicated cells led to a significant up-regulation of IL-1β and IL-10, while debris only stimulated the expression of IL-1β. TLR-2 and -4 are not described from salmonid fishes, however TLR-3, -5 and -9 are described in this evolutionary lineage of the bony fishes. The expression of TLR-3 and -9 receptors were significantly up-regulated following physical damage of muscle tissue as well as in stimulated fibroblasts, where LPS induced both TLR-3 and -9, supernatant from sonicated cells only TLR-9 while debris caused no induction. The present study reinforce the idea that fibroblasts are able to react to PAMPs and DAMPs and that non-immune cell-types play an important role in the inflammatory reaction per se. From an evolutionary perspective the facilitation of an inflammatory response through recognition of PAMPs and DAMPs by non immune cells seems plausible.