Activation of membrane-bound and soluble Toll-like Receptors 5 in Salmo salar depends on the MyD88 signalling pathway

Background

TLR5M and TLR5S are Toll-like Receptors (TLRs), expressed in teleost fish, that recognize flagellin as a ligand, in the same way as the TLR5 ortholog in mammals. However, it has not been demonstrated whether the signalling pathway induced by these TLRs depends on MyD88 to generate a pro-inflammatory response in Salmo salar.

Methods

A mathematical model was constructed using the CellDesigner software, which represented the signalling pathways of the all TLRs in S. salar. It was used to make predictions which were corroborated experimentally in Salmo salar Head Kidney Leukocytes (HKLs) treated with flagellin and a MyD88 peptide inhibitor.

Results

The in silico model consisted of 135 species, 221 nodes and 279 bridges; MyD88 was one of the nodes with the highest betweenness centrality. The model simulations predicted that inhibition of MyD88 or TLR5S would cause a delay in response to stimulation with flagellin. The stimulation of HKLs with flagellin demonstrated a kinetic of relative expression of genes concordant with a positive feedback mechanism between TLR5M, MyD88 and TLR5S. Furthermore, MyD88 inhibition induced a significant decrease in the relative expression of pro-inflammatory genes downstream of the TLR5M signalling pathway.

Conclusions and general significance

In S. salar, activation of TLR5M and TLR5S is dependent on MyD88 as an adaptor protein after stimulation with flagellin. A sequential mechanism of activation, amplification and attenuation of the TLR5M/flagellin signalling pathway is proposed for this species. Our mathematical model is a robust predictive tool for generating new hypotheses about TLRs in S. salar.