Transcriptional and in silico analyses of MIF cytokine and TLR signalling interplay in the LPS inflammatory response of Ciona robusta

The close phylogenetic relationship between Ciona robusta and vertebrates makes it a powerfulmodel for studying innate immunity and the evolution of immune genes. To elucidate the nature anddynamics of the immune response, the molecular mechanisms by which bacterial infection is detectedand translated into inflammation and how potential pattern recognition receptors (PRRs) are involvedin pathogen recognition in tunicate C. robusta (formerly known as Ciona intestinalis), we appliedan approach combining bacterial infections, next-generation sequencing, qRT-PCR, bioinformaticsand in silico analyses (criteria of a p-value < 0.05 and FDR < 0.05). A STRING analysis indicated afunctional link between components of the Tlr/MyD88-dependent signalling pathway (Tlr2, MyD88,and Irak4) and components of the Nf-κB signalling pathway (Nf-κB, IκBα, and Ikkα) (p-value < 0.05,FDR < 0.05). A qRT-PCR analysis of immune genes selected from transcriptome data revealed Mif asmore frequently expressed in the inflammatory response than inflammation mediator or effectormolecules (e.g., Il-17s, Tnf-α, Tgf-β, Mmp9, Tlrs, MyD88, Irak4, Nf-κB, and galectins), suggesting closeinterplay between Mif cytokines and Nf-κB signalling pathway components in the biphasic activationof the inflammatory response. An in silico analyses of the 3′-UTR of Tlr2, MyD88, IκBα, Ikk, and Nf-κBtranscripts showed the presence of GAIT elements, which are known to play key roles in the regulationof immune gene-specific translation in humans. These findings provide a new level of understandingof the mechanisms involved in the regulation of the C. robusta inflammatory response induced byLPS and suggest that in C. robusta, as in humans, a complex transcriptional and post-transcriptionalcontrol mechanism is involved in the regulation of several inflammatory genes.