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

Abstract

The close phylogenetic relationship between Ciona robusta and vertebrates makes it a powerful model for studying innate immunity and the evolution of immune genes. To elucidate the nature and dynamics of the immune response, the molecular mechanisms by which bacterial infection is detected and translated into inflammation and how potential pattern recognition receptors (PRRs) are involved in pathogen recognition in tunicate C. robusta (formerly known as Ciona intestinalis), we applied an approach combining bacterial infections, next-generation sequencing, qRT-PCR, bioinformatics and in silico analyses (criteria of a p-value < 0.05 and FDR < 0.05). A STRING analysis indicated a functional 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 as more frequently expressed in the inflammatory response than inflammation mediator or effector molecules (e.g., Il-17s, Tnf-α, Tgf-β, Mmp9, Tlrs, MyD88, Irak4, Nf-κB, and galectins), suggesting close interplay between Mif cytokines and Nf-κB signalling pathway components in the biphasic activation of the inflammatory response. An in silico analyses of the 3′-UTR of Tlr2, MyD88, IκBα, Ikk, and Nf-κB transcripts showed the presence of GAIT elements, which are known to play key roles in the regulation of immune gene-specific translation in humans. These findings provide a new level of understanding of the mechanisms involved in the regulation of the C. robusta inflammatory response induced by LPS and suggest that in C. robusta, as in humans, a complex transcriptional and post-transcriptional control mechanism is involved in the regulation of several inflammatory genes.

ceRNA Network Regulation of TGF-β, WNT, FOXO, Hedgehog Pathways in the Pharynx of Ciona robusta

“…C. robusta specimens were collected from Sciacca harbor (Sicily, Italy) and were acclimatized and maintained as reported in Arizza et al [ 33 ]…”

“…Fragments of pharynx tissue (200 mg) were immediately soaked in RNAlater tissue collection solution (AMBION, Austin, TX, USA) and Total RNA extraction was performed as reported in in Arizza et al [ 33 ].…”

“…High-quality RNA samples (A260/A280 = 1.9–2.1, RIN ≥ 7) were used for cDNA library construction. RNA sequencing (RNA-Seq) was performed by BMR Genomics (Padua, Italy) on an Illumina platform in a single-end format 75 bp (1 × 75 bp) containing ~40 million ± 10% of reads/sample [ 33 ].…”

“…In C. robusta Mif2 show cis-acting gamma interferon inhibitor of translation element (GAIT) and a cytoplasmic polyadenylation element (CPE) in 3′-UTR, which is not present in the 3′-UTR of Mif1 , suggesting the presence of both transcriptional and post-transcriptional control mechanisms involved in the gene expression of the Mif genes ( Mif1 , Mif2 ) in innate immune response [ 31 ]. Furthermore, in C. robusta the presence of cis-acting GAIT elements in the 3′-UTR of Tlr2 , MyD88 , IκB , Ikkα , and Nf-κB mRNA confirmed that mechanisms involved in post-transcriptional regulation control of immune genes in human resulted highly conserved [ 33 ]. In C. robusta the signaling pathways and their post-transcriptional regulation in hematopoiesis are poorly studied and only some authors have described haematopoietic nodules in the pharynx in proliferation zones.…”

Role of the Macrophage Migration Inhibitory Factor in the Pathophysiology of Pre-Eclampsia

“…Interestingly, MIF positively regulates the expression of TLR4 in macrophages promoting the recognition of LPS, a cell wall constituent of most Gram-negative bacteria, by the innate immune system [ 79 , 87 , 88 ]. A very recent report showed that Mif gene expression is activated by LPS in Ciona Robusta , a marine invertebrate model, suggesting MIF as a universal signaling mediator for the defense against pathogens [ 89 ].…”