Enhanced expression of a cloned and sequenced Ciona intestinalis TNFα-like (CiTNFα) gene during the LPS-induced inflammatory response

Abstract

A tumor necrosis factor-alpha (TNFα)-like gene from Ciona intestinalis (CiTNFα-like) body wall challenged with bacterial lipopolysaccharide (LPS) was cloned and sequenced 4 h after LPS inoculation. An open reading frame of 936 bp encoding a propeptide of 312 amino acids (35.4 kDa) displaying a transmembrane domain from positions 7 to 29, a TACE cleavage site, and a mature peptide domain of 185 amino acids (20.9 kDa), was determined with a predicted isoelectric point of 9.4. The phylogenetic tree based on deduced amino acid sequences of invertebrate TNF-like protein and vertebrate TNFs supported the divergence between the ascidian and vertebrate TNF families, whereas D. melanogaster Eiger A and B TNF-like sequences were distinctly separated from the chordate TNFs. Thus, the ascidian TNFα-like cytokine was upregulated by in vivo LPS challenge supporting its proinflammatory role. In the pharynx, increased expression levels were found following analysis by real-time polymerase chain reaction, whereas in situ hybridization assay showed positive hemocytes both in the tissue and in circulating hemocytes. Finally, Western blot with monoclonal antibodies disclosed human TNFα epitopes in a 15-kDa protein component of the hemolymph serum and in a 43-kDa protein contained in the hemocyte lysate supernatant prepared in the presence of detergents. Both soluble and hemocyte-bound CiTNFα-like protein therefore appeared to be modulated by the LPS challenge.

Inducible galectins are expressed in the inflamed pharynx of the ascidian Ciona intestinalis

“…As previously described [21,23], 100 mg LPS in 100 ml MS per specimen were inoculated through the body wall (middle region of the longitudinal axis) just under the tunic to allow the local spread into the pharynx tissue. Then, at various time points, a constant amount of pharynx tissue was excised from the body wall at the injection site.…”

“…CiLgalsa-a and CiLgals-b genes expression was detected by realtime PCR using the Taqman method as previously described [23]. The expression was performed using CiLgals-a probe (AACTTCGCT-CACCGTTT), forward primer (5 0 -TGTTGAATGGCTTCCACTTGTT-3 0 ) and reverse primers (5 0 -TCGGATGTTACGCAGAGGTTT-3 0 ), CiLgalsb probe (CTTCTGCGACTTCACA), forward primer (5 0 -CTCGGTGTATGT-GAACGATGTTC-3 0 ) and reverse primers (5 0 -CGGTAGAGCGGCA-CCTTGT-3 0 ), actin probe (TCGACAATGGATCCGGT), actin forward (5 0 -TGATGTTGCCGCACTCGTA-3 0 ) and reverse (5 0 -TCGACAATGGAT-CCGGT-3 0 ) primers.…”

“…Histological sections were examined under a Leica DMRE microscope and tissues identified as reported in previous papers [21,23].…”

Molecular characterisation, evolution and expression analysis of g-type lysozymes in Ciona intestinalis

“…They are proto-chordates which possess an innate immune system, including inflammatory humoral and cellular responses. An inflammatory response induced by LPS injection in the body wall of C. intestinalis is a well-established model for the analysis of regulator and effector inducible host defense molecules of the innate immune system (Bonura et al, 2009;Parrinello et al, 2008Parrinello et al, , 2010Vizzini et al, 2012Vizzini et al, , 20132015a, 2015b2016). In the present paper, we report on the identification, characterization and expression of C. intestinalis g-type lysozymes.…”

MIF from mussel: Coding sequence, phylogeny, polymorphism, 3D model and regulation of expression

“…The presence of cytokine-like activity or of related mRNA have also been observed in nearly all invertebrate species, including tunicates, echinoderms, annelids, nematodes, arachnids and insects (reviewed by (Beschin et al, 2001)). A recent paper reveals that the expression of a TNF a-like gene is involved in ascidian inflammatory responses (Parrinello et al, 2008). In mollusks, first indirect evidences of cytokines were obtained by cross-reactions with anti-cytokine antibodies, in vitro activities of vertebrate cytokines on invertebrate cells and activity of invertebrate sera on vertebrate cells (reviewed by (Canesi et al, 2006;Ottaviani, 2006)).…”