Elevated cortisol modulates hsp70 and hsp90 gene expression and protein in sea bass head kidney and isolated leukocytes

In fish, interactions between Hsps and cortisol are involved in stress modulated physiological processes including innate immune responses. Cortisol exerts a role in the regulation of Hsps synthesis. Fish head kidney is a lymphomieloid and endocrine organ releasing cortisol, and it is the central organ for immune-endocrine interactions. In sea bass, cortisol intraperitoneal injection and in vitro treatment of head kidney cells show that inducible Hsp70 and Hsp90 are modulated by this hormone. However, an inverse relationship between mRNA expression (real-time PCR) and Hsp70 and Hsp90 protein levels (densitometric band analysis) was found. Time-course assays indicate a cortisol-mediated regulation. Furthermore, Hsp70 gene modulation appears to be more susceptible to the cortisol action and the mRNA was transcribed within 3. h post-injection. The restoration of the homeostatic conditions was observed at a week p.i., when plasma cortisol baseline was reached. Although fish manipulation and injection exerted stressing effects as indicated by serological parameters, differences between cortisol treated specimens compared to untreated or sham fish are statistically significant. Similar results were found by examining in vitro total cells and isolated leukocytes from head kidney cultured for 3. h with increasing cortisol concentration. Finally, MTT test and DNA fragmentation experiments showed that the apoptotic effect expected in cortisol-treated cells could be counteracted by high Hsp70 intracellular levels

Transforming growth factor β (CiTGF-β) gene expression is induced in the inflammatory reaction of Ciona intestinalis

Transforming growth factor (TGF-β) is a well-known component of a regulatory cytokines superfamily that has pleiotropic functions in a broad range of cell types and is involved, in vertebrates, in numerous physiological and pathological processes. In the current study, we report on Ciona intestinalis molecular characterisation and expression of a transforming growth factor β homologue (CiTGF-β). The gene organisation, phylogenetic tree and modelling supported the close relationship with the mammalian TGF suggesting that the C. intestinalis TGF-β gene shares a common ancestor in the chordate lineages. Functionally, real-time PCR analysis showed that CiTGF-β was transcriptionally upregulated in the inflammatory process induced by LPS inoculation, suggesting that is involved in the first phase and significant in the secondary phase of the inflammatory response in which cell differentiation occurs. In situ hybridisation assays revealed that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune system, and expressed by cluster of hemocytes inside the pharynx vessels. These data supported the view that CiTGF-β is a potential molecule in immune defence systems against bacterial infection

Phenoloxidases of different sizes are modulated by LPS inoculation into Ciona intestinalis tunic and pharynx

In the present study, to further characterize the pro-phenoloxidase (proPO) and active phenoloxidase (PO) involved in the Ciona intestinalis inflammatory response, tunic and pharynx homogenate supernatants were separated on high pressure liquid chromatography and fractions were assayed for the PO activity before and after LPS inoculation, as well as before and after trypsin treatment which activates proPO. The LPS inoculation per se did not significantly change the basal PO activity of the tunic homogenate supernatant (THS) and pharynx homogenate supernatant (PHS) restricted in two confluent peaks, whereas a significant enhancement was observable after the trypsin treatment. This trypsin effect suggests that proPO is the main component of the HPLC separated fractions, and indicates that LPS inoculation mainly challenges the pro-enzyme production by tunic cells and hemocytes, as well as the activation of the serine-protease pathway. The protein size analysis and DOPA-MBTH assay, disclose two active proteins of 90.0 and 170.0 kDa differently contained in the two main chromatographic peaks. Due to the SDS activating effect on the proenzyme analyzed by SDS-PAGE, the size of proPO could not be shown, whereas modulation of an oligomerization process of the 90 kDa component is suggested

LPS Challenge Regulates Gene Expression and Tissue Localization of a Ciona intestinalis Gene through an Alternative Polyadenylation Mechanism

subtractive hybridization strategy for the identification of differentially expressed genes was performed between LPSchallenged and naive Ciona intestinalis. This strategy allowed the characterization of two transcripts (Ci8short and Ci8long) generated by the use of two Alternative Polyadenylation sites. The Ci8long transcript contains a protein domain with relevant homology to several components of the Receptor Transporting Protein (RTP) family not present in the Ci8short mRNA. By means of Real Time PCR and Northern Blot, the Ci8short and Ci8long transcripts showed a different pattern of gene expression with the Ci8short mRNA being strongly activated after LPS injection in the pharynx. In situ hybridization analysis demonstrated that the activation of the APA site also influenced the tissue localization of the Ci8short transcript. This analysis showed that the Ci8long mRNA was expressed in hemocytes meanwhile the Ci8short mRNA was highly transcribed also in vessel endothelial cells and in the epithelium of pharynx. These findings demonstrated that regulation of gene expression based on different polyadenylation sites is an ancestral powerful strategy influencing both the level of expression and tissue distribution of alternative transcripts

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

Although ascidians belong to a key group in chordate phylogenesis, amino acid sequences of Ciona intestinalis galectin-CRDs (CiLgals-a and -b) have been retained too divergent from vertebrate galectins. In the present paper, to contribute in disclosing Bi-CRD galectin evolution a novel attempt was carried out on CiLgals-a and -b CRDs phylogenetic analysis, and their involvement in ascidian inflammatory responses was shown. CiLgals resulted aligned with Bi-CRD galectins from vertebrates (Xenopus tropicalis, Gallus gallus, Mus musculus, Homo sapiens), cephalochordates (Branchiostoma floridae), echinoderms (Strongylocentrotus purpuratus) and a mono-CRD galectin from the ascidian Clavelina picta. The CiLgalsa N-terminal and C-terminal CRDs contain the signature sequence involved in carbohydrate binding, whereas the CiLgals-b C-CRD presents only three out of seven key aminoacids and it could not be suitable as sugar binding motif. Sequence similarity between clusters suggests an evolutionary model based on CRD domain gene duplication and sequence diversification. In particular CiLgals-b N-CRD and C-CRD were similar to each other and both grouped with the ascidian C. picta mono-CRD. Homology modeling process shows a CiLgals molecular structure superimposed to chicken and mouse galectins. The CiLgalsa and CiLgals-b genes were upregulated by LPS inoculation suggesting that they are inducible and expressed in the inflamed pharynx as revealed by real-time PCR analysis. Finally, in situ hybridization and immunohistochemical assays showed their localization in the inflamed tissues, while immunoblotting analysis indicated that CiLgals can form oligomer

Ciona intestinalis peroxinectin is a novel component of the peroxidase– cyclooxygenase gene superfamily upregulated by LPS

Peroxinectins function as hemoperoxidase and cell adhesion factor involved in invertebrate immune reac- tion. In this study, the ascidian (Ciona intestinalis) peroxinectin gene (CiPxt) and its expression during the inflammatory response have been examined. CiPxt is a new member of the peroxidase–cyclooxygenase gene superfamily that contains both the peroxidase domain and the integrin KGD (Lys-Gly-Asp) binding motif. A phylogenetic tree showed that CiPxt is very close to the chordate group and appears to be the out-group ofmammalianMPO, EPO and TPO clades. The CiPxtmolecular structuremodel resulted superimposable to the human myeloperoxidase. The CiPxt mRNA expression is upregulated by LPS inoculation suggesting it is involved in C. intestinalis inflammatory response. The CiPxt was expressed in hemocytes (compartment/morula cells), vessel epithelium, and unilocular refractile granulocytes populating the inflamed tunic matrix and in the zones 7, 8 and 9 of the endostyle, a special pharynx organs homolog to the vertebrate thyroid glan

The Ciona intestinalis immune-related galectin genes (CiLgals-a and CiLgals-b) are expressed by the gastric epithelium

The transcription of two Ciona intestinalis galectin genes (CiLgals-a and CiLgalseb) is uparegulated by LPS in the pharynxis (hemocytes, vessel epithelium, endostilar zones) which is retained the main organ of the immunity. In this ascidian, for the first time we show, by immunohistochemistry and in situ hybridization methods, that these two immune-related genes are expressed in the gastric epithelium of na\uefve ascidians, whereas the galectins appear to be only contained in the intestine columnar epithelium. In addition, according to previous results on the pharynx, the genes are also expressed and galectins produced by hemocytes scattered in the connective tissue surrounding the gut. The genes expression and galectin localization in several tissues, including the previous findings on the transcription upregulation, the constitutive expression of these genes by endostylar zones and by the gastric epithelium suggest a potential multifunctional role of these galectins. In this respect, it is of interest to define where the CiLgals are normally found as related to the tissue functions. Such an approach should be a starting point for further investigations

Inflamed adult pharynx tissues and swimming larva of Ciona intestinalis share CiTNFα-producing cells

In situ hybridisation and mmunohistochemistry analyses have shown that the Ciona intestinalis tumour necrosis factor alpha gene (CiTNFα), which has been previously cloned and sequenced, is expressed either during the inflammatory pharynx response to lipopolysaccharide (LPS) or during the swimming larval phase of development. Granulocytes with large granules and compartment/morula cells are CiTNFα-producing cells in both inflamed pharynx and larvae. Pharynx vessel endothelium also takes part in the inflammatory response. Haemocyte nodules in the vessel lumen or associated with the endothelium suggest the involvement of CiTNFα in recruiting lymphocyte-like cells and promoting the differentiation of inflammatory haemocytes. Specific antibodies against a CiTNFα peptide have identified a 43-kDa cell-bound form of the protein. Observations of pharynx histological sections (at 4 and 8 h post-LPS inoculation) from naive and medium-inoculated ascidians have confirmed the CiTNFα-positive tissue response. Larval histological sections and whole-mount preparations have revealed that CiTNFα is expressed by trunk mesenchyme,preoral lobe and tunic cells, indicating CiTNFα-expressing cell immigration events and an ontogenetic role