Xbp1-u and Xbp1-s from Nile tilapia (Oreochromis niloticus): Transcriptional profiling upon Streptococcus agalactiae infection and the potential role in B cell activation and differentiation.

X-box protein 1 (Xbp1), an essential transcription factor including an unstable form (Xbp1-u) and a stable form (Xbp1-s), plays an vital role in B cell activation and differentiation to plasma cells. In this study, we cloned and identified Xbp1-u gene from Nile tilapia (Oreochromis niloticus), containing 783 bp of nucleotide sequence encoding 260 amino acids. The deduced protein possesses a basic region leucine zipper domain (bZIP) and 26 ribonucleotides of OnXbp1-u transcript. Transcription analysis revealed OnXbp1-u and OnXbp1-s were widely distributed in all examined tissues, with a high expression in immune-related tissues. When stimulated with Streptococcus agalactiae in vivo, the expressions of OnXbp1-u and OnXbp1-s were significantly up-regulated in liver, spleen, head kidney, blood, skin and intestine. After in vitro challenge upon S.agalactiae, the similar up-regulations of OnXbp1-u and OnXbp1-s were also demonstrated in head kidney leukocytes. Moreover, the OnXbp1-u and OnXbp1-s could get involved in LPS-inducible B cell activation and (r)OnIL6-inducible B cell differentiation. Taken together, the results indicated that OnXbp1-u and OnXbp1-s might not only involved in the immune response against S. agalactiae challenge, but also in the B cell activation and differentiation in Nile tilapia.

Identification and functional characterization of three caspases in Takifugu obscurus in response to bacterial infection.

Caspases are evolutionarily conserved proteases, which are inextricably linked with the apoptosis and immune system in mammals. However, the expression pattern and function of some caspases remain largely unknown in pufferfish. In this study, three different pufferfish caspases (caspase-2 (Pfcasp-2), caspase-3 (Pfcasp-3), and caspase-8 (Pfcasp-8)) were characterized, and their expression patterns and functions were determined following Aeromonas hydrophila infection. The open reading frames of Pfcasp-2, -3, and -8 are 1,320, 846, and 1455 bp, respectively. Analyses of sequence alignment and phylogenetic tree showed that casp-2, -3, and -8 share 52%-65%, 33%-40%, 63%-78% overall sequence identities with those of other vertebrates, respectively. 3D structures of Pfcasp-2, -3, and -8 enjoy conservation in core area together, while each owns a distinctive profile. Comparisons of deduced amino acid sequences indicated that Pfcaspases possessed the caspase domain and conserved active sites like 'HG' and 'QACXG' (X for R or G). qRT-PCR results revealed that Pfcasp-2, -3, and -8 were expressed constitutively in a wide range of organs, especially in immune-related organs including whole blood and kidney. In vitro, the expressions of the three caspases (Pfcasp-2, 3, and -8) and immune-related genes (IgM and IL-8) were significantly up-regulated in kidney leukocytes after A. Hydrophila challenge and inhibitors treatment. The expressions of Pfcasp-2 and Pfcasp-3 were successfully inhibited in the kidney leukocytes by Ac-DEVD-CHO (an inhibitor to caspase-3), but the expression of Pfcasp-8 was not affected. Cellular localization analysis showed that the distribution of Pfcasp-2, -3, and -8 was in cytoplasm. Further, overexpression of Pfcasp-2, -3, or -8 was found to cause DNA damage and apoptosis, suggesting that three caspases may be related to apoptosis and mediate different apoptosis pathways in pufferfish. Moreover, the expressions of these caspases were also up-regulated in whole blood and kidney after A. hydrophila challenge, indicating their possible involvement in the immune response against A. hydrophia stimulation. Taken together, the results of this study suggest that the caspase-2,-3, and -8 may play an important role in the apoptosis and immune response in pufferfish.

Molecular characterization and expression analysis of chemokine (CXCL12) from Nile tilapia (Oreochromis niloticus).

Chemokines are a class of small molecular weight cytokines of 6-14 kDa, exerting important roles in the regulation of various inflammatory diseases and immune cell migration. In this study, we have identified the CXCL12 gene from Nile tilapia (Oreochromis niloticus), including CXCL12a (OnCXCL12a) and CXCL12b (OnCXCL12b). The open reading frames of OnCXCL12a and OnCXCL12b are 309 and 297 bp, encoding 102 and 98 amino acids, respectively. Multiple alignment showed that OnCXCL12a and OnCXCL12b have characteristics of CXC chemokines and share high identity with CXCL12 amino acid sequences from the known species. Tissue distribution in the healthy fish indicated that OnCXCL12a and OnCXCL12b expressed in all examined tissues, with the highest expression in muscle and anterior kidney, respectively. After challenged by Streptococcus agalactiae, Poly(I:C) and LPS in vivo and in vitro, OnCXCL12 is transcriptionally up-regulated in immune tissues and cells significantly. The recombinant OnCXCL12 proteins, (r)OnCXCL12a and (r)OnCXCL12b, enhance the release of nitric oxide and increase the expression of inflammatory cytokines (TNF-α, IL-6, and IL-10) in anterior kidney leukocytes, as well as exhibit chemotactic activity for leukocytes from anterior kidney. Summarizing, these results indicate that OnCXCL12 is involved in the immune response of Nile tilapia against pathogen infection and may play an important role in mediating inflammatory response.

A microfibril-associated glycoprotein 4 (MFAP4) from Nile tilapia (Oreochromis niloticus) possesses agglutination and opsonization ability to bacterial pathogens.

Microfibril-associated glycoprotein 4 (MFAP4), a pattern recognition-like molecule with a fibrinogen-like domain (FBG), has the ability to combine and agglutinate pathogens, playing an essential role in the first line of innate immune defense. In this study, the sequence of Nile tilapia (Oreochromis niloticus) microfibril-associated glycoprotein 4 (OnMFAP4) open reading frame (ORF) was amplified and identified. The ORF of OnMFAP4 is 720 bp of nucleotides and codes for 239 amino acids. Spatial mRNA encoding analysis indicated that OnMFAP4 was highly produced in liver, intestine and head kidney in healthy tilapia, and with the lowest expression in muscle. After challenges with Streptococcus agalactiae (S. agalactiae) and Aeromonas hydrophila (A. hydrophila), the expression of OnMFAP4 mRNA was prominently produced in the liver, spleen and head kidney. The up-regulation of OnMFAP4 expression was also presented in head kidney monocytes/macrophages (MO/MΦ) and hepatocytes. Recombinant OnMFAP4 ((r)OnMFAP4) could bind and agglutinate both bacterial pathogens. Moreover, (r)OnMFAP4 could take part in the modulation of inflammation and phagocytosis. In conclusion, this study revealed that OnMFAP4 might take effect in host defense against bacterial infection in Nile tilapia, with agglutination and opsonization capability to bacterial pathogens.

Molecular and functional characterization of IL-6 receptor (IL-6R) and glycoprotein 130 (gp130) in Nile tilapia (Oreochromis niloticus).

Interleukin 6 (IL-6) is a pleiotropic cytokine that exerts its biological functions through interaction with its receptor system consisting of a ligand-specific IL-6 receptor (IL-6R) and a common signal-transducing receptor (gp130). In this study, OnIL-6R and Ongp130 genes from Nile tilapia (Oreochromis niloticus) were identified, and their roles in bacterial or viral infection and in regulation of inflammatory response involved in IL-6 were investigated. The open reading frames (ORFs) of OnIL-6R and Ongp130 are 2019 bp and 2679 bp, encoding 672 and 892 amino acids, respectively. Domain analysis of the deduced amino acid sequences of OnIL-6R and Ongp130 showed that both of them contained a conserved Ig-like domain, FNIII domains, and a WSXWS motif. The transcripts of OnIL-6R and Ongp130 were widely expressed in all examined tissues. Following in vivo challenges with Streptococcus agalactia, Poly I: C and lipopolysaccharide (LPS), the mRNAs of OnIL-6R and Ongp130 were notably induced in liver, head kidney and spleen. The transcriptional up-regulations of OnIL-6R and Ongp130 were also detected in Nile tilapia monocytes/macrophages and lymphocytes after in vitro stimulations with S. agalactiae, Poly I: C and LPS. Besides, increasing mRNA levels of the inflammation-related cytokines (IL-1ß, TNF-α, IL-6, IL-10, and MIF) induced by recombinant OnIL-6 could be further enhanced by co-treatment with recombinant soluble OnIL-6R in lymphocytes. Furthermore, recombinant soluble Ongp130 suppressed the induction of expression of these cytokines in lymphocytes when co-stimulated with (r)OnIL-6 and (r)sOnIL-6R. Taken together, these results indicated that OnIL-6R and Ongp130 were likely involved in the resistance to bacterial or viral infection in Nile tilapia. Moreover, soluble OnIL-6R and soluble Ongp130 have an agonistic effect or antagonistic effect in the inflammation response involved in OnIL-6.

CD38 play roles in T cell-dependent response and B cell differentiation in nile tilapia (Oreochromis niloticus).

CD38 is a multifunctional cell surface molecule that plays a crucial role in B cell activation, differentiation, and maturation in mammals with an increased expression in B cell maturation. In this study, a CD38-like molecule (OnCD38) was cloned and identified from Nile tilapia (Oreochromis niloticus), and its functional characterization was investigated. The open reading frame of OnCD38 is 828 bp of the nucleotide sequence, encoding a polypeptide of 275 amino acids. The deduced amino acid sequence of OnCD38 is highly homologous to other teleost fish and similar to mammals, containing extracellular, intracellular and transmembrane regions. Subcellular localization studies revealed that OnCD38 molecules were presented on the surface of B cells. Three healthy tilapia were used in each experimental group and control group. Following keyhole limpet hemocyanin (KLH) challenge in vivo, the mRNA expression of OnCD38 was significantly up-regulated in peripheral blood, spleen, and head kidney, with an earlier up-regulation in the second challenge than the first one. The up-regulation of OnCD38 expression was also detected in head kidney leukocytes after stimulation with LPS, recombinant HomoIL-10 ((r)HomoIL-10), (r)OnIL-10, and LPS plus (r)OnIL-10 in vitro. Furthermore, the OnCD38 expression increased with the differentiation of B cells, reaching a high level (10.1 fold higher than resting mature B cells) at the plasma-like B cells. Taken together, in this study, these results indicate that the OnCD38 is likely involved in the T cell-dependent response and plays roles in B cell differentiation in Nile tilapia.

Complement C1q subunit molecules from Xenopus laevis possess conserved function in C1q-immunoglobulin interaction.

Complement component 1q (C1q), together with C1r and C1s to form C1, recognize and bind immune complex to initiate the classical complement pathway. In this study, C1q subunit molecules (XlC1qA, XlC1qB, XlC1qC) were cloned and analyzed from Xenopus laevis (X. laevis). The open reading frame (ORF) of XlC1qA is 819 bp of nucleotide sequence encoding 272 amino acids, the ORF of XlC1qB is 711 bp encoding 236 aa, and the XlC1qC is consists of 732 bp encoding 243 aa. The deduced amino acid sequences contain a collagen-like region (CLR), Gly-X-Y repeats in the N-terminus and a C1q family domain at the C-terminus. Phylogenetic analysis revealed that the XlC1qs are clustered with the amphibian clade. Expression analysis indicated that the XlC1qs exhibited constitutive expression in all examined tissues, with the highest expression in liver. Additionally, XlC1q could interact with heat-aggregated mouse IgG and IgM, Xenopus IgM and Nile tilapia IgM, respectively, indicating the functional conservation of XlC1q binding to immunoglobulins. Further, XlC1qs can inhibit C1q-dependent hemolysis of sensitized sheep red blood cells with concentration-dependent manner. These data collectively suggest that the function of C1qs in X. laevis may be conserved in interaction with immunoglobulins, as that of mammals and teleosts.

Blimp-1 is involved in B cell activation and maturation in Nile tilapia (Oreochromis niloticus).

B lymphocyte-induced maturation protein 1 (Blimp-1), a transcription factor containing zinc finger, is required and sufficient to trigger terminal differentiation of B cells in mammals. The Blimp-1 (OnBlimp-1) from Nile tilapia (Oreochromis niloticus) was identified and characterized its expression pattern during B cell activation and maturation. The cDNA of OnBlimp-1 open reading frame is 2547 bp encoding a protein of 848 amino acids and the predicted molecular weight is 93.36 kDa. OnBlimp-1 contains a SET domain and five ZnF_C2H2 domains, which shares high homology with that of other species. OnBlimp-1 transcription was detected in all examined tissues with high expression in the spleen (SPL). Analysis of sorted lymphocyte populations, including IgM+ and IgM- cells from peripheral blood (PBL), SPL and anterior kidney (AK), indicated that the OnBlimp-1 transcription was highly expressed in the IgM+ B cells. Upon LPS stimulation, OnBlimp-1 expression was up-regulated in tissues of PBL, SPL and AK significantly. The expression of OnBlimp-1, as well as the secreted IgM, was significantly up-regulated in the SPL and AK leukocytes stimulated with anti-OnIgM monoclonal antibody and LPS in vitro, respectively. Above results suggest that OnBlimp-1, a cytokine regulating the terminal differentiation of activated B cells to antibody-secreting cells, is likely to play important roles in B cell activation and maturation in Nile tilapia.