Lead induced structural and functional damage and microbiota dysbiosis in the intestine of crucian carp (Carassius auratus).

Lead (Pb) is a hazardous pollutant in water environments that can cause significant damage to aquatic animals and humans. In this study, crucian carp (Carassius auratus) were exposed to waterborne Pb for 96 h; then, histopathological analysis, quantitative qPCR analysis, and 16S high-throughput sequencing were performed to explore the effects of Pb on intestinal bioaccumulation, structural damage, oxidative stress, immune response, and microbiota imbalance of C. auratus. After Pb exposure, the intestinal morphology was obviously damaged, including significantly increasing the thickness of the intestinal wall and the number of goblet cells and reducing the depth of intestinal crypts. Pb exposure reduced the mRNA expressions of Claudin-7 and villin-1 while significantly elevated the level of GST, GSH, CAT, IL-8, IL-10, IL-1, and TNF-α. Furthermore, 16S rRNA analysis showed that the Shannon and Simpson indices decreased at 48 h after Pb exposure, and the abundance of pathogenic bacteria (Erysipelotrichaceae, Weeksellaceae, and Vibrionaceae) increased after Pb exposure. In addition, the correlation network analysis found that Proteobacteria were negatively correlated with Firmicutes and positively correlated with Bacteroidetes. Functional prediction analysis of bacteria speculated that the change in intestinal microbiota led to the PPAR signaling pathway and peroxisome function of the intestine of crucian carp was increased, while the immune system and membrane transport function were decreased. Finally, canonical correlation analysis (CCA) found that there were correlations between the intestinal microbiota, morphology, antioxidant factors, and immune factors of crucian carp after Pb exposure. Taken together, our results demonstrated that intestinal flora dysbiosis, morphological disruption, oxidative stress, and immune injury are involved in the toxic damage of Pb exposure to the intestinal structure and function of crucian carp. Meanwhile, Pb exposure rapidly increased the abundance of pathogenic bacteria, leading to intestinal disorders, further aggravating the damage of Pb to intestinal structure and function. These findings provide us a basis for the link between gut microbiome changes and heavy metal toxicity, and gut microbiota can be used as biomarkers for the evaluation of heavy metal pollution in future.

Mannose-binding lectin suppresses macrophage proliferation through TGF-ß1 signaling pathway in Nile tilapia.

Mannose-binding lectin (MBL) is a multifunctional pattern recognition molecule, which not only mediates the recognition of pathogenic microorganisms and their products, playing an important role in innate immune defense, but also participates in adaptive immune responses of mammalian. However, it's related immune mechanism remains limited, especially the regulation of cell proliferation in early vertebrates. In this study, OnMBL was found to bind to kidney macrophages (MФ) from Nile tilapia (Oreochromis niloticus). Interestingly, OnMBL was able to reduce the proliferation of activated-MФ by regulating the cell cycle, arresting a large number of cells in the G0/G1 phase, and increasing the probability of apoptosis. More importantly, we found that the inhibition of cell proliferation by OnMBL was closely related to the evolutionarily conserved canonical transforming growth factor-beta 1 (TGF-ß1) signaling pathway. Mechanistically, OnMBL could significantly increase the expression of TGF-ß1, activate and regulate the downstream Smad-dependent pathway to reduce the MФ proliferation, thereby maintaining cellular homeostasis in the body's internal environment. This study represents the first description regarding the regulatory mechanisms of the MBL on cell proliferation in teleost fish, which provides a novel perspective on the understanding of the multiple function and evolutionary origins of C-type lectins in the immune system.

Major histocompatibility complex class IIα and IIß of pufferfish (Takifugu obscurus): Identification and functional characterization in response to bacterial challenge.

Classical major histocompatibility complex (MHC) class II molecules play an essential role in immune system. In this study, MHC IIα (Pf-MHC IIα) and MHC IIß (Pf-MHC IIß) homology genes from pufferfish (Takifugu obscurus) were cloned and their functional characterization in response to bacterial challenge was identified. The nucleotide sequences of the open reading frames (ORFs) of pufferfish Pf-MHC IIα and Pf-MHC IIß were 708 bp and 750 bp, encoding 235 aa and 249 aa, respectively. The structure of Pf-MHC IIα or Pf-MHC IIß contained a signal peptide, an α1/ß1 domain, an α2/ß2 domain, a transmembrane region and a cytoplasmic region. Multiple sequence alignment and phylogenetic analysis showed that Pf-MHC IIα and Pf-MHC IIß molecules had the highest similarity with Fugu rubripes (Takifugu rubripes). Cellular localization analysis indicated that the distribution of Pf-MHC IIα and Pf-MHC IIß was in the lymphocyte membrane and cytoplasm. qRT-PCR results showed that Pf-MHC IIα and Pf-MHC IIß expressed relatively high in skin, gills and gut. In addition, after stimulation challenge in vitro (lipopolysaccharide, or polyinosinic: polycytidylic acid) and in vivo (A. hydrophila), the mRNA expressions of Pf-MHC IIα and Pf-MHC IIß were significantly up-regulated in lymphocytes and in tissues of skin, gills, gut and head kidney. Moreover, Pf-MHC IIα or Pf-MHC IIß neutralization reduced the ability of A. hydrophila to induce the expressions of lymphocyte cytokines (TNF-α, IL-1ß and IL-10). Overall, it is speculated that Pf-MHC IIα and Pf-MHC IIß may play an important role in the host response against A. hydrophila in pufferfish.

Identification and Functional Analysis of Tartrate-Resistant Acid Phosphatase Type 5b (TRAP5b) in Oreochromis niloticus.

Tartrate-resistant acid phosphatase type 5 (TRAP5) is an enzyme that is highly expressed in activated macrophages and osteoclasts and plays important biological functions in mammalian immune defense systems. In the study, we investigated the functions of tartrate-resistant acid phosphatase type 5b from Oreochromis niloticus (OnTRAP5b). The OnTRAP5b gene has an open reading frame of 975 bp, which encodes a mature peptide consisting of 302 amino acids with a molecular weight of 33.448 kDa. The OnTRAP5b protein contains a metallophosphatase domain with metal binding and active sites. Phylogenetic analysis revealed that OnTRAP5b is clustered with TRAP5b of teleost fish and shares a high amino acid sequence similarity with other TRAP5b in teleost fish (61.73-98.15%). Tissues expression analysis showed that OnTRAP5b was most abundant in the liver and was also widely expressed in other tissues. Upon challenge with Streptococcus agalactiae and Aeromonas hydrophila in vivo and in vitro, the expression of OnTRAP5b was significantly up-regulated. Additionally, the purified recombinant OnTRAP5b ((r)OnTRAP5) protein exhibited optimal phosphatase activity at pH 5.0 and an ideal temperature of 50 °C. The Vmax, Km, and kcat of purified (r)OnTRAP5b were found to be 0.484 µmol × min-1 × mg-1, 2.112 mM, and 0.27 s-1 with respect to pNPP as a substrate, respectively. Its phosphatase activity was differentially affected by metal ions (K+, Na+, Mg2+, Ca2+, Mn2+, Cu2+, Zn2+, and Fe3+) and inhibitors (sodium tartrate, sodium fluoride, and EDTA). Furthermore, (r)OnTRAP5b was found to promote the expression of inflammatory-related genes in head kidney macrophages and induce reactive oxygen expression and phagocytosis. Moreover, OnTRAP5b overexpression and knockdown had a significant effect on bacterial proliferation in vivo. When taken together, our findings suggest that OnTRAP5b plays a significant role in the immune response against bacterial infection in Nile tilapia.

Lead exposure induces structural damage, digestive stress, immune response and microbiota dysbiosis in the intestine of silver carp (Hypophthalmichthys molitrix).

Lead (Pb) is one of the most common trace metals in water, and its high concentration in the environment can cause harm to aquatic animals and humans. In the present study, the effects of Pb exposure (3.84 mg/kg) on the morphology, digestive enzyme activity, immune function and microbiota structure of silver carp (Hypophthalmichthys molitrix) intestines within 96 h were detected. Moreover, the correlation between them was analyzed. The results showed that Pb exposure on the one hand severely impaired the intestinal morphology, including significantly shortening the intestinal villi's length, increasing the goblet cells' number, causing the intestinal leukocyte infiltration, and thickening the intestinal wall abnormally, on the other hand, increasing the activity of intestinal digestive enzyme (trypsin and lipase). In addition, the mRNA expressions of structure-related genes (Claudin-7 and villin-1) were down-regulated, and the immune factors genes (IL-8, IL-10 and TNF-α) were up-regulated after Pb exposure. Furthermore, data of the MiSeq sequencing showed that the abundance of membrane transport, immune system function and digestive system of silver carp intestinal microbiota all decreased, while cellular antigens increased. Finally, the canonical correlation analysis (CCA) showed that there were correlations between silver carp's intestinal microbiota and intestinal morphology and immune factors. In conclusion, it is speculated that the entry of Pb into the intestine leads the microbiota dysbiosis, affects the intestinal immunity and digestive function, and further damages the intestinal barrier of silver carp.

Affinity-Driven Site-Specific High Mannose Modification Determines the Structural Polymerization and Function of Tetrameric IgM in a Primitive Vertebrate.

Teleost tetramer IgM is the predominant Ig in the immune system and plays essential roles in host defense against microbial infection. Due to variable disulfide polymerization of the monomeric subunits, tetrameric IgM possesses considerable structural diversity. Previous work indicated that the teleost IgM H chain was fully occupied with complex-type N-glycans. However, after challenge with trinitrophenyl (TNP) Ag, the complex N-glycans in the Asn-509 site of Oreochromis niloticus IgM H chain transformed into high mannose. This study, therefore, was conducted to examine the functional roles of the affinity-related high-mannose modification in tilapia IgM. The TNP-specific IgM Ab affinity maturation was revealed in tilapia over the response. A positive correlation between TNP-specific IgM affinity and its disulfide polymerization level of isomeric structure was demonstrated. Mass spectrometric analysis indicated that the relationship between IgM affinity and disulfide polymerization was associated with the Asn-509 site-specific high-mannose modification. Furthermore, the increase of high mannose content promoted the combination of IgM and mannose receptor (MR) on the surface of phagocytes. Moreover, the increased interaction of IgM and MR amplified the phagocytic ability of phagocytes to Streptococcus agalactiae. To our knowledge, this study demonstrates that site-specific high-mannose modification associates with IgM Ab affinity and its structural disulfide polymerization and amplifies the phagocytosis of phagocytes by the combination of IgM and MR. The present study provides evidence for understanding the association of IgM structure and function during the evolution of the immune system.

Toxicological damages on copper exposure to IgM+ B cells of Nile tilapia (Oreochromis niloticus) and mitigation of its adverse effects by ß-glucan administration.

Present investigation was carried out to study toxicological damages of copper exposure and mitigation of its adverse effects with ß-glucan administration in IgM+ B cells which processes multiple roles similar to macrophages in Nile tilapia (Oreochromis niloticus). IgM+ B cells were pretreated with ß-glucan (25 µg/mL) for 24 h before exposed to cupric oxide nanoparticles (CuO NPs) or cupric chloride (Cu ions) at the doses of 0, 5, 10, and 20 µg/mL for 24 h, respectively. Our results demonstrated that ß-glucan increased reduced glutathione (GSH) to against oxidative damage from CuO NPs and Cu ions exposure in IgM+ B cells. The apoptosis process through mitochondrial signaling pathway was depressed in IgM+ B cells since the mitochondrial membrane potential (ΔΨm) was protected from copper exposure by ß-glucan treatment. Furthermore, the inhibition on phagocytic abilities of IgM+ B cells caused by copper exposure could be enhanced with ß-glucan treatment via evaluation of microspheres and bioparticles uptake and LPS-induced NO production. Importantly, ß-glucan might participate in immunomodulation in IgM+ B cells through B cell antigen receptor (BCR) to suppress toxicological effect derived from copper exposure. Taken together, this study provides more information on the toxicological damages in IgM+ B cells upon copper exposure and explains the molecular mechanism to reverse adverse effects caused by copper exposure with ß-glucan administration.

Fiber connectivity density mapping in end-stage renal disease patients: a preliminary study.

Abnormal brain structural connectivity of end-stage renal disease(ESRD) is associated with cognitive impairment. However, the characteristics of cortical structural connectivity have not been investigated in ESRD patients. Here, we study structural connectivity of the entire cerebral cortex using a fiber connectivity density(FiCD) mapping method derived from diffusion tensor imaging(DTI) data of 25 ESRD patients and 20 healthy controls, and between-group differences were compared in a vertexwise manner. We also investigated the associations between these abnormal cortical connectivities and the clinical variables using Pearson correlation analysis and multifactor linear regression analysis. Our results demonstrated that the mean global FiCD value was significantly decreased in ESRD patients. Notably, FiCD values were significantly changed(decreased or increased) in certain cortical regions, which mainly involved the bilateral dorsolateral prefrontal cortex(DLPFC), inferior parietal cortex, lateral temporal cortex and middle occipital cortex. In ESRD patients, we found a trend of negative correlation between the increased FiCD values of bilateral middle frontal gyrus and serum creatinine, urea, parathyroid hormone(PTH) levels and dialysis duration. Only the white matter hyperintensity(WMH) scores were significantly negatively correlated with the global FiCD value in multifactor regression analysis. Our results suggested that ESRD patients exhibited extensive impaired cortical structural connectivity, which was related to the severity of WMHs. A compensation mechanism of cortical structural recombination may play a role in how the brain adapts to maintain optimal network function. Additionally, the serum creatinine, urea and PTH levels may be risk factors for brain structural network decompensation in ESRD patients.

Functional characterization of TNF-α in pufferfish (Takifugu obscurus) in immune response and apoptosis against Aeromonas hydrophila.

Tumour necrosis factor-α (TNF-α) is a multifunctional cytokine involved in immune system homeostasis, antimicrobial defence, regulation of apoptosis, cell proliferation and differentiation. Although the pro-inflammatory property of TNF-α has been made new progress, detailed research on host defence against bacterial infection and inducing apoptosis remains to be revealed in early vertebrates. Here, we reported the TNF-α homologue (ToTNF-α) from pufferfish (Takifugu obscurus). The open reading frame (ORF) of ToTNF-α was 753 bp, encoding a protein of 250 aa contained the TNF family signature and conserved cysteine residues. The mRNA expression of ToTNF-α had a wide range of tested tissues, with the highest expression in the skin. After Aeromonas hydrophila infection, the mRNA expression of ToTNF-α was significantly up-regulated both in vivo and in vitro experiments. After stimulation by recombinant protein of ToTNF-α ((r)ToTNF-α), the relative expressions of endogenous TNF-α, caspase 8, caspase 3, p53, and Bax inhibitor-1 in head kidney leucocytes were all notably up-regulated. These results showed that ToTNF-α might induce apoptosis depend on pro- and anti-apoptotic proteins at mRNA level. Moreover, flow cytometry analysis indicated that the (r)ToTNF-α can induce apoptosis of head kidney leucocytes. Taken together, these characteristics suggest that ToTNF-α can participate in immune response against A. hydrophila and induce apoptosis at mRNA and cellular level, which will help to understand the mechanism of apoptosis and immune response in teleost fish.

Garlic (Allium sativum) and Fu-ling (Poria cocos) mitigate lead toxicity by improving antioxidant defense mechanisms and chelating ability in the liver of grass carp (Ctenopharyngodon idella).

The heavy metal lead (Pb) is a contaminant widely distributed in the food chain. In this study, eight weeks of feeding containing Garlic (Allium sativum) or Fu-ling (Poria cocos) or both, markedly increased the growth index, enzyme activity, and serum index and significantly decreased muscle Pb level in grass carp (Ctenopharyngodon idella). Upon Pb exposure, the feeding Garlic or Fu-ling or both possessed the similar effects on improving the function of the antioxidant system and chelating ability. Further, the gene expressions of metal binding proteins (TF and MT-2) in the liver of the three experimental groups were significantly higher than those of the control group, which were all highly up-regulated after Pb exposure. At the same time, the activities of antioxidant enzymes (SOD and CAT) and the content of non-enzymatic substance (GSH) in the liver of the Garlic group, Fu-ling group and mixed group were stable compared to the control group after Pb exposure. Moreover, the reduction of Pb toxicity was manifested by the decrease of Pb content in the muscle, and the stable expression of heat stress proteins (HSP30 and HSP60) and immune-related genes (TNF-α and IL-1ß). Taken together, the study preliminarily shows that the Garlic and Fu-ling play a role in mitigating the toxicity of Pb in grass carp.

Identification and functional characterization of CD154 in T cell-dependent immune response in Nile tilapia (Oreochromis niloticus).

CD154, a member of the TNF superfamily, is a multifunctional molecule highly expressed in activated T cells, and plays important roles in T cell-dependent humoral immune response. In this study, CD154 of Nile tilapia (Oreochromis niloticus) was identified, and its functions in the T cell-dependent immune response were demonstrated. The open reading frame (ORF) of OnCD154 is 699 bp, encoding a protein of 232 amino acids with a 23 amino acid transmembrane region. Amino acid sequence of OnCD154 is highly homologous to that of other teleost fish, especially rainbow trout. Quantitative real-time PCR (qRT-PCR) demonstrated that mRNA of OnCD154 is highly expressed in immune organs, especially in spleen, thymus, gills, head kidney, etc. In addition, the anti-OnCD154 polyclonal antibody (anti-(r)OnCD154) was successfully prepared, and it can react with natural protein in head kidney leukocytes. Following two immunizations with keyhole limpet hemocyanin (KLH) in vivo, the significantly up-regulated expression level of OnCD154 mRNA appeared earlier (fifth day) and higher (42.9 folds) in the second challenge than the first on in head kidney. Further, after stimulation with KLH in vitro, the expressions of T cell-dependent immune response-related molecules (activated T cell specific surface molecules CD3ε and CD154) and B cell differentiation-related molecules (Blimp1 and sIgM) and CD40 were significantly up-regulated in head kidney leukocytes. Moreover, the up-regulated expressions of these molecules were blocked with the treatment of anti-(r)OnCD154 antibody. Taken together, these results indicate that OnCD154 might get involved in T cell-dependent immune response, and provide a new insight into the humoral immune response of teleost fish.

Molecular cloning, characterization and expression analysis of major histocompatibility complex class I alpha gene of pufferfish (Takifugu obscurus).

The major histocompatibility complex (MHC) genes play a key role in immune response in vertebrates. In this study, an MHC I alpha homolog gene (PfMHC Ⅰα) from pufferfish (Takifugu obscurus) was identified and its subcellular localization and expression patterns of PfMHC Ⅰα after challenge in vivo and in vitro were analysed. The open reading frame of PfMHC Ⅰα was 1,089 bp in length, encoding 362 aa. The immunofluorescence result revealed that PfMHC Ⅰα was presented on the membrane of lymphocytes. qRT-PCR analysis indicated that PfMHC Ⅰα was expressed in all examined tissues, with the highest expression in skin, followed by the expression in gills and whole blood. After challenge of Aeromonas hydrophila or polyinosinic: polycytidylic acid (Poly I:C) in vitro, the expression levels of PfMHC Ⅰα on pufferfish kidney lymphocytes were significantly up-regulated, with the highest expression level at 48 hr post-challenge. After infection with A. hydrophila or Poly I:C in vivo, the expression levels of PfMHC Ⅰα in the skin, whole blood and kidneys were significantly up-regulated. Taken together, it is speculated that PfMHC Ⅰα associates with resistance to both intracellular and extracellular antigens and plays an important role in the host response against pathogen infection in pufferfish.

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.

A Siglec-1-like lectin from Nile tilapia (Oreochromis niloticus) possesses functions of agglutination and mediation of macrophage phagocytic activity.

Siglec-1, one of the sialic acid-binding immunoglobulin-type lectins, is closely related to the recognition of host-pathogen and cell-cell interactions in the adaptive and innate immune systems. In this communication, a Siglec-1-like gene (OnSiglec-1-like) from Nile tilapia (Oreochromis niloticus) was analyzed. Relative expression revealed that the OnSiglec-1-like was expressed in all tested tissues, and the highest expression was found in the anterior kidney. Upon Streptococcus agalactiae (S. agalactiae) infection, the expression of OnSiglec-1-like was up-regulated in anterior kidney and spleen significantly in vivo. Additionally, the same phenomenon was observed in anterior kidney leukocytes upon LPS and S. agalactiae challenges as well in vitro. Western-blotting and ELISA analyses revealed that recombinant OnSiglec-1-like protein possessed high binding activity to LTA, LPS and S. agalactiae. Further, the recombinant OnSiglec-1-like was able to agglutinate S. agalactiae. Moreover, with the digestion of specific sialidase, the phagocytic ability of macrophages to S. agalactiae was greatly enhanced. Taken together, these results indicated that the Siglec-1-like possesses conserved functions of agglutination and promotion of macrophage phagocytic activity in Nile tilapia.

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.

Long-Lived Plasma Cells Secrete High-Affinity Antibodies Responding to a T-Dependent Immunization in a Teleost Fish.

The recent discovery of long-lived plasma cells (LLPCs) in mammals, which provide a constant expression of specific high-affinity antibodies that mediate humoral memory, has caused a dramatic paradigm shift in the study of immunity and vaccine development. In teleost fish, there are few studies regarding the association between LLPCs and antibody production, and the affinity of the antibodies secreted by the LLPCs is poorly understood. In the present study, channel catfish (Ictalurus punctatus) were immunized with trinitrophenylated-keyhole limpet hemocyanin (TNP-KLH) to examine TNP-specific antibody titers, affinities, antibody-secreting cell (ASC) dynamic changes, and especially the affinity of secreted antibodies by LLPCs post-immunization. We demonstrated that TNP-specific LLPCs were generated starting at week 4 post-immunization, achieved a maximal number at week 8, and maintained a comparable level throughout the 18-week post-immunization period, which was correlated with the dynamics of serum antibody titers and affinity maturation in the response. The LLPCs appeared to mostly reside within, or migrate to, the anterior kidney (bone marrow-like tissue in mammals), but a small portion was also located in the spleen and peripheral blood. The antibodies produced by the LLPCs possessed high affinities, indicating that the generation and development of LLPCs were driven by affinity selection in teleosts. Collectively, the results of this study provide insights toward the evolutionary understanding of the affinity-dependent mechanism of LLPC generation and development.

Comparative transcriptome analysis of the transcriptional heterogeneity in different IgM+ cell subsets from peripheral blood of Nile tilapia (Oreochromis niloticus).

In teleost fish, IgM+ B cells play important roles in innate and adaptive immunity. Different IgM+ B cells are detected in teleost, named IgMlo and IgMhi B cell subsets, according to the distinct expression levels of membrane IgM (mIgM). However, the study on the heterogeneity in IgM+ B cell subsets remains poorly understood. In this study, the comparative transcriptomic profiles of IgM-, IgMlo and IgMhi from peripheral blood of Nile tilapia (Oreochromis niloticus) were carried out by using RNA-sequencing technique. A total of 6045 and 5470 differentially expressed genes (DEGs) were detected in IgMlo and IgMhi cells, respectively, as compared with IgM- lymphocytes, whereas 3835 genes were differentially expressed when IgMlo compared to IgMhi cells. Analysis of the KEGG database indicated that the DEGs were enriched in immune system categories and signaling transduction and interaction in IgM- vs IgMhi, IgM- vs IgMlo and IgMlo vs IgMhi. Comparatively, in IgMlo vs IgMhi, GO enrichment analysis indicated that the DEGs enriched in nucleic acid binding transcription factor activity. Analysis of crucial transcription factors for B cell differentiation indicated that IgMlo and IgMhi cell clusters belonged to the different B cell subsets. The data generated in this study may provide insights into understanding the heterogeneity of IgM+ cells in teleost, and suggest that IgM+ B cells play a crucial role in innate immunity.

The key differentially expressed genes and proteins related to immune response in the spleen of pufferfish (Takifugu obscurus) infected by Aeromonas hydrophila.

The immune mechanism elicited in pufferfish (Takifugu obscurus) against the invasion of Aeromonas hydrophila is still poorly understood. We examined the spleen of pufferfish at the transcriptome and proteome levels by using Illumina-seq and TMT coupled mass spectrometry after 12 h infection by A. hydrophila, respectively. A total of 2,339 genes (1,512 up-regulated and 827 down-regulated) and 537 (237 up-regulated and 300 down-regulated) proteins were identified. GO and KEGG analyses revealed that the responses to stimulus were the main biological processes, intestinal immune network for IgT production and calcium signaling pathway. Fourteen genes (8 up-regulated and 6 down-regulated) and proteins (5 up-regulated and 9 down-regulated) involved immune responses or signal transduction were validated by qRT-PCR and parallel reaction monitoring to confirm the reliability of the transcriptomic and proteomic analyses, respectively. Moreover, qRT-PCR and flow cytometry were used to detect dynamics of the genes in calcium signaling pathway and changes of concentration of cytoplasm Ca2+ in spleen cells within a 72 h challenge. This study provides the findings regarding immune response, especially intestinal immune network for IgT production pathway and calcium signaling pathway at the molecular, protein and cellular in pufferfish after infection by A. hydrophila. These results would provide a new insight and molecular targets into the response to pathogenic infection in pufferfish.

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.