IL-2 Signaling Couples the MAPK and mTORC1 Axes to Promote T Cell Proliferation and Differentiation in Teleosts.

IL-2 is a pleiotropic cytokine that is critical for T cell immunity. Although the IL-2-mediated regulation of T cell immunity in mammals is relatively well understood, it remains largely unknown whether and how IL-2 regulates T cell immunity in lower vertebrates. To address this knowledge gap, we investigated the role played by IL-2 in the regulation of T cell response, as well as the associated underlying mechanisms in a teleost fish, large yellow croaker (Larimichthys crocea). We found that large yellow croaker (L. crocea) IL-2 (LcIL-2) significantly promoted T cell proliferation both in vivo and in vitro; significantly induced the differentiation of Th1, Th2, regulatory T, and cytotoxic T cells while inhibiting Th17 differentiation; and participated in the elimination of invading pathogenic bacteria. Mechanistically, the binding of LcIL-2 to its heterotrimer receptor complex (LcIL-15Rα/LcIL-2Rß/Lcγc) triggered the conserved JAK-STAT5 pathway, which in turn regulated the expression of genes involved in T cell expansion, differentiation, and biological function. The MAPK and mammalian target of rapamycin complex 1 (mTORC1) axes, which are involved in TCR-mediated signaling, were also required for LcIL-2-mediated T cell response. Collectively, our results demonstrated that fish IL-2 plays a comprehensive regulatory role in T cell response and highlighted the complex and delicate network regulating T cell-driven immune response. We propose that T cell immunity is regulated by the interplay between TCR signaling and cytokine signaling, and that this basic strategy evolved before the emergence of the tetrapod lineage. Our findings provide valuable insights into the regulatory mechanisms underlying T cell response in teleosts.

Whole genome sequencing of a snailfish from the Yap Trench (~7,000 m) clarifies the molecular mechanisms underlying adaptation to the deep sea.

Hadal environments (depths below 6,000 m) are characterized by extremely high hydrostatic pressures, low temperatures, a scarce food supply, and little light. The evolutionary adaptations that allow vertebrates to survive in this extreme environment are poorly understood. Here, we constructed a high-quality reference genome for Yap hadal snailfish (YHS), which was captured at a depth of ~7,000 m in the Yap Trench. The final YHS genome assembly was 731.75 Mb, with a contig N50 of 0.75 Mb and a scaffold N50 of 1.26 Mb. We predicted 24,329 protein-coding genes in the YHS genome, and 24,265 of these genes were successfully functionally annotated. Phylogenetic analyses suggested that YHS diverged from a Mariana Trench snailfish approximately 0.92 million years ago. Many genes associated with DNA repair show evidence of positive selection and have expanded copy numbers in the YHS genome, possibly helping to maintain the integrity of DNA under increased hydrostatic pressure. The levels of trimethylamine N-oxide (TMAO), a potent protein stabilizer, are much higher in the muscles of YHS than in those of shallow-water fish. This difference is perhaps due to the five copies of the TMAO-generating enzyme flavin-containing monooxygenase-3 gene (fmo3) in the YHS genome and the abundance of trimethylamine (TMA)-generating bacteria in the YHS gut. Thus, the high TMAO content might help YHS adapt to high hydrostatic pressure by improving protein stability. Additionally, the evolutionary features of the YHS genes encoding sensory-related proteins are consistent with the scarce food supply and darkness in the hadal environments. These results clarify the molecular mechanisms underlying the adaptation of hadal organisms to the deep-sea environment and provide valuable genomic resources for in-depth investigations of hadal biology.

Large yellow croaker (Lrimichthys crocea) IL-2 modulates humoral immunity via the conserved JAK-STAT5 signal pathway.

The terminal differentiation of B cells into plasma cells is central to the generation of protective, long-lived humoral immune responses. In mammals, interleukin-2 (IL-2) has been shown to play a role in B cell proliferation and differentiation. However, it remains unclear whether fish IL-2 is involved in B cell proliferation and differentiation. To this end, we investigated the regulatory role of IL-2 in B cell proliferation and differentiation in large yellow croaker (Larimichthys crocea). We found that L. crocea IL-2 (LcIL-2) significantly increased IgM+ B cells proliferation both in vivo and in vitro and facilitated IgM+ B cells differentiation into plasma cells. Furthermore, LcIL-2 increased the production of specific antibodies after immunization with the Vibrio alginolyticus subunit vaccine, recombinant dihydrolipoamide dehydrogenase (rDLD); simultaneous administration of LcIL-2 and rDLD prior to challenge with Vibrio parahaemolyticus or V. alginolyticus significantly increased relative percent survival. Mechanistically, LcIL-2 promoted B cell proliferation and regulated B cell differentiation by triggering the JAK-STAT5 signaling pathway. Collectively, our results demonstrated that LcIL-2 improved B cell proliferation and specific antibody production via the conserved JAK-STAT5 signaling pathway in large yellow croaker, providing valuable insights into the mechanisms underlying the IL-2-mediated regulation of the humoral immune response in fish.

Antimicrobial activity and mechanisms of a derived antimicrobial peptide TroNKL-27 from golden pompano (Trachinotus ovatus) NK-lysin.

NK-lysin, a homologue of granulysin among human, is predominantly found in natural killer cells and cytotoxic T-lymphocytes, which plays a pivotal part in innate immune responses against diverse pathogenic bacteria. Nonetheless, in teleosts, the research on antimicrobial activity and mechanisms of NK-lysin are seldom reported. In this study, we determined the antimicrobial activity of the truncated peptide TroNKL-27 that derived from golden pompano (Trachinotus ovatus) NK-lysin, and investigated its antimicrobial mechanisms. The results showed that TroNKL-27 had considerable antimicrobial potency against both Gram-positive (Staphylococcus aureus, Streptococcus agalactiae) and Gram-negative bacteria (Vibrio harveyi, V. alginolyticus, Escherichia coli, Edwardsiella tarda). Cytoplasmic membrane depolarization and propidium iodide (PI) uptake assay manifested that TroNKL-27 could induce the bacterial membrane depolarization and change its membrane permeability, respectively. In the light of scanning electron microscopy (SEM) observation, TroNKL-27 was capable of altering morphological structures of bacteria and leading to leakage of cellular contents. Moreover, the results of gel retardation assay indicated TroNKL-27 had the ability to induce the degradation of bacterial genomic DNA. As regards in vivo assay, TroNKL-27 could reduce the replication of V. harveyi in tissues of golden pompano, protect the tissue from pathological changes. Moreover, TroNKL-27 in vivo could significantly increase the expression of the immune genes (such as IL1ß, TNFα, IFN-γ, C3 and Mx) in presence or absence of V. harveyi infection. All of these results suggest that TroNKL-27 is a novel antimicrobial peptide possessing antibacterial and immunoregulatory function in vivo and in vitro, and the observed effects of TroNKL-27 will lay a solid foundation for the development of new antimicrobial agents used in aquaculture.

CC chemokine 1 protein from Cromileptes altivelis (CaCC1) promotes antimicrobial immune defense.

Chemokines are a family of small signaling proteins that are secreted by various cells. In addition to their roles in immune surveillance, localization of antigen, and lymphocyte trafficking for the maintenance of homeostasis, chemokines also function in induce immune cell migration under pathological conditions. In the present study, a novel CC chemokine gene (CaCC1) from humpback grouper (Cromileptes altivelis) was cloned and characterized. CaCC1 comprised a 435 bp open reading frame encoding 144 amino acid residues. The putative molecular weight of CaCC1 protein was 15 kDa CaCC1 contains four characteristic cysteines that are conserved in other known CC chemokines. CaCC1 also shares 11.64%-90.28% identity with other teleost and mammal CC chemokines. Phylogenetic analysis revealed that CaCC1 is most closely related to Epinephelus coioides EcCC1, both of which are in a fish-specific CC chemokine clade. CaCC1 was constitutively expressed in all examined C. altivelis tissues, with high expression levels in skin, heart, liver, and intestine. Vibrio harveyi stimulation up-regulated CaCC1 expression levels in liver, spleen, and head-kidney. Functional analyses revealed that the recombinant protein (rCaCC1) could induce the migration of head-kidney lymphocytes from C. altivelis. Moreover, rCaCC1 significantly enhanced phagocytosis in head-kidney macrophages from C. altivelis. In addition, rCaCC1 exhibited antimicrobial activities against Staphylococcus aureus, Edwardsiella tarda, and V. harveyi. In vivo, CaCC1 overexpression improved bacterial clearance in V. harveyi infected fish. Conversely, CaCC1 knockdown resulted in a significant decrease of bacterial clearance. These results demonstrate the important roles that CaCC1 plays in homeostasis and in inflammatory response to bacterial infection.

Molecular characterization of the interferon regulatory factor (IRF) family and functional analysis of IRF11 in the large yellow croaker (Larimichthys crocea).

Interferon regulatory factors (IRFs) are a family of transcription factors involved in regulating interferon (IFN) responses and immune cell development. A total of 11 IRFs have been identified in teleost fish. Here, a complete repertoire of 11 IRFs (LcIRFs) in the large yellow croaker (Larimichthys crocea) was characterized with the addition of five newly identified members, LcIRF2, LcIRF5, LcIRF6, LcIRF10, and LcIRF11. These five LcIRFs possess a DNA-binding domain (DBD) at the N-terminal that contains five to six conserved tryptophan residues and an IRF-association domain (IAD) or IAD2 at the C-terminal that is responsible for interaction with other IRFs or co-modulators. Phylogenetic analysis showed that the 11 LcIRFs were divided into four clades including the IRF1 subfamily, IRF3 subfamily, IRF4 subfamily, and IRF5 subfamily. These are evolutionarily related to their respective counterparts in other fish species. The 11 LcIRFs were constitutively expressed in all examined tissues, although at different expression levels. Upon polyinosinic: polycytidylic acid (poly (I:C)) stimulation, the expression of all 11 LcIRFs was significantly induced in the head kidney and reached the highest levels at 6 h post-stimulation (except LcIRF4). LcIRF1, LcIRF3, LcIRF7, LcIRF8, and LcIRF10 were more strongly induced by poly (I:C) than the other LcIRFs. Significant induction of all LcIRFs was observed in the spleen, with LcIRF2, LcIRF5, LcIRF6, LcIRF7, LcIRF9, and LcIRF11 reaching their highest levels at 48 h LcIRF3 and LcIRF11 showed a stronger response to poly (I:C) in the spleen than the other LcIRFs. In addition, LcIRF1, LcIRF3, LcIRF7, LcIRF9, LcIRF10, and LcIRF11 were significantly induced by Vibro alginolyticus in both the spleen and the head kidney, with LcIRF1 strongly induced. Thus, LcIRFs exhibited differential inducible expression patterns in response to different stimuli in different tissues, suggesting that LcIRFs have different functions in the regulation of immune responses. Furthermore, overexpression of LcIRF11 activated the promoters of LcIFNc, LcIFNd, and LcIFNh, and differentially induced the expression levels of LcIFNs and IFN-stimulated genes (ISGs). Overexpression of LcIRF11 in epithelioma papulosum cyprinid (EPC) cells inhibited the replication of viral genes after infection of spring viremia of carp virus (SVCV). These data suggested that LcIRF11 may function as a positive regulator in regulating the cellular antiviral response through induction of type I IFN expression. Taken together, the present study reported molecular characterization and expression analysis of 11 IRFs in the large yellow croaker, and investigated the role of LcIRF11 in the antiviral response, which laid a good foundation for further study on the evolution and functional characterization of fish IRFs.

Identification and bioactivity of a granulocyte colony-stimulating factor a homologue from large yellow croaker (Larimichthys crocea).

Granulocyte colony-stimulating factor (GCSF) is a growth factor that drives the proliferation and differentiation of granulocytes and monocytes/macrophages. Currently, two copies of GCSF, named GCSFa and GCSFb, have been identified in teleost fish, but data on the functions and signal pathways of these fish GCSFs are still limited. In the present study, a GCSFa homologue (LcGCSFa) was identified from large yellow croaker (Larimichthys crocea). The open reading frame (ORF) of LcGCSFa is 636 bp long and encodes a protein of 211 amino acids (aa), with a 19-aa signal peptide and a typical IL-6 domain, conserved in fish GCSF sequences. The phylogenetic analysis showed that LcGCSFa clustered with other fish GCSFa homologues. LcGCSFa was constitutively expressed in all tissues tested and significantly up-regulated in head kidney and spleen by Vibrio alginolyticus or poly(I:C). LcGCSFa transcripts were also detected in primary head kidney leucocytes (PKL), primary head kidney macrophages (PKM), and primary head kidney granulocytes (PKG), and significantly up-regulated in PKL and PKG by LPS or poly(I:C). These data indicated that LcGCSFa may be involved in the immune responses induced by bacterium and virus. The recombinant LcGCSFa protein (rLcGCSFa) produced in Pichia pastoris promoted the proliferation of PKL both in vivo and in vitro. Furthermore, rLcGCSFa significantly increased both transcription and phosphorylation levels of the signal transducers and activators of transcription (STAT) proteins (LcSTAT3 and LcSTAT5) in PKL, which are required for the GCSF-dependent proliferation. These results showed that LcGCSFa may promote the proliferation of PKL via the activation of LcSTAT3 and LcSTAT5, suggesting a conserved role across vertebrate GCSFs.

Characterization and function of a group II type I interferon in the perciform fish, large yellow croaker (Larimichthys crocea).

Teleost fish possess two groups of type I interferons (IFNs) with two (group I IFNs) or four (group II IFNs) conserved cysteines, which are further classified into seven subgroups. In our previous study, two group I type I IFNs, LcIFNd and LcIFNh (a new subgroup member), were identified in the perciform fish, large yellow croaker (Larimichthys crocea). Here, we identified a group II type I IFN, LcIFNc, in this species. The deduced LcIFNc contained six cysteines, four of which are highly conserved (C1: C28, C2:C53, C3: C130, and C4:C159) in the fish group II type I IFNs, and a typical type I IFN signature motif was also found in it. Phylogenetic analysis indicated that LcIFNc belongs to the IFNc subgroup of fish group II type I IFNs. LcIFNc was constitutively expressed in all examined tissues, and was rapidly up-regulated in spleen and head kidney by poly(I:C) and Aeromonas hydrophila. Recombinant LcIFNc protein (rLcIFNc) could increase the expression of antiviral genes, Mx1, PKR and ISG15, in large yellow croaker peripheral blood leukocytes (PBLs). The rLcIFNc also exhibited obvious antiviral activity based on less cytopathic effect (CPE) and decreased expression levels of several viral genes in the rLcIFNc-treated grouper spleen (GS) cells following Singapore grouper iridovirus (SGIV) infection. Additionally, rLcIFNc was able to induce the expression of LcIFNc, as well as LcIFNd and LcIFNh in the PBLs and primary head kidney cells (HKCs) from large yellow croaker. These results therefore indicated that LcIFNc not only had antiviral activity, but also mediated the regulation of type I IFN response.

Molecular characterization of a new fish specific chemokine CXCL_F6 in large yellow croaker (Larimichthys crocea) and its role in inflammatory response.

Chemokines are a superfamily of structurally related chemotactic cytokines exerting significant roles in regulating cell migration and activation. Currently, five subgroups of fish specific CXC chemokines, named CXCL_F1-CXCL_F5, have been identified in teleost fish. However, understanding of the functions of these fish specific CXC chemokines is still limited. Here, a new member of fish specific CXC chemokines, LcCXCL_F6, was cloned from large yellow croaker Larimichthys crocea. Its open reading frame (ORF) is 369 nucleotides long, encoding a peptide of 122 amino acids (aa). The deduced LcCXCL_F6 protein contains a 19-aa signal peptide and a 103-aa mature polypeptide, which has four conserved cysteine residues (C28, C30, C56, and C72), as found in other known CXC chemokines. Phylogenetic analysis showed LcCXCL_F6 formed a separate clade with sequences from other fish species, tentatively named CXCL_F6, distinct from the clades formed by fish CXCL_F1-5 and mammalian CXC chemokines. The LcCXCL_F6 transcripts were constitutively expressed in all examined tissues and significantly up-regulated in the spleen and head kidney tissues by poly (I:C) and Vibrio alginolyticus. Its transcripts were also detected in primary head kidney leukocytes (HKLs), peripheral blood leucocytes (PBLs), and large yellow croaker head kidney (LYCK) cell line, and significantly up-regulated by poly(I:C), lipopolysaccharide (LPS), and peptidoglycan (PGN) in HKLs. Recombinant LcCXCL_F6 protein (rLcCXCL_F6) could not only chemotactically attract monocytes/macrophages and lymphocytes from PBLs, but also enhance NO release and expression of proinflammatory cytokines (TNF-α, IL-1ß, and CXCL8) in monocytes/macrophages. These results indicate that LcCXCL_F6 plays a role in mediating the inflammatory response.

Identification and bioactivity of a granulocyte colony-stimulating factor b homologue from large yellow croaker (Larimichthys crocea).

Granulocyte colony-stimulating factor (GCSF) is a pleiotropic cytokine that plays a key role in regulation of hematopoiesis, innate and adaptive immune responses in mammals. However, bioactivity of GCSF in teleost fish remains largely unknown. In this study, a GCSFb homologue from large yellow croaker (Larimichthys crocea) (LcGCSFb) was cloned by RACE-PCR techniques. The open reading frame (ORF) of LcGCSFb is 603 bp long and encoded a protein precursor of 200 amino acids (aa), with a 19-aa signal peptide and a 181-aa mature peptide. In healthy fish, the LcGCSFb was constitutively expressed in all examined tissues, with the highest levels in mucous tissues, such as gills, intestine, and stomach. Its transcripts in head kidney, spleen, gills, intestine and stomach were significantly induced by Vibrio alginolyticus challenge. LcGCSFb transcripts were also detected in primary head kidney leukocytes (PKL), primary head kidney macrophages (PKM), primary head kidney granulocytes (PKG) and head kidney cell line (LYCK), and markedly upregulated by inactivated V. alginolyticus. These data suggested that LcGCSFb may play a role in immune response against bacterial infection. In vivo administration of recombinant LcGCSFb protein (rLcGCSFb) significantly upregulated the expression levels of the inflammatory cytokines (IL-6 and TNFα), and transcription factor C/EBPß, which is required for proliferation of neutrophils. Furthermore, rLcGCSFb showed an ability to strengthen the phagocytosis of PKL in vitro. Taken together, LcGCSFb may be involved in antibacterial immunity via promoting the inflammatory response and the phagocytic activity of leukocytes. To our knowledge, this is the first report on immunoregulatory roles of GCSF in teleost.

Development of monoclonal antibody against IgM of large yellow croaker (Larimichthys crocea) and characterization of IgM+ B cells.

In the present study, a monoclonal antibody (mAb) against large yellow croaker IgM was produced by immunizing mice with purified large yellow croaker serum IgM. Western blotting showed that this mAb could specifically react with the heavy chain of large yellow croaker serum IgM. Indirect immunofluorescence assay (IFA) analysis suggested that the resulting mouse anti-IgM mAb could recognize membrane-bound IgM (mIgM) molecules of large yellow croaker. This mouse anti-IgM mAb also can be used for sorting of large yellow croaker IgM+ B cells through the magnetic-activated cell sorting (MACS) method, which was further confirmed by RT-PCR analysis of specific marker genes for B cells. Flow cytometry analysis showed that the percentages of IgM+ B cells in head kidney, spleen and peripheral blood lymphocytes were 29.00 ±â€¯1.58%, 33.00 ±â€¯1.64%, and 16.50 ±â€¯2.39%, respectively. Additionally, the phagocytosis rates of IgM+ B cells for 0.5 µm beads in head kidney, spleen and peripheral blood were calculated to be 7.56 ±â€¯0.58%, 4.053 ±â€¯0.62% and 23.17 ±â€¯2.26%, respectively, while only 2.36 ±â€¯0.23%, 1.16 ±â€¯0.44% and 6.41 ±â€¯0.45 of IgM+ B cells in these three tissues ingested 1 µm beads. Taken together, our data demonstrated that the mouse anti-IgM mAb produced in this study could be used as a tool to characterize IgM+ B cells and to study functions of IgM in large yellow croaker.

Molecular cloning and bioactivity of an IL-2 homologue in large yellow croaker (Larimichthys crocea).

Interleukin-2 (IL-2), an important immunomodulatory cytokine, plays a crucial role in promoting the proliferation, activation and differentiation of T cells. Here, the cDNA of an IL-2 homologue (LcIL-2) in large yellow croaker (Larimichthys crocea) was cloned by RACE-PCR techniques. The open reading frame (ORF) of LcIL-2 gene is 426 bp long and encoded a precursor protein of 141 amino acids (aa), with a 20-aa signal peptide and a 121-aa mature peptide containing two putative N-glycosylation sites at Asn77 and Asn101. The LcIL-2 is preferentially expressed in lymphocytes-rich tissues, such as spleen and blood, and is increased in head kidney and spleen upon inactivated trivalent bacterial vaccine or poly(I:C) stimulation. LcIL-2 expression could also be detected in primary head kidney leukocytes (PKL), primary head kidney macrophages (PKM) and primary head kidney granulocytes (PKG), with the highest level in PKL. In addition, the expression level of LcIL-2 in PKL was slightly induced by LPS or poly(I:C), while markedly induced by PHA or Con-A. The recombinant LcIL-2 protein produced in Pichia pastoris could increase the expression of genes involved in Th1 (IL-2, IFN-γ and T-bet) and Th2 (IL-4/13A, IL-4/13B and GATA3) development and differentiation, and of the IL-2 downstream transcription factor STAT5B gene, but inhibit the expression of genes related to Th17 (IL-17A/F2 and IL-17A/F3) development and differentiation. Taken together, our results indicated that LcIL-2 possesses similar structural and functional characteristics to other vertebrate IL-2s, and may play a role in T cell development and differentiation.

Molecular characterization and functional activity of CXCL8_L3 in large yellow croaker Larimichthys crocea.

CXCL8, also called interleukin-8, is a typical CXC chemokine that plays a key role in promoting inflammation. Phylogenetically, fish CXCL8 chemokines can be divided into three subgroups, CXCL8_L1, CXCL8_L2, and CXCL8_L3, of which CXCL8_L3 is a new subgroup. The CXCL8_L3 gene sequences have been reported in many fish species, but their function remains unknown. Here, a CXCL8_L3 (LycCXCL8_L3) gene was cloned from large yellow croaker Larimichthys crocea. Its open reading frame (ORF) was 309 nucleotides long, encoding a peptide of 102 amino acids. The deduced LycCXCL8_L3 protein contains an 18-aa signal peptide and an 84-aa mature polypeptide, which has four conserved cysteine residues (C30, C32, C57, and C73) as found in other known CXCL8 chemokines. Phylogenetic analysis showed LycCXCL8_L3 formed a major clade with CXCL8_L3 sequences from other fish species. The LycCXCL8_L3 transcript was constitutively expressed in all examined tissues and significantly up-regulated in the spleen and head kidney tissues by inactivated trivalent bacterial vaccine. The LycCXCL8_L3 transcript was also detected in peripheral blood leukocytes (PBLs), primary head kidney macrophages (PKM), and large yellow croaker head kidney cell line (LYCK), with the highest levels in PKM. Recombinant LycCXCL8_L3 (rLycCXCL8_L3) protein could not only chemotactically attract lymphocytes and eosinophils in PBLs, but also enhance the respiratory burst activity of PKM. These results indicate that LycCXCL8_L3 may play an important role in the inflammatory response of large yellow croaker. To our knowledge, this is the first report on functional study of the CXCL8_L3 in fish.

Identification of two IL-4/13 homologues in large yellow croaker (Larimichthys crocea) revealed their similar roles in inducing alternative activation of monocytes/macrophages.

Mammalian interleukin-4 (IL-4) and -13 (IL-13), two anti-inflammatory T helper cell type 2 (Th2) cytokines, play the central roles in mediating the alternative activation of monocytes/macrophages (MO/Mφs). However, exact functions in MO/Mφs polarization of IL-4/13 homologues in teleost fish remain largely unknown. In this study, we identified two IL-4/13 homologues from large yellow croaker Larimichthys crocea, LcIL-4/13A and LcIL-4/13B, which share low amino acid sequence identities to the known fish IL-4/13 molecules. Phylogenetic analysis showed that LcIL-4/13A is evolutionarily closely related to Dicentrarchus labrax IL-4/13A, and LcIL-4/13B to Takifugu rubripes IL-4/13B. The two LcIL-4/13 genes were constitutively expressed in all examined tissues, but with different expression levels. Both LcIL-4/13A and LcIL-4/13B were up-regulated by inactivated trivalent bacterial vaccine in the head kidney, and LcIL-4/13B appeared more responsive to bacterial vaccine than LcIL-4/13A. Recombinant LcIL-4/13A and LcIL-4/13B proteins (rLcIL-4/13A and rLcIL-4/13B) produced in Escherichia coli could significantly decrease production of reactive oxygen species (ROS) and nitrogen oxide (NO) in the head kidney MO/Mφs from large yellow croaker. Furthermore, rLcIL-4/13A and rLcIL-4/13B obviously down-regulated expression of pro-inflammatory cytokine (IL-1ß and TNF-α) and inducible NO synthase (iNOS) genes in MO/Mφs, while they increased mRNA expression of anti-inflammatory cytokines (TGF-ß and VEGF) and arginase-2. Additionally, the phagocytic activity of MO/Mφs was also inhibited by rLcIL-4/13A or rLcIL-4/13B. All these results therefore indicated that both LcIL-4/13A and LcIL-4/13B, although exhibiting a lower degree of sequence identity of 15.6% and differential expression pattern, have the similar roles in promoting alternative activation of head kidney MO/Mφs.

Genome sequencing of the perciform fish Larimichthys crocea provides insights into molecular and genetic mechanisms of stress adaptation.

The large yellow croaker Larimichthys crocea (L. crocea) is one of the most economically important marine fish in China and East Asian countries. It also exhibits peculiar behavioral and physiological characteristics, especially sensitive to various environmental stresses, such as hypoxia and air exposure. These traits may render L. crocea a good model for investigating the response mechanisms to environmental stress. To understand the molecular and genetic mechanisms underlying the adaptation and response of L. crocea to environmental stress, we sequenced and assembled the genome of L. crocea using a bacterial artificial chromosome and whole-genome shotgun hierarchical strategy. The final genome assembly was 679 Mb, with a contig N50 of 63.11 kb and a scaffold N50 of 1.03 Mb, containing 25,401 protein-coding genes. Gene families underlying adaptive behaviours, such as vision-related crystallins, olfactory receptors, and auditory sense-related genes, were significantly expanded in the genome of L. crocea relative to those of other vertebrates. Transcriptome analyses of the hypoxia-exposed L. crocea brain revealed new aspects of neuro-endocrine-immune/metabolism regulatory networks that may help the fish to avoid cerebral inflammatory injury and maintain energy balance under hypoxia. Proteomics data demonstrate that skin mucus of the air-exposed L. crocea had a complex composition, with an unexpectedly high number of proteins (3,209), suggesting its multiple protective mechanisms involved in antioxidant functions, oxygen transport, immune defence, and osmotic and ionic regulation. Our results reveal the molecular and genetic basis of fish adaptation and response to hypoxia and air exposure. The data generated by this study will provide valuable resources for the genetic improvement of stress resistance and yield potential in L. crocea.

Molecular characterization and expression analysis during embryo development of CD4-1 homologue in large yellow croaker Larimichthys crocea.

CD4+ helper T (Th) cells are a master component of the adaptive immune response. CD4 is one of the most effective surface markers for identifying Th cells. In the present study, we cloned and characterized a CD4-1 homologue, LycCD4-1, from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCD4-1 is 1695 bp long, encoding a protein of 462 amino acids. The deduced LycCD4-1 protein has a typical domain architecture as found in mammalian CD4 molecules, including a signal peptide, four extracellular immunoglobulin-like (Ig-like) domains, a transmembrane region, and a CXC signaling motif in the cytoplasmic tail. Four N-glycosylation sites and 10 cysteine residues were also found in LycCD4-1, which may be essential for its tertiary structure and succeeding function. Homology comparison showed that LycCD4-1 has 27.9-58.4% identity to other teleost fish CD4-1 molecules, and 16.4-20% identity to those of higher vertebrates. Genomic analysis revealed that the LycCD4-1 gene consisted of nine exons and eight introns and exhibited a similar exon-intron organization to other species CD4 genes except for a different intron length. Phylogenetic analysis showed that LycCD4-1 form a cluster with CD4-1 molecules in other fish species. The LycCD4-1 was constitutively expressed in all tissues tested, with a higher expression in gills and spleen. LycCD4-1 mRNA expression in the spleen and head kidney tissue was increased by poly (I:C) at 48 h, whereas its expression levels were somewhat down-regulated at 6 h and 72 h after bacterial vaccine induction in spleen. Unexpectedly, LycCD4-1 mRNA could be detected in each stage of early embryo development since fertilized eggs, with a higher level before mid-gastrula and the highest level in high blastocysts. These results will be helpful for better understanding molecular characteristics of CD4-1 and tracing origin of CD4-1+ cell precursors in fish.

Functional activities of interferon gamma in large yellow croaker Larimichthys crocea.

Interferon gamma (IFN-γ) is a T helper cell type 1 (Th1) cytokine that plays important roles in almost all phases of immune and inflammatory responses. Although IFN-γ gene in large yellow croaker Larimichthys crocea has been reported, little is known about its bioactivity. In this study, large yellow croaker IFN-γ (LycIFN-γ) gene was found to be constitutively expressed in all tissues tested, with the highest levels in blood and heart. Based on stimulation with polyinosinic-polycytidylic acid [poly (I:C)] or inactivated trivalent bacterial vaccine, LycIFN-γ mRNA was significantly increased in spleen and head kidney tissues. LycIFN-γ transcripts were also detected in head kidney granulocytes, primary head kidney macrophages (PKM), head kidney leukocytes, and large yellow croaker head kidney cell line (LYCK), and were significantly up-regulated by poly(I:C) or lipopolysaccharide (LPS) in head kidney leukocytes. Recombinant LycIFN-γ protein (rLycIFN-γ) produced in Escherichia coli could enhance respiratory burst responses in PKM. Furthermore, rLycIFN-γ not only induced the expression of iNOS gene and release of NO, but also up-regulated the expression of proinflammatory cytokines TNF-α and IL-1ß in PKM. These findings therefore indicated that LycIFN-γ has a role in mediating inflammatory response. In addition, rLycIFN-γ could significantly up-regulate expression of IFN-γ receptor CRFB13, signal transduction factor STAT1, transcription factors IRF1 and T-bet, and Th1-related cytokines IFN-γ and IL-2 in head kidney leukocytes, suggesting that LycIFN-γ may have the potential to promote Th1 immune response in large yellow croaker. Taken together, our results show that LycIFN-γ may be involved in inflammatory response and promote Th1 immune response as its mammalian counterpart.

Induction of type I interferons in response to bacterial stimuli in large yellow croaker Larimichthys crocea.

In addition to the crucial roles in coordinating antiviral immune responses, type I interferons (IFNs) also play a role in the host immunity against bacterial pathogens. Our previous study identified two type I IFNs from large yellow croaker Larimichthys croaea(Lc), LcIFNd and LcIFNh, and showed their strong induction by poly(I:C) and antiviral activities. In the present study, both LcIFNd and LcIFNh were found to be rapidly induced in head kidney and spleen by mixed bacteria of Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas hydrophila. In the head kidney primary cells (HKCs), expression of these two LcIFN genes was increased by peptidoglycan (PGN) from Bacillus subtilis and lipopolysaccharide (LPS) from Escherichia coli. Consistently, Lc IFN-regulatory factor (LcIRF) 3 and LcIRF7, two key transcription factors of type I IFN expression, were also induced by these three bacteria, PGN, and LPS. These observations strongly suggested that large yellow croaker type I IFNs are involved in the immune response against bacterial infection. Luciferase assays showed that promoters of both LcIFNd and LcIFNh were activated by PGN, LPS, and genomic DNA of A. hydrophila, and A. hydrophila DNA was more potent than PGN and LPS in activating LcIFNd and LcIFNh promoters. Furthermore, the induction of LcIFNd promoter by these bacterial stimuli was further enhanced by the overexpression of LcIRF7 or LcIRF7 along with LcIRF3, while that of LcIFNh promoter was increased following the overexpression of LcIRF3 alone, suggesting that the induction of these two large yellow croaker IFNs by bacterial stimuli may be regulated via distinct manners. These results therefore revealed novel aspects of the functional regulation of teleost type I IFNs.

Identification of a fish specific chemokine CXCL_F2 in large yellow croaker (Larimichthys crocea) reveals its primitive chemotactic function.

Chemokines are a superfamily of cytokines regulating immune cell migration under both inflammatory and normal physiological conditions. Currently, a number of fish specific CXC chemokines, named as CXCL_F1-5, have been identified in several species. However, understanding of their functional characteristics is still limited. In this study, we identified a fish specific chemokine CXCL_F2 (LycCXCL_F2) from large yellow croaker (Larimichthys crocea). The open reading frame (ORF) of LycCXCL_F2 is 348 nucleotides long, encoding a protein of 115 amino acids (aa). The deduced LycCXCL_F2 protein contains a 20-aa signal peptide and a 95-aa mature polypeptide. Phylogenetic analysis showed that LycCXCL_F2 fell into a major clade formed by CXCL_F2 sequences and was separated from CXCL_F1 and CXCL_F3-5 subgroups. LycCXCL_F2 mRNA transcript was constitutively expressed in various tissues, with the highest levels in spleen and head kidney. After stimulation with inactivated trivalent bacterial vaccines, LycCXCL_F2 mRNA transcription was significantly increased in both spleen and head kidney. Moreover, recombinant LycCXCL_F2 protein exhibited obvious chemotaxis to monocytes, lymphocytes and eosnophils of PBLs isolated from large yellow croaker, but could not induce the respiratory burst of macrophages. These results indicate that this fish specific CXC chemokine LycCXCL_F2 possesses primitive chemotactic activity and may play a role in immune response in large yellow croaker.

A cystatin F homologue from large yellow croaker (Larimichthys crocea) inhibits activity of multiple cysteine proteinases and Ii chain processing in vitro.

Cystatin F, a member of the family II cystatins, plays important roles in immune response-related processes through inhibiting specific enzyme targets. In this study, a cystatin F homologue, LycCysF, was identified and characterized from large yellow croaker (Larimichthys crocea). The deduced LycCysF protein exhibits a typical structural feature of type II cystatins, including three evolutionally conserved motifs, Gly(35), QVVRG(79-83) and PW(130-131). Tissue expression analysis showed that LycCysF mRNA was expressed in all tissues examined, albeit at different levels. Recombinant LycCysF (rLycCysF) produced in Pichia pastoris could inhibit the activity of multiple cysteine proteases, including papain, legumain and recombinant large yellow croaker cathepsin B, L and S. Moreover, rLycCysF could inhibit the Ii chain processing by recombinant cathepsin S in vitro. These data suggest that LycCysF may participate in regulation of cathepsins and MHC-II associated Ii chain processing. In addition, mammalian cystatin F is produced as an inactive dimer, becoming activated by proteolysis in the endo/lysosome of immune cells and then exerts its function of regulating downstream proteases activity. However, the N-terminal extension and two additional cysteine residues responsible for dimer formation are absent in LycCysF and cystatin F from other fish species, reptiles and Aves, indicating that these proteins can not form dimer and may regulate the proteases activity via an alternate pathway distinct from mammalian cystatin F. To our knowledge, this is the first report on molecular characteristics of a teleost cystatin F and its role in Ii chain processing.