Molecular characterization, expression and functional analysis of large yellow croaker (Larimichthys crocea) peroxisome proliferator-activated receptor gamma.

The peroxisome proliferator-activated receptor gamma (PPARγ) are nuclear receptors with distinct roles in energy metabolism and immunity. Although extensively studied in mammals, immunomodulatory roles of this molecule in teleost fish remain to be investigated. In this study, large yellow croaker (Larimichthys crocea) PPARγ (LcPPARγ) sequence was cloned, which encodes a polypeptide of 541 amino acids that include signature domains belonging to the nuclear receptor superfamily. Phylogenetically, LcPPARγ was most closely related to PPARγ derived from European sea bass (Dicentrarchus labrax). Quantitative analysis shown a ubiquitous expression of this molecule, with highest expression level detected in the intestine. The expression of LcPPARγ was decreased in the intestine, muscle, body kidney, spleen and head kidney-derived monocytes/macrophages (MO/MФs) over the course of Vibrio alginolyticus (V. alginolyticus) infection. In contrast, an up-regulation of LcPPARγ was observed in head kidney-derived MO/MФs following docosahexaenoic acid (DHA) treatment. This increase in LcPPARγ leads to an up-regulation of LcCD11b and LcCD18 and an enhancement of complement-mediated phagocytosis. Furthermore, cytokine secretions of V. alginolyticus-stimulated MO/MФs were modulated following LcPPARγ activations that up-regulated the expression of LcIL-10, while decreased the expression of LcIL-1ß, LcTNF-α and LcTGF-ß1. Overall, our results indicated that LcPPARγ plays a role in regulating functions of MO/MФs and likely contribute to MO/MФs polarization.

CXCR4s in Teleosts: Two Paralogous Chemokine Receptors and Their Roles in Hematopoietic Stem/Progenitor Cell Homeostasis.

Hematopoietic stem/progenitor cells (HSPCs) generate the entire repertoire of immune cells in vertebrates and play a crucial role during infection. Although two copies of CXC motif chemokine receptor 4 (CXCR4) genes are generally identified in teleosts, the function of teleost CXCR4 genes in HSPCs is less known. In this study, we identified two CXCR4 genes from a teleost, ayu (Plecoglossus altivelis), named PaCXCR4a and PaCXCR4b. PaCXCR4b was constitutively expressed in ayu HSPCs, whereas PaCXCR4a was induced by LPS treatment. The stromal-derived factor-1-binding activity of CXCR4b was significantly higher than that of CXCR4a, whereas the LPS-binding activity of CXCR4a was significantly higher than that of CXCR4b in the teleosts ayu, large yellow croaker (Larimichthys crocea), and tiger puffer (Takifugu rubripes). CXCR4a+ HSPCs were mobilized into blood by LPS, whereas CXCR4b+ HSPCs were mobilized by leukocyte cell-derived chemotaxin-2. PaSDF-1 and PaCXCR4b, but not PaCXCR4a, inhibited HSPC proliferation by regulating reactive oxygen species levels. Compared with PaCXCR4b+ HSPCs, PaCXCR4a+ HSPCs preferentially differentiated into myeloid cells in ayu by maintaining high stem cell leukemia expression. These data suggest that the two copies of CXCR4s achieve a division of labor in the regulation of teleost HSPC homeostasis, supporting the concept that subfunctionalization after gene duplication in teleosts may stabilize the immune system.

Two ACTH analogs exert differential effects on monocytes/macrophages function regulation in ayu (Plecoglossus altivelis).

Adrenocorticotropic hormone (ACTH), a bioactive peptide of the family of melanocortins, is generated from pro-opiomelanocortin (POMC). So far, the research on the specific functions of ACTH in the immune system of teleosts is limited. We determined two complementary DNA (cDNA) sequences of POMC in ayu (Plecoglossus altivelis), termed PaPOMC-A and PaPOMC-B. PaPOMCs transcripts occurred in all examined tissues, and their expression in immune tissues changed following experimental infection with Vibrio anguillarum. PaACTH-B, but not PaACTH-A, suppressed the phagocytosis of monocytes/macrophages (MO/MФ). Two isoforms of PaACTH increased the bactericidal capacity of MO/MФ. PaACTH-A increased anti-inflammatory cytokine expression, while PaACTH-B decreased pro-inflammatory cytokine expression in MO/MФ. Compared with PaACTH-B treatment, the PaACTH-A treatment improved survival rate and reduced the bacterial load in V. anguillarum-infected ayu through interleukin (IL)-10. Our results indicate that the two PaACTH isoforms exert different effects in the host defense against bacterial infection.

The interleukin-6 regulates the function of monocytes/macrophages (MO/MФ) via the interleukin-6 receptor ß in ayu (Plecoglossus altivelis).

Interleukin-6 (IL-6) is one of the most pleiotropic cytokines because of its wide range of effects on cells of the immune and non-immune systems in the body. However, the role of IL-6 in fish monocytes/macrophages (MO/MФ) is poorly understood. In this study, we cloned the cDNA sequence of the IL-6 gene from ayu (Plecoglossus altivelis) and demonstrated using a tissue distribution assay that ayu interleukin-6 (PaIL-6) mRNA is expressed in all tested tissues. Changes in expression were observed in immune tissues as well as in MO/MФ after a Vibrio anguillarum infection; subsequently, PaIL-6 was expressed and purified to prepare anti-PaIL-6 antibodies. Recombinant PaIL-6 protein (rPaIL-6) treatment enhanced pro-inflammatory cytokine expression. Ayu interleukin-6 receptor ß (PaIL-6Rß) knockdown resulted in decreased pro-inflammatory cytokine expression in MO/MФ treated with rPaIL-6, whereas no significant changes were observed after ayu interleukin-6 receptor α (PaIL-6Rα) knockdown in MO/MФ. PaIL-6 and PaIL-6Rß knockdown in MO/MФ inhibited the phosphorylation of signal transducer and activator of transcription 1. Moreover, PaIL-6Rß knockdown inhibited the phagocytic and bactericidal ability of ayu MO/MФ treated with rPaIL-6. These data indicate that PaIL-6 may be able to regulate the function of ayu MO/MФ.

Glucocorticoid receptor in ayu (Plecoglossus altivelis): Genomic and non-genomic effects on monocytes/macrophages function.

The glucocorticoid receptor (GR) is an important feedback regulator of the hypothalamic-pituitary-interrenal (HPI) axis. However, there are a limited number of studies focused on host-pathogen interactions in which an association between GR and immune response has been evaluated in monocytes/macrophages (MO/MФ) after being challenged with highly pathogenic bacteria. Here, we cloned the cDNA sequence of the glucocorticoid receptor (PaGR) gene from ayu fish. The PaGR transcript was expressed in all tissues, and changes in expression were observed in immune tissues and MO/MФ after live Vibrio anguillarum infection. Subsequently, PaGR was expressed and purified to prepare anti-PaGR antibodies. We analyzed the subcellular localization of PaGR. PaGR was expressed not only in the intracellular space but also in the plasma membrane. PaGR activation decreased the expression of pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokines. However, PaGR activation suppressed the phagocytosis activity of V. anguillarum-infected ayu MO/MФ via a non-genomic pathway. Interestingly, PaGR activation could enhance MO/MФ bacterial killing capability and apoptosis. Therefore, PaGR may modulate the immune response in ayu MO/MФ by genomic and non-genomic pathways.

CXCR3.1 and CXCR3.2 Differentially Contribute to Macrophage Polarization in Teleost Fish.

The study of multiple copies of chemokine receptor genes in various teleosts has long appealed to investigators seeking to understand the evolution of the immune system. The CXCR CXCR3 gene has two isoforms, CXCR3.1 and CXCR3.2, which are both expressed in macrophages. The distinct roles of teleost CXCR3s have not been identified previously. In this article, we found that CXCR3.1 and CXCR3.2 differentially contributed to macrophage polarization in the teleosts: ayu (Plecoglossus altivelis), grass carp (Ctenopharyngodon idella), and spotted green pufferfish (Tetraodon nigroviridis). In ayu macrophages, the P. altivelis CXCR3.1 (PaCXCR3.1) gene was constitutively expressed, whereas the P. altivelis CXCR3.2 (PaCXCR3.2) gene was induced postinfection with Escherichia coli Upon E. coli infection, PaCXCR3.1+ and PaCXCR3.2+ macrophages showed an M1 and an M2 phenotype, respectively. CXCL9-11-like proteins mediated M1 and M2 polarization by interacting with the PaCXCR3.1 and PaCXCR3.2 proteins on macrophages, respectively. The transcription factors P. altivelis STAT1 and P. altivelis STAT3 were activated in PaCXCR3.1+ and PaCXCR3.2+ macrophages, respectively. Furthermore, the prognosis of septic ayu adoptively transferred with PaCXCR3.2+ macrophages was improved. Our data reveal a previously unknown mechanism for macrophage polarization, suggesting that redundant genes may regulate crucial functions in the teleost immune system.

Full-length and a smaller globular fragment of adiponectin have opposite roles in regulating monocyte/macrophage functions in ayu, Plecoglossus altivelis.

Adiponectin (ADP), a regulator of the innate immune system, plays a role in the progression of inflammation and metabolic disorders in mammals. However, the role of ADP in fish is poorly understood. Here, we cloned the cDNA sequence of a ADP homolog (PaADP) gene from ayu. Multiple sequence alignment revealed that PaADP presented typical characteristics of ADPs. Phylogenetic tree analysis showed that PaADP was most closely related to that of rainbow trout. In healthy ayu, the transcripts of PaADP were detected in most of the tested tissues and cells, with the highest level in the adipose tissue. Upon V. anguillarum infection, the mRNA expression of PaADP was significantly up-regulated in the tissues and cells except adipose tissue. Subsequently, the full-length mature PaADP (fPaADP) and the globular domain fragment (gPaADP) were prokaryotically expressed in bacteria and purified, and anti-PaADP antibodies were produced. Western blot analysis revealed that three fragments including fPaADP and gPaADP were existed in ayu serum. The recombinant fPaADP (rfPaADP) had an anti-inflammatory effect on ayu MO/MФ by upregulating anti-inflammatory cytokine expressions, downregulating pro-inflammatory cytokine expressions, inhibiting the phagocytosis and subsequent bacterial killing. In contrast, the recombinant gPaADP (rgPaADP) presented a pro-inflammatory effect on ayu MO/MФ by upregulating pro-inflammatory cytokine expression, downregulating anti-inflammatory cytokine expressions, enhancing the phagocytosis and subsequent bacterial killing. These results suggested that fPaADP and gPaADP have opposite roles in the regulation of MO/MФ functions in ayu.

Molecular characterization of a CC motif chemokine 19-like gene in ayu (Plecoglossus altivelis) and its role in leukocyte trafficking.

The CC motif chemokine 19 (CCL19) functions in acute inflammation by recruiting lymphocytes and other cells. However, CCL19 has only been investigated in few fish species. In this study, we characterized a CCL19-like molecule (PaCCL19l) in ayu (Plecoglossus altivelis), a teleost fish. Sequence analysis revealed that PaCCL19l was most closely related to Atlantic salmon (Salmon salar) CCL19l1, which belonged to the fish CCL19a.1 subcluster. PaCCL19l was constitutively expressed in the tested ayu tissues and peripheral blood mononuclear cells (PBMCs), with the highest transcript level in PBMCs. Upon infection with Vibrio anguillarum, the expressions of PaCCL19l in the head kidney, liver, spleen, PBMCs, and monocytes/macrophages (MO/MΦ) were dramatically up-regulated. Recombinant PaCCL19l (rPaCCL19l) exhibited a significant effect on the chemotaxis of lymphocytes and MO/MΦ in vitro and in vivo. Meanwhile, rPaCCL19l exerted a high chemotaxic activity for lipopolysaccharide (LPS)-stimulated MO/MΦ (M1-type), but not for cyclic adenosine monophosphate (cAMP)-stimulated MO/MΦ (M2-type). When ayu MO/MΦ was treated with rPaCCL19l along with Vibrio anguillarum infection, the mRNA expression of proinflammatory cytokines (IL-1ß, TNFα, IL-6, IL-12b, and IFN-γ) was up-regulated, while that of anti-inflammatory cytokines (IL-10, TGFß, and IL-22) was down-regulated. Ayu MO/MΦ treated with anti-PaCCL19l IgG gave the opposite result. These results implicated that PaCCL19l is involved in the selective chemotaxis of ayu immune cells and promotes the host at a pro-inflammatory state.

Molecular identification and functional analysis of KLF2 in Plecoglossus altivelis (ayu): It's regulatory role in monocyte/macrophage activation.

Monocytes/macrophages (MO/MФ) play an important role in the response to infection in Plecoglossus altivelis (ayu). However, the role of transcription factors in the function of ayu MO/MФ is poorly understood. Here, we cloned the cDNA sequence of the Kruppel-like factor 2 (PaKLF2) gene from ayu. Phylogenetic analysis indicated that PaKLF2 was closest to that of Atlantic salmon (Salmo salar). Real time quantitative PCR (RT-qPCR) revealed that the PaKLF2 mRNA level was highest in the peripheral blood mononuclear cells among all tested tissues. The mRNA expression of PaKLF2 was upregulated in the head kidney, liver, spleen, and brain after Listonella anguillarum infection. Subsequently, PaKLF2 was expressed and purified to prepare anti-PaKLF2 antibodies. After L. anguillarum challenge, the PaKLF2 mRNA and protein levels were significantly upregulated in ayu MO/MФ. Moreover, PaKLF2 knockdown in MO/MФ resulted in the enhancement of cytokine production as well as phagocytotic and bactericidal capability. Therefore, PaKLF2 may modulate the immune response in ayu by suppressing the function of MO/MФ.

The protection effect of LEAP-2 on the mudskipper (Boleophthalmus pectinirostris) against Edwardsiella tarda infection is associated with its immunomodulatory activity on monocytes/macrophages.

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a cationic peptide that plays an important role in the host's innate immune system. However, the mechanism by which LEAP-2 modulates/regulates the host defense against pathogens remains largely unknown. In this study, we identified a cDNA sequence encoding LEAP-2 homolog (BpLEAP-2) in the mudskipper, Boleophthalmus pectinirostris. Sequence analysis revealed that BpLEAP-2 belonged to the fish LEAP-2A cluster and that it was closely related to ayu LEAP-2. BpLEAP-2 mRNA was detected in a wide range of tissues, with the highest level of transcripts found in the liver. Upon infection with Edwardsiella tarda, BpLEAP-2 mRNA expression was significantly increased in the liver, kidney, spleen, and gill, but decreased in the intestine. Chemically synthesized BpLEAP-2 mature peptide did not exhibit antibacterial activity against E. tarda in vitro. Intraperitoneal injection of BpLEAP-2 (1.0 or 10.0 µg/g) resulted in significantly improved survival rate and reduced tissue bacterial load in E. tarda-infected mudskippers. In E. tarda-infected fish, BpLEAP-2 (0.1, 1.0, or 10.0 µg/g) eliminated E. tarda-induced tissue mRNA expression of BpTNF-α and BpIL-1ß. In monocytes/macrophages (MO/MФ), BpLEAP-2 (1.0 or 10.0 µg/ml) induced chemotaxis, enhanced respiratory burst, and inhibited E. tarda-induced mRNA expression of BpTNF-α and BpIL-1ß. At a concentration of 10.0 µg/ml, BpLEAP-2 also significantly enhanced the bacterial killing efficiency of MO/MФ. No significant effect was seen in the phagocytic activity of MO/MФ upon treatment with BpLEAP-2. Our study provides evidence, for the first time, that LEAP-2 exhibited immunomodulatory effects on immune cells, and protected the host from pathogenic infections independent of direct bacterial killing function.

Identification and functional characterization of the CSF1R gene from grass carp Ctenopharyngodon idellus and its use as a marker of monocytes/macrophages.

Colony-stimulating factor 1 receptor (CSF1R) is an important regulator of monocytes/macrophages (MO/MΦ). Although CSF1R gene has been identified and functionally studied in many fish, the precise role of CSF1R in grass carp (Ctenopharyngodon idellus) remains unclear. In this study, we determined the cDNA sequence of CSF1R (CiCSF1R) from a teleost fish, grass carp. Sequence comparison and phylogenetic tree analysis showed that CiCSF1R was most closely related to the CSF1R of zebrafish. The CiCSF1R transcript was mainly expressed in the spleen, head kidney, and head kidney-derived MO/MΦ, and its expression was altered in various tissues upon Aeromonas hydrophila infection. We prepared antibodies for neutralization of CiCSF1R on grass carp MO/MΦ. CiCSF1R neutralization or knockdown led to anti-inflammatory status in MO/MΦ upon A. hydrophila infection. CiCSF1R neutralization or knockdown also decreased the phagocytic activity of MO/MΦ. Flow cytometric analysis showed that more than 85% of grass carp MO/MΦ were CiCSF1R-positive cells. The percentage of CiCSF1R-positive cells in the head kidney of grass carp was above 10%, whereas it was only 5% and 4% in the spleen and liver, respectively. In conclusion, CSF1R is a specific surface marker of grass carp MO/MΦ, and it regulates the functions of MO/MΦ.

Molecular characterization and functional analysis of a novel C-type lectin receptor-like gene from a teleost fish, Plecoglossus altivelis.

C-type lectin-like receptors (CLRs) are important pathogen pattern recognition molecules that recognize carbohydrate structures. However, the functions of these receptors in fish keep less known. In this study, we characterized a novel CLR from a teleost fish, Plecoglossus altivelis (ayu), tentatively named PaCD209L. The cDNA of PaCD209L is 1464 nucleotides (nts) in length, encoding a polypeptide of 281 amino acid residues with a calculated molecular weight of 31.5 kDa. Multiple alignment of the deduced amino acid sequences of PaCD209L and other related fish CLRs revealed that the PaCD209L sequence had typical characteristics of fish CLRs, but without Ca(2+)-binding sites. Sequence comparison and phylogenetic tree analysis showed that PaCD209L shared the highest amino acid identity (44%) with rainbow trout (Oncorhynchus mykiss) CD209 aE PaCD209L transcripts were detected in all of the tissues examined, mainly expressed in the brain and heart. Upon Vibrio anguillarum infection, PaCD209L transcripts were upregulated in all tested tissues and in monocytes/macrophages (MO/MΦ). We prepared recombinant PaCD209L (rPaCD209L) by prokaryotic expression and raised antiserum against PaCD209L. Western blot analysis revealed that native PaCD209L was glycosylated, and its protein expression significantly increased in ayu MO/MΦ upon V. anguillarum infection. In addition, rPaCD209L was able to bind Gram-positive and Gram-negative bacteria in the absence of Ca(2+). After PaCD209L was blocked by anti-PaCD209L IgG, the phagocytosis and bacterial killing activity of MO/MΦ significantly decreased. These results suggest that PaCD209L plays an important role in the regulation of MO/MΦ functions in ayu.

Cathelicidin modulates the function of monocytes/macrophages via the P2X7 receptor in a teleost, Plecoglossus altivelis.

Cathelicidins (CATHs) are a family of endogenous antimicrobial peptides that are capable of both direct bacteria-killing and immunomodulatory effects. P2X7 receptor (P2X7R) is a mediator of CATH in mammalian immune cells. Here, we studied the function and regulation of CATH in head kidney-derived monocytes/macrophages (MO/MФ) from ayu, Plecoglossus altivelis. We investigated the chemotaxis of MO/MФ in response to ayu CATH (PaCATH), and found that PaCATH had a dose-dependent effect on MO/MФ chemotaxis with the optimal concentration of 10.0 µg/ml. The qPCR and Western blot analysis revealed that PaCATH inhibited the expression of ayu P2X7R (PaP2X7R) at both mRNA and protein levels. Knockdown of the PaP2X7R expression in ayu MO/MФ by RNA interference not only significantly inhibited the chemotactic effect of PaCATH on MO/MФ, but also obviously reduced the effect of PaCATH on the phagocytosis, bacteria-killing, respiratory burst, and cytokine expression of ayu MO/MФ. Our study revealed that the immunomodulatory effect of fish CATH is mediated by P2X7R.

A novel lipopolysaccharide-binding protein (LBP) gene from sweetfish Plecoglossus altivelis: molecular characterization and its role in the immune response of monocytes/macrophages.

Lipopolysaccharide-binding protein (LBP) belongs to the lipid transfer/LBP (LT-LBP) family, and plays a crucial role in the recognition of bacterial components that modulate cellular signals in phagocytic cells. Although several LBPs have been identified in teleosts, the effects of LBP homologs on teleost phagocytic cells are still obscure. Here, we report the cloning a novel full-length cDNA sequence of LBP-like protein (paLBP) gene from sweetfish, Plecoglossus altivelis. The paLBP cDNA encoded a 464 aa polypeptide, which was closest to that of rainbow smelt (Osmerus mordax). paLBP mRNA was detected mainly in the spleen, liver, and head kidney and levels dramatically increased in various tissues after Listonella anguillarum infection. In contrast to mammalian studies, paLBP mRNA could also be detected in sweetfish monocytes/macrophages. Recombinant paLBP showed LPS-binding activity and Western blot results revealed a significant increase of paLBP in the supernatant of sweetfish monocytes/macrophages challenged with L. anguillarum. Moreover, paLBP neutralization led to up-regulation of IL-1ß and TNF-α mRNA as well as respiratory burst activity in sweetfish monocytes/macrophages in response to L. anguillarum or LPS challenge. Therefore, these results suggest that paLBP is an inducible acute-phase protein mediating the immune response of sweetfish monocytes/macrophages upon bacterial challenge.

LECT2 improves the outcomes in ayu with Vibrio anguillarum infection via monocytes/macrophages.

Leukocyte cell-derived chemotaxin 2 (LECT2) is reported to be a cytokine involved in the immune response against pathogenic microorganisms in fish. However, its accurate function in whole fish remains unclear. In this study, we provide the first report on the effect of LECT2 on fish defenses against pathogens in vivo. The administration of recombinant LECT2 improved the survival rate of Vibrio anguillarum infected ayu. The bacterial burden of V. anguillarum infected ayu was decreased in LECT2-treated ayu blood, liver, spleen, and kidney compared with saline control. In bacteria-infected ayu, LECT2 treatment altered the mRNA expression of cytokines, including TNFα, IL-1ß, and IL-10, which are all important for the inflammatory response in fish. LECT2 treatment also reduced histological damage in bacteria-infected ayu, and increased peritoneal monocytes/macrophages in both healthy and infected ayu at 12 h post infection. When ayu monocytes/macrophages were depleted by clodronate-liposomes treatment, LECT2 treatment did not increase the survival rate of bacteria-infected fish compared with healthy control fish. Thus our results suggest that LECT2 can modulate host defense in ayu and mediate antibacterial protection against V. anguillarum through monocytes/macrophages.

Molecular characterization and functional analysis of two distinct liver-expressed antimicrobial peptide 2 (LEAP-2) genes in large yellow croaker (Larimichthys crocea).

Liver-expressed antimicrobial peptide 2 (LEAP-2) plays a vital role in the host innate immune system. In the present study, two LEAP-2 genes (LcLEAP-2A and LcLEAP-2C) from large yellow croaker (Larimichthys crocea) were cloned, both of which consist of 3 exons and 2 introns. The LcLEAP-2A transcripts were expressed in a wide range of tissues, with the highest mRNA levels found in the liver and intestine, while LcLEAP-2C transcripts showed obvious lower mRNA levels in all tested tissues compared to LcLEAP-2A. Upon infection by Vibrio alginolyticus, LcLEAP-2A transcripts were significantly up-regulated in liver, trunk kidney, spleen, head kidney, and gill, but down-regulated in intestine. In addition, significant up-regulation of LcLEAP-2C transcripts were also detected in all tissues tested, including intestine. The LcLEAP-2A and LcLEAP-2C mature peptides were chemically synthesized and found to exhibit selective antimicrobial activity in vitro against various species of bacteria. LcLEAP-2C, but not LcLEAP-2A, had antimicrobial activity against V. alginolyticus. Moreover, LcLEAP-2C treatment at low concentrations was evaluated and found to improve survival rate in V. alginolyticus-infected large yellow croaker, resulting in a decrease in bacterial load and expression of inflammatory cytokines. These results suggest that LcLEAP-2 isoforms play an important role in innate immunity by killing bacteria and inhibiting early inflammatory response in large yellow croaker.

Passive protective effect of chicken egg yolk immunoglobulins against experimental Vibrio anguillarum infection in ayu (Plecoglossus altivelis).

Oral administration of chicken egg yolk immunoglobulins (IgY) has attracted much attention as a means for controlling infectious diseases caused by microorganisms. This study evaluated the protective effect of IgY against Vibrio anguillarum infection in ayu, Plecoglossus altivelis. IgY was isolated from egg yolks laid by hens initially immunized with formalin-inactivated V. anguillarum. Lower mortality of ayu was observed in groups treated with anti-V. anguillarum IgY (aVIgY), compared with those treated with saline or with nonspecific IgY (nspIgY). All fish in saline-treated groups died within seven days after bacterial inoculation. The bacterial load in blood, liver, and spleen was significantly lower in fish treated with aVIgY than in fish treated with nspIgY. aVIgY treatment significantly reduced tumor necrosis factor-α (PaTNF-α), interleukin-1ß (PaIL-1ß), transforming growth factor-ß (PaTGF-ß), and leukocyte cell-derived chemotaxin-2 (PaLECT2) transcript levels in the head kidney, spleen, and liver of ayu challenged by V. anguillarum, compared with nspIgY treatment. The phagocytic activity of macrophages for V. anguillarum in the presence of specific IgY was significantly higher than that seen for nonspecific IgY. These results suggest that passive immunization by oral intubation with pathogen-specific IgY may provide a valuable treatment for V. anguillarum infection in ayu.

TFPI from erythroblasts drives heme production in central macrophages promoting erythropoiesis in polycythemia.

Bleeding and thrombosis are known as common complications of polycythemia for a long time. However, the role of coagulation system in erythropoiesis is unclear. Here, we discover that an anticoagulant protein tissue factor pathway inhibitor (TFPI) plays an essential role in erythropoiesis via the control of heme biosynthesis in central macrophages. TFPI levels are elevated in erythroblasts of human erythroblastic islands with JAK2V617F mutation and hypoxia condition. Erythroid lineage-specific knockout TFPI results in impaired erythropoiesis through decreasing ferrochelatase expression and heme biosynthesis in central macrophages. Mechanistically, the TFPI interacts with thrombomodulin to promote the downstream ERK1/2-GATA1 signaling pathway to induce heme biosynthesis in central macrophages. Furthermore, TFPI blockade impairs human erythropoiesis in vitro, and normalizes the erythroid compartment in mice with polycythemia. These results show that erythroblast-derived TFPI plays an important role in the regulation of erythropoiesis and reveal an interplay between erythroblasts and central macrophages.

Molecular characterization of an IL-1ß gene from ayu, Plecoglossus altivelis.

IL-1ß plays a crucial role as a prototypical proinflammatory cytokine in immune responses and has been shown to affect macrophage functions. However, the effects of putative IL-1ß homologs on fish macrophages are still less known. Here, we cloned the full-length cDNA sequence of IL-1ß (aIL-1ß) gene from ayu, Plecoglossus altivelis. Phylogenetic analysis indicated that aIL-1ß was closest to that of Atlantic salmon (Salmo salar). Real-time quantitative PCR (RT-qPCR) revealed that aIL-1ß transcript was mainly expressed in spleen, head kidney and gill, and dramatically increased in various tissues after Listonella anguillarum infection. Subsequently, aIL-1ß was prokaryotic expressed and purified to prepare anti-aIL-1ß antibody. After L. anguillarum challenge, the aIL-1ß mRNA and protein levels were significantly up-regulated in ayu monocytes/macrophages. Moreover, aIL-1ß neutralization did not change phagocytic capability, but reduced bacterial killing capability in ayu head kidney-derived monocytes/macrophages. Therefore, aIL-1ß may play an important role in immune response of ayu, especially, contributing to bacterial killing of monocytes/macrophages.