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.

Identification and characterization of two novel antimicrobial peptides from Japanese sea bass (Lateolabrax japonicus) with antimicrobial activity and MO/MФ activation capability.

Piscidins participate in the innate immune response of fish, which aims to eliminate recognized foreign microbes and restore the homeostasis of immune system. We characterized two piscidin-like antimicrobial peptides (LjPL-3 and LjPL-2) isolated from Japanese sea bass (Lateolabrax japonicus). LjPL-3 and LjPL-2 showed different expression patterns in tissues. After Vibrio harveyi infection, the mRNA expression of LjPL-3 and LjPL-2 was upregulated in the liver, spleen, head kidney, and trunk kidney. The synthetic mature peptides LjPL-3 and LjPL-2 exhibited different antimicrobial spectra. Furthermore, LjPL-3 and LjPL-2 treatments decreased inflammatory cytokine production while promoting chemotaxis and phagocytosis in monocytes/macrophages (MO/MФ). LjPL-2, but not LjPL-3, displayed bacterial killing capability in MO/MФ. LjPL-3 and LjPL-2 administration increased Japanese sea bass survival after V. harveyi challenge, which was accompanied by a decline in bacterial burden. These data suggested that LjPL-3 and LjPL-2 participate in immune response through direct bacterial killing and MO/MФ activation.

Meteorin links the bone marrow hypoxic state to hematopoietic stem/progenitor cell mobilization.

Hematopoietic stem/progenitor cells (HSPCs) are supported and regulated by niche cells in the bone marrow with an important characterization of physiological hypoxia. However, how hypoxia regulates HSPCs is still unclear. Here, we find that meteorin (Metrn) from hypoxic macrophages restrains HSPC mobilization. Hypoxia-induced factor 1α and Yin Yang 1 induce the high expression of Metrn in macrophages, and macrophage-specific Metrn knockout increases HSPC mobilization through modulating HSPC proliferation and migration. Mechanistically, Metrn interacts with its receptor 5-hydroxytryptamine receptor 2b (Htr2b) to regulate the reactive oxygen species levels in HSPCs through targeting phospholipase C signaling. The reactive oxygen species levels are reduced in HSPCs of macrophage-specific Metrn knockout mice with activated phospholipase C signaling. Targeting the Metrn/Htr2b axis could therefore be a potential strategy to improve HSPC mobilization for stem cell-based therapy.

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.

Two paralogs of CXCR4 in the Japanese sea bass (Lateolabrax japonica) are involved in the immune response of B lymphocytes.

CXC chemokine receptor 4 (CXCR4), a member of the G-protein-coupled receptor family, plays an important role in host immune responses. Within the teleost lineage, there are two paralogs of CXCR4; however, the role of CXCR4 in teleost B cells is poorly understood. In this study, we determined the cDNA sequences of the two CXCR4 paralogs from the Japanese sea bass (Lateolabrax japonica; LjCXCR4a and LjCXCR4b). Sequence and phylogenetic tree analyses revealed that LjCXCR4a and LjCXCR4b are most closely related to CXCR4a and CXCR4b, respectively, in the large yellow croaker (Larimichthys crocea). CXCR4 transcripts were mainly expressed in the gills, and their expression in different tissues was altered upon infection with Vibrio harveyi. LjCXCR4a and LjCXCR4b protein levels were upregulated in infected B cells. Knockdown of LjCXCR4a and LjCXCR4b in B cells by RNA interference, the phagocytic activity of B cells was not affected. Furthermore, knockdown of LjCXCR4a, not of LjCXCR4b, was observed to inhibit LjIgM expression in lipopolysaccharide-stimulated B cells. In addition, knockdown of LjCXCR4a, not of LjCXCR4b, was found to reduce reactive oxygen species levels in B cells. Our results indicate that LjCXCR4a and LjCXCR4b modulate the immune response of Japanese sea bass B cells against bacterial infection, albeit via different pathways.

Matrix metalloproteinase-25 from Japanese sea bass (Lateolabrax japonicus) is involved in pro-inflammatory responses.

Matrix metalloproteinases (MMPs) are highly expressed in leukocytes and macrophages, which play a role in the innate immune response. Here, the cDNA sequence of MMP25 from Japanese sea bass (Lateolabrax japonicus) (LjMMP25) was identified. Phylogenetic analysis revealed that LjMMP25 was most closely related to large yellow croaker MMP25. Multiple sequence alignment of LjMMP25 with MMP25 sequences from other teleosts revealed that regions of known functional importance were highly conserved. Expression analysis revealed that LjMMP25 was highly expressed in the head kidney and widely expressed in other tissues including gill, spleen, and liver. LjMMP25 was found to regulate inflammatory cytokine production and promote phagocytosis and bacterial killing in monocytes/macrophages (MO/MФ). Furthermore, LjMMP25 regulated the inflammatory response by modulating NF-κB signaling. These findings reveal new information about the role of LjMMP25 in regulating pro-inflammatory responses in this species.

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.

Characterization of large yellow croaker (Larimichthys crocea) osteoprotegerin and its role in the innate immune response against to Vibrio alginolyticus.

Osteoprotegerin (OPG) is a member of the tumor necrosis factor receptor superfamily, contributing to inflammation, apoptosis, and differentiation. However, the function of OPG in the host immune system of teleosts remains unclear. Here, we cloned the cDNA of the LcOPG gene from large yellow croaker. LcOPG mRNA was expressed in all analyzed tissues and was upregulated by Vibrio alginolyticus infection in immune tissues and monocytes/macrophages (MO/MФ). Subsequently, the LcOPG protein was expressed and purified using a prokaryotic expression system. Recombinant LcOPG protein (rLcOPG) treatment suppressed V. alginolyticus-induced pro-inflammatory cytokine and enhanced V. alginolyticus-induced anti-inflammatory cytokine mRNA expression. Furthermore, rLcOPG decreased V. alginolyticus-induced MO/MФ apoptosis. Therefore, the results indicate that LcOPG might play a role in the immune response of V. alginolyticus-infected large yellow croaker.

Pharmacological inhibition of KDM5A for cancer treatment.

Lysine-specific demethylase 5A (KDM5A, also named RBP2 or JARID1A) is a demethylase that can remove methyl groups from histones H3K4me1/2/3. It is aberrantly expressed in many cancers, where it impedes differentiation and contributes to cancer cell proliferation, cell metastasis and invasiveness, drug resistance, and is associated with poor prognosis. Pharmacological inhibition of KDM5A has been reported to significantly attenuate tumor progression in vitro and in vivo in a range of solid tumors and acute myeloid leukemia. This review will present the structural aspects of KDM5A, its role in carcinogenesis, a comparison of currently available approaches for screening KDM5A inhibitors, a classification of KDM5A inhibitors, and its potential as a drug target in cancer therapy.

Hypoxia-inducible factor-1α involved in macrophage regulation in ayu (Plecoglossus altivelis) under hypoxia.

Hypoxia-inducible factor-1α (HIF-1α) plays a critical role in immune and inflammatory responses and is important in controlling a variety of processes in monocytes and macrophages. However, the role of HIF-1α in the teleost immune system remains less known. In this study, we cloned the cDNA sequence of HIF-1α from the ayu (Plecoglossus altivelis, PaHIF-1α). Sequence and phylogenetic tree analysis showed that PaHIF-1α clustered within the fish HIF-1α tree and was closely related to that of Northern pike (Esox lucius). PaHIF-1α was expressed in all tested tissues and expression increased in liver, head kidney, and body kidney upon Vibrio anguillarum infection. PaHIF-1α was found to regulate the expression of cytokines in ayu monocytes/macrophages (MO/MФ). PaHIF-1α mediated hypoxia-induced enhancement of MO/MФ phagocytic and bactericidal activities to enhance host defenses. Compared with the control, intermittent hypoxia further increased the expression of PaHIF-1α mRNA, improved the survival rate, and reduced the bacterial load of V. anguillarum-infected ayu. Therefore, PaHIF-1α may play a predominant role in the modulation of ayu MO/MФ function.

Two transcription factors PU.1a and PU.1b have different functions in the immune system of teleost ayu.

Transcription factor PU.1 is a regulator of macrophage function, however, the specific function of PU.1 in teleost monocytes/macrophages (MO/MФ) remains unknown. We determined the cDNA sequence of two PU.1 genes from ayu (Plecoglossus altivelis; PaPU.1a and PaPU.1b). Sequence comparisons showed that PaPU.1 were most closely related to the PU.1 of rainbow smelt (Osmerus mordax). The PU.1 transcripts were mainly expressed in the spleen, and their expression was altered in various tissues upon infection with Vibrio anguillarum. PaPU.1a and PaPU.1b proteins were upregulated in MO/MФ, after infection. RNA interference was employed to knockdown PaPU.1a and PaPU.1b to investigate their function in MO/MФ. The expression of inflammatory cytokines was regulated by PaPU.1a, but not PaPU.1b, in ayu MO/MФ upon V. anguillarum infection. Both PaPU.1a and PaPU.1b knockdown lowered the phagocytic activity of MO/MФ. Furthermore, PaPU.1b knockdown attenuated MO/MФ bacterial killing capability. Our results indicate that two PaPU.1 genes differentially modulate the immune response in ayu MO/MФ against bacterial infection.

The emerging role of KDM5A in human cancer.

Histone methylation is a key posttranslational modification of chromatin, and its dysregulation affects a wide array of nuclear activities including the maintenance of genome integrity, transcriptional regulation, and epigenetic inheritance. Variations in the pattern of histone methylation influence both physiological and pathological events. Lysine-specific demethylase 5A (KDM5A, also known as JARID1A or RBP2) is a KDM5 Jumonji histone demethylase subfamily member that erases di- and tri-methyl groups from lysine 4 of histone H3. Emerging studies indicate that KDM5A is responsible for driving multiple human diseases, particularly cancers. In this review, we summarize the roles of KDM5A in human cancers, survey the field of KDM5A inhibitors including their anticancer activity and modes of action, and the current challenges and potential opportunities of this field.

Characterization of ayu (Plecoglossus altivelis) urocortin: The function of an endocrine factor in monocyte/macrophage regulation.

Urocortin (UCN) is a hormone in the hypothalamic-pituitary-adrenal axis that is expressed in various immune cells. However, the function of teleost UCN in the immune system remains unclear. In this study, we cloned the cDNA sequence of UCN from ayu Plecoglossus altivelis (PaUCN). Sequence and phylogenetic tree analyses showed that PaUCN clustered within the fish UCN 1 group and was most related to the rainbow trout (Oncorhynchus mykiss) UCN. PaUCN was expressed in all tested tissues and its expression increased in the liver, spleen, head kidney, and gill upon Vibrio anguillarum infection. Mature PaUCN protein (mPaUCN) treatment affected the phagocytosis and bacterial killing of monocytes/macrophages (MO/MФ). mPaUCN reduced pro-inflammatory cytokine expression in MO/MФ, which was partially mediated via interaction with ayu interleukin-6. mPaUCN reduced bacterial load and increased the survival of V. anguillarum-infected ayu. Overall, UCN as an endocrine factor regulates the immune response of ayu after infection by activating MO/MФ, thus contributing to enhance fish survival.

Identification and characterization of a tumor necrosis factor receptor like protein encoded by Cyprinid Herpesvirus 2.

Virus-encoded tumor necrosis factor receptors (vTNFRs) facilitate viral escape from the host immune response during viral propagation. Cyprinid Herpesvirus-2 (CyHV-2) is a double-stranded DNA virus of alloherpesviridae family that causes great economic losses in the aquaculture industry. The present study identified and characterized a novel TNFR homolog termed ORF4 in CyHV-2. ORF4 was identified as a secreted protein and a homolog of herpesvirus entry mediator (HVEM). ORF4 localized to the cytoplasm in infected GiCF cells. ORF4 overexpression enhanced viral propagation, while downregulation of ORF4 via siRNA decreased viral propagation. ORF4 overexpression promoted GiCF proliferation, and its downregulation suppressed CyHV-2-induced apoptosis. GST-pulldown and LC-MS/MS assays identified 44 conditional binding proteins that interact with ORF4 protein, while the GST pulldown test did not support the idea that ORF4 interact with histone H3.3. Taken together, our results contribute to our understanding of the vTNFR function in alloherpesviridae pathogenesis and host immune regulation.

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.

Beta-adrenergic receptor stimulation influences the function of monocytes/macrophages in ayu (Plecoglossus altivelis).

Adrenergic receptors (ARs) are members of the G-protein-coupled receptor superfamily that can be categorized into αARs and ßARs. The specific function of ARs in teleost monocytes/macrophages (MO/MФ) remains unknown. We determined the cDNA sequence of ARs from ayu (Plecoglossus altivelis; PaαAR and PaßAR). Sequence comparisons showed that PaαAR was most closely related to the αAR of the Japanese flounder and Nile tilapia, while PaßAR was most closely related to the ßAR of Atlantic salmon. The AR transcripts were mainly expressed in the spleen, and their expression was altered in various tissues upon infection with Vibrio anguillarum. PaαAR and PaßAR proteins were upregulated in MO/MФ after infection, and PaßAR knockdown resulted in a pro-inflammatory status in ayu MO/MФ upon V. anguillarum infection and lowered the phagocytic activity of MO/MФ. Our results indicate that PaßAR plays the role of an anti-inflammatory mediator in the immune response of ayu 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.

Specific function and modulation of teleost monocytes/macrophages: polarization and phagocytosis.

Macrophages exist in most tissues and play a variety of functions in vertebrates. Teleost fish species are found in most aquatic environments throughout the world and are quite diverse for a group of vertebrate animals. Due to whole genome duplication and environmental adaptation, teleost monocytes/macrophages possess a variety of different functions and modulations compared with those of mammals. A deeper understanding of teleost monocytes/macrophages in the immune system will not only help develop teleost-specific methods of disease prevention but will also help improve our understanding of the various immune mechanisms in mammals. In this review, we summarize the differences in polarization and phagocytosis of teleost and mammalian macrophages to improve our understanding of the various immune mechanisms in vertebrates.

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.

A Novel Lipopolysaccharide Recognition Mechanism Mediated by Internalization in Teleost Macrophages.

Macrophages in teleosts are less sensitive to lipopolysaccharide (LPS) compared to mammals. The functional equivalent of the mammalian LPS surface receptor in teleost macrophages for the pro-inflammatory response is either non-existent or replaced by negative regulation. LPS signaling in teleost macrophages remains unclear. Here, we found a scavenger receptor class B 2a (PaSRB2a) that played a crucial role in LPS signaling in teleost macrophages. The internalization of LPS and subsequent pro-inflammatory responses in macrophages were mediated by PaSRB2a, which is a novel isoform of the mammalian SRB2 gene. LPS internalization by PaSRB2a is dependent on its C-terminal intracellular domain. Following LPS internalization, it interacts with the ayu intracellular receptors nucleotide-binding oligomerization domain protein 1 (PaNOD1) and PaNOD2. Moreover, LPS pre-stimulation with sub-threshold concentrations reduced the effect of secondary LPS treatment on pro-inflammatory responses that were mediated by PaSRB2a. The pro-inflammatory responses in LPS-treated ayu were down-regulated upon PaSRB2a knockdown by lentivirus siRNA delivery. In grass carp and spotted green pufferfish, SRB2a also mediated LPS internalization and pro-inflammatory responses. Our work identifies a novel LPS signaling pathway in teleosts that differs from those in mammals, and contributes to our understanding of the evolution of pathogen recognition in vertebrates.