Primary Head Kidney Macrophages Research Articles

Effects of laminarin on growth performance and resistance against Pseudomonas plecoglossicida of large yellow croaker （Larimichthys crocea）

Laminarin (LAM) is widely used as an immunopotentiator in aquaculture, but its protective mechanism is still unclear. In this study, the effects of LAM on the growth performance and resistance against Pseudomonas plecoglossicida of large yellow croaker were studied in vitro and in vivo. The 42 d-feeding trial in large yellow croaker showed that dietary LAM could obviously promote the fish growth by improving the weight gain rate (WGR), specific growth rate (SGR), and feed conversion rate (FCR). Dietary LAM could also improve the survival rate of large yellow croakers subjected to P. plecoglossicida infection, and 500 mg/kg LAM produced the highest relative percent survival (RPS) of 35.00 %. LAM improved fish antioxidant level by enhancing serum total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity, and reducing malondialdehyde (MDA) content. In addition, LAM also improved fish innate immunity by increasing serum acid phosphatase (ACP) and alkaline phosphatase (AKP) activities and complement 3 (C3) content under P. plecoglossicida infection. What is more, on 9 d post P. plecoglossicida challenge, LAM could significantly decrease the bacteria load in head kidneys, spleens and livers of fish, and the lowest bacterial load was found in 500 mg/kg LAM group. In vitro, LAM exerted a protective role against inactivated P. plecoglossicida-triggered inflammatory injury in primary head kidney macrophages (PKM) of large yellow croaker by recovering cell viability, suppressing NO production, and reversing pro-inflammatory cytokine expression (IL-1β, IL-6, and IL-8). All these findings therefore will provide insights into the protection mechanism of LAM in fish, facilitating its application in prevention and control of fish bacteriosis.

Ferroptosis preceded the onset of oxidative stress under acute ammonia exposure and quercetin relieved ammonia-induced ferroptosis of yellow catfish (Pelteobagrus fulvidraco)

Ammonia is the most common pollutant and stressor in the aquaculture environment and can cause massive fish mortality. Its toxicity in fish is mainly related to the generation of oxidative stress, but the mechanisms are not well understood. Ferroptosis is a unique cell death pathway characterized by iron-dependent lipid peroxidation. However, little information is available regarding the association between oxidative stress and ferroptosis under ammonia stress and mitigation measures in fish. This study was divided into two parts. In the first part of the study, yellow catfish were exposed to three levels of ammonia (0 mg L−1, 5.7 mg L−1, 28.5 mg L−1) for 96 h. Results showed that ammonia poisoning could result in a decrease in survival rates, elevation of blood ammonia levels, ferroptosis activation (iron accumulation, reduced glutathione depletion, upregulation of transferrin receptor 1 and 15B-lipoxygenase, first increased then a decreased expression of glutamate/cystine antiporter system xc– and glutathione peroxidase 4) and oxidative stress (first increased and then decreased expression of superoxide dismutase and catalase). In the second part of the study, primary head-kidney macrophages were used for investigating the underlying mechanism of quercetin in protecting against ammonia-induced ferroptosis in vitro. Results showed that quercetin could decrease the production of reactive oxygen species, increase GSH contents, suppress the expression of TFR1 and LOX-15B, and upregulate the expression levels of FER, system xc– as well as GPX4. The results of this study suggested that oxidative stress induced by ferroptosis might be a prominent mechanism behind ammonia-induced cytotoxicity and quercetin exhibited good protective effects against macrophage ferroptosis under ammonia stress.

Astragalus polysaccharides protect against inactivated Vibrio alginolyticus-induced inflammatory injury in macrophages of large yellow croaker

As an effective immunostimulant, Astragalus polysaccharides (APS) have been widely used in fish aquaculture, however, their action mechanisms remain poorly understood. In the present paper, the inflammatory macrophage model of large yellow croaker (Larimichthys crocea) was constructed by using formalin-inactivated Vibrio alginolyticus. Inactivated V. alginolyticus could cause cellular damage of primary head kidney macrophages (PKM) by decreasing cell activity and inducing reactive oxygen species (ROS) production and cell apoptosis. When PKM were pretreated with APS, the depressed cell activity induced by inactivated V. alginolyticus was significantly improved, and ROS overproduction and cell apoptosis were inhibited. Then the protection mechanism of APS was investigated by transcriptome analysis. After treated with inactivated V. alginolyticus, the expression of immune-related genes (TLR5s, TLR13, Clec4e, IKK, IκB, BCL-3, NF-κB2, REL, IL-1β, and IL-6) and pyroptosis-related genes (caspase-1, NLRP3, and NLRC3) in PKM were significantly up-regulated. However, APS pretreatment reversed the up-regulation of most of the above-mentioned genes, where TLR5s, BCL-3, REL, caspase-1, NLRP12, IL-1β, and IL-6 were significantly down-regulated compared with inactivated V. alginolyticus-treated group. These results suggested that APS could protect large yellow croaker PKM against inactivated V. alginolyticus-induced inflammatory injury, and may exert their protection effects by activating NF-κB and pyroptosis signaling pathways. These findings therefore advance our understanding of the immune regulation mechanism of APS in fish, and facilitate the application of APS in prevention and control of fish bacteriosis.

Molecular characterization, expression analysis and cellular location of IL-4/13 receptors in large yellow croaker (Larimichthys crocea)

Interleukin (IL)-4 and IL-13 are closely related class I cytokines that play key roles in the T helper (Th)-2 immune response via heterodimeric receptors. IL-4 signals via both the type I (IL-4Rα/γc) and type II (IL-4Rα/IL-13Rα1) receptor complexes, while IL-13 signals only via the type II receptor complex. IL-13Rα2 is traditionally considered a “decoy” receptor for IL-13. However, the IL-4/13 system and its response to pathogenic infection are still not fully understood in fish. In this study, we identified four IL-4/13 receptor subunit genes in the large yellow croaker (Larimichthys crocea): LcIL-4Rα1, LcIL-4Rα2, LcIL-13Rα1, and LcIL-13Rα2. Sequence analysis showed that these receptors possessed typical characteristic domains, including a signal peptide, two fibronectin type III (FN III)-like domains, and a transmembrane domain, but their cytoplasmic regions were not well conserved. The mRNA and protein of the four IL-4/13 receptors were constitutively expressed in all examined tissues of large yellow croaker. Their mRNAs were also detected in primary head kidney macrophages (PKMs), primary head kidney granulocytes (PKGs), and primary head kidney lymphocytes (PKLs). Immunofluorescence assay further showed that LcIL-4Rα and LcIL-13Rα1 were expressed on the membrane of IgM + B cells. After stimulation by Vibrio alginolyticus and poly (I:C) (a viral dsRNA mimic), the mRNA levels of LcIL-4/13 receptors were significantly upregulated in the head kidney and spleen. Their mRNA levels were also upregulated in head kidney leukocytes in response to poly (I:C) and lipopolysaccharide (LPS) treatment. Moreover, both recombinant LcIL-4/13A and LcIL-4/13B upregulated LcIL-4Rα1 and LcIL-4Rα2 in primary leukocytes, but only recombinant LcIL-4/13A upregulated LcIL-13Rα1 and LcIL-13Rα2. These results indicated that LcIL-4/13 receptors, containing conserved functional domains, may be involved in the IL-4/13-mediated immune response to pathogenic infections in the large yellow croaker.

ATG12 is involved in the antiviral immune response in large yellow croaker (Larimichthys crocea)

ATG12, a core autophagy protein, forms a conjugate with ATG5 to promote the formation of autophagosome membrane, and plays an important role in antiviral immunity. However, little is known about the function of ATG12 in fish. Here, we cloned the open reading frame (ORF) of large yellow croaker (Larimichthys crocea) ATG12 (LcATG12), which is 354 nucleotides long and encodes a protein of 117 amino acids. The deduced LcATG12 possesses a conserved APG12 domain (residues 31 to 117), and shares 91.45% identities with ATG12 in orange-spotted grouper (Epinephelus coioides). LcATG12 was constitutively expressed in all examined tissues, with the highest level in intestine. Its transcript was also detected in primary head kidney granulocytes (PKG), primary head kidney macrophages (PKM), primary head kidney lymphocytes (PKL), and large yellow croaker head kidney (LYCK) cell line, and was significantly up-regulated by poly(I:C). LcATG12 was regularly distributed in both cytoplasm and nucleus of LYCK and epithelioma papulosum cyprinid (EPC) cells. Overexpression of LcATG12 in EPC cells significantly inhibited the replication of spring viremia of carp virus (SVCV). Further studies reveled that LcATG12 could induce the occurrence of autophagy in LYCK cells. Furthermore, overexpression of LcATG12 in LYCK cells increased the expression levels of large yellow croaker type I interferons (IFNs, IFNc, IFNd, and IFNh), IFN regulatory factors (IRF3 and IRF7), and IFN-stimulated genes (PKR, Mx, and Viperin). All these data indicated that LcATG12 plays a role in the antiviral immunity possibly by inducing both autophagy and type I IFN response in large yellow croaker.

Integrated analysis of mRNA and long non-coding RNA expression profiles reveals the potential roles of lncRNA-mRNA network in carp macrophage immune regulation

Long non-coding RNAs (lncRNAs) have emerged as a hot topic in research as mounting evidence has indicated their transcriptional or post-transcriptional regulatory potential in multiple biological processes. Previous studies have revealed the involvement of lncRNAs in the immunoregulation of mammalian macrophages by changing mRNA expression; however, studies on the lncRNAs in fish macrophages and their potential roles in the immune system remain unknown. Primary macrophages were isolated from the head kidney (HK) of red common carp (Cyprinus carpio) and high-throughput lncRNA-mRNA sequencing was performed using the Illumina HiSeq platform. The results revealed that the most highly expressed mRNAs in primary HK macrophages were mainly involved in immune-related signal pathways. Furthermore, the most enriched immune-related GO term and KEGG pathway of the mRNAs were "immune system development" and "chemokine signaling pathway," respectively. A total of 20,333 lncRNAs, composed of 10,512 known and 9821 novel lncRNAs, were identified, and functional enrichment analysis of the lncRNA-mRNA network indicated that the expressed lncRNAs in primary HK macrophages could be associated with the regulation of multiple immune-related signaling pathways. In addition, the expressions of several selected lncRNAs and their related mRNAs were determined in carp macrophages following a 6-h exposure to lipopolysaccharide (LPS) and Poly(I: C), the results of which confirmed the co-expression regulation of lncRNAs and target mRNAs in the immune response of carp macrophages. These results suggest the correlative of the lncRNA-mRNA network in fish macrophage immune response, which may further affect the cross-talk of various signaling pathways by interaction with other network genes. Here, we provided fundamental data about the transcriptome profiles of primary HK macrophages from red common carp by analysis of the lncRNA-mRNA network, and ultimately suggest the potential roles of lncRNA-mRNA networks in immune regulation in teleost fish.

Molecular characterization and role in virus infection of Beclin-1 in large yellow croaker (Larimichthys crocea)

Beclin-1, the ortholog of yeast autophagy-related gene 6 (Atg6), has a central role in autophagy, which has been linked to diverse biological processes including immunity, development, tumor suppression, and lifespan extension. However, understanding of function of fish Beclin-1 is limited now. In this study, the complete Beclin-1 cDNA of large yellow croaker Larimichthys crocea (LcBeclin-1) was cloned, whose open reading frame (ORF) is 1344 bp long and encodes a protein of 447 amino acids (aa). The deduced LcBeclin-1 possesses a typical Bcl-2 homology domain 3(BH3) and an APG6 domain that contains a central coiled-coil domain (CCD, residues 174 to 231) and a C-terminal evolutionarily conserved domain (ECD, residues 241 to 334). LcBeclin-1 shared a high amino acid identity of 81.66–98.66% with reported Beclin-1 molecules from other vertebrate species. LcBeclin-1 gene was constitutively expressed in all tissues tested, with the highest levels in heart. LcBeclin-1 transcripts were also detected in primary head kidney granulocytes (PKGs), primary head kidney macrophages (PKMs), primary head kidney leukocytes (PKLs), and large yellow croaker head kidney cell line (LYCK), and were significantly upregulated by poly (I:C) in PKMs and LYCK cells. Subcellular localization showed that LcBeclin-1 was evenly distributed in the cytoplasm and nucleus of LYCK cells. Overexpression of LcBeclin-1 significantly increased the replication of SVCV, as evidenced by increased severity of the cytopathic effects, enhanced viral titre, and upregulated transcriptional levels of viral genes. Further studies showed that LcBeclin-1 induced the occurrence of autophagy in LYCK cells. Additionally, LcBeclin-1 also decreased the expression levels of large yellow croaker interferons (IFNs; IFNc, IFNd, and IFNh), interferon regulatory factor 3 (IRF3) and IRF7, IFN-stimulated genes (ISGs; Mx, PKR, and Viperin) in LYCK cells. All these data suggest that LcBeclin-1 promoted the viral replication possibly by inducing autophagy or negatively modulating IFN response, which will help us to further understand the function of fish Beclin-1.

Immunomodulatory and antioxidant effects of Astragalus polysaccharide liposome in large yellow croaker (Larimichthys crocea)

Astragalus polysaccharides (APS) have been widely used as immunopotentiators in aquaculture, however, the best way of their administration remains to be explored. In the present study, APS liposome (APSL) was prepared by film dispersion-ultrasonic method. The optimal conditions of APSL preparation were determined by response surface methodology, with a ratio of 10:1 (w/w) for soybean lecithin to APS and 8:1 (w/w) for soybean lecithin to cholesterol, and an ultrasound time of 15 min, which produced an encapsulation efficiency of 73.88 ± 0.88% of APSL. In vivo feeding experiments in large yellow croaker showed that both APS and APSL could enhance the contents of serum total protein (TP) and albumin (ALB), activities of serum non-specific immune enzymes such as acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM), and phagocytic activity of head kidney macrophages. Meanwhile, they both increased the activities of serum antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and reduced the content of final lipid peroxidation product malondialdehyde (MDA) in serum, thus exhibiting the antioxidant effects. In vitro experiments on primary head kidney macrophages (PKM) showed that both APS and APSL inhibited ROS production, but obviously enhanced NO production and phagocytic activity of PKM. Furthermore, expression levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α), IFN-γ, and iNOS in PKM were significantly up-regulated after APS and APSL treatments, but no expression change of IFN-h was observed. Taken together, our results showed that both APS and APSL could improve several immune parameters and antioxidant ability of large yellow croaker either in vivo or in vitro, and the efficacy of APSL was markedly better than APS. These findings therefore indicated that the immunomodulatory and antioxidant activities of APS could be enhanced after encapsulated with liposome, and APSL may represent a potential drug delivery system of APS for development of immunoenhancers in aquaculture.

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.

Functional characterisation of interleukin 34 in grass carp Ctenopharyngodon idella

Interleukin (IL) 34 plays an important role in regulating macrophage functions and inflammation process. IL-34 homologues have recently been discovered in fish but the functions have not been studied. In this study, an IL-34 homologue was identified in grass carp Ctenopharyngodon idella and its bioactivities were investigated. The grass carp IL-34 was constitutively expressed in tissues, with the highest expression detected in spleen. It could be up-regulated in spleen after infection with F. cloumnare and grass carp reovirus II, and in primary head kidney leucocytes by recombinant IL-4/13B. The recombinant IL-34 produced in bacteria and HEK293T cells showed stimulatory effect on the expression of IL-1β, IL-6 and IL-8 but inhibited expression of IL-10 and TGF-β1 in primary head kidney macrophages. The results demonstrate that IL-34 is a proinflammatory cytokine in grass carp.

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/EBPβ, 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.

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.

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-1β 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.

Characterisation of ZBTB46 and DC-SCRIPT/ZNF366 in rainbow trout, transcription factors potentially involved in dendritic cell maturation and activation in fish

ZBTB46 and DC-SCRIPT/ZNF366 are two zinc finger transcription factors that play important roles in regulating differentiation of dendritic cells in mammals. In this study, the ZBTB46 and DC-SCRIPT/ZNF366 homologues were identified in rainbow trout Oncorhynchus mykiss and their expression analysed in vivo and in vitro. As transcription factors, they are well conserved in sequence, genomic organisation and gene synteny. Their expression was differentially modulated by bacterial and viral PAMPs in the monocyte/macrophage-like cell line RTS-11, in primary head kidney (HK) macrophages, and in HK macrophages cultured with IL-4/13A. In the RTS-11 cells and primary HK macrophages, all the ZBTB46 and DC-SCRIPT/ZNF366 homologues were down-regulated by interferon gamma (type II IFN) but unaffected by IFN2 (type I IFN), administered as recombinant proteins to cell cultures. In fish gills, infection with amoebae (Paramoebae perurans) resulted in reduction of ZBTB46 and DC-SCRIPT/ZNF366 expression in Atlantic salmon Salmo salar, whilst infection with Yersinia ruckeri induced gene expression in rainbow trout.

Identification and expression analysis of two interleukin-23α (p19) isoforms, in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar

Interleukin (IL)-23 is a heterodimeric IL-12 family cytokine composed of a p19 α-chain, linked to a p40 β-chain that is shared with IL-12. IL-23 is distinguished functionally from IL-12 by its ability to induce the production of IL-17, and differentiation of Th17 cells in mammals. Three isoforms of p40 (p40a, p40b and p40c) have been found in some 3R teleosts. Salmonids also possess three p40 isoforms (p40b1, p40b2 and p40c) although p40a is missing, and two copies (paralogues) of p40b are present that have presumably been retained following the 4R duplication in this fish lineage. Teleost p19 has been discovered recently in zebrafish, but to date there is limited information on expression and modulation of this molecule.In this report we have cloned two p19 paralogues (p19a and p19b) in salmonids, suggesting that a salmonid can possess six potential IL-23 isoforms. Whilst Atlantic salmon has two active p19 genes, the rainbow trout p19b gene may have been pseudogenized. The salmonid p19 translations share moderate identities (22.8–29.9%) to zebrafish and mammalian p19 molecules, but their identity was supported by structural features, a conserved 4 exon/3 intron gene organisation, and phylogenetic tree analysis. The active salmonid p19 genes are highly expressed in blood and gonad. Bacterial (Yersinia ruckeri) and viral infection in rainbow trout induces the expression of p19a, suggesting pathogen-specific induction of IL-23 isoforms. Trout p19a expression was also induced by PAMPs (poly IC and peptidoglycan) and the proinflammatory cytokine IL-1β in primary head kidney macrophages. These data may indicate diverse functional roles of trout IL-23 isoforms in regulating the immune response in fish.

Identification of IL-34 in teleost fish: Differential expression of rainbow trout IL-34, MCSF1 and MCSF2, ligands of the MCSF receptor

The mononuclear phagocyte system is composed of monocytes, macrophages and dendritic cells and has crucial roles in inflammation, autoimmunity, infection, cancer, organ transplantation and in maintaining organismal homeostasis. Interleukin-34 (IL-34) and macrophage colony stimulating factor (MCSF), both signalling through the MCSF receptor, regulate the mononuclear phagocyte system. A single IL-34 and MCSF gene are present in tetrapods. Two types of MCSF exist in teleost fish which is resulted from teleost-wide whole genome duplication. In this report, we first identified and sequence analysed six IL-34 genes in five teleost fish, rainbow trout, fugu, Atlantic salmon, catfish and zebrafish. The fish IL-34 molecules had a higher identity within fish group but low identities to IL-34s from birds (27.2–33.8%) and mammals (22.2–31.4%). However, they grouped with tetrapod IL-34 molecules in phylogenetic tree analysis, had a similar 7 exon/6 intron gene organisation, and genes in the IL-34 loci were syntenically conserved. In addition, the regions of the four main helices, along with a critical N-glycosylation site were well conserved. Taken together these data suggest that the teleost IL-34 genes described in this report are orthologues of tetrapod IL-34.Comparative expression study of the three trout MCSFR ligands revealed that IL-34, MCSF1 and MCSF2 are differentially expressed in tissues and cell lines. The expression of MCSF1 and MCSF2 showed great variance in different tissues and cell lines, suggesting a role in the differentiation and maintenance of specific macrophage lineages in specific locations. The relatively high levels of IL-34 expression across different tissues suggests a homeostatic role of IL-34 for the macrophage lineage in fish. One striking observation in the present study was the lack of induction of MCSF1 and MCSF2 expression but the quick induction of IL-34 expression by PAMPs and inflammatory cytokines in cell lines and primary head kidney macrophages in rainbow trout. In a parasitic proliferative kidney disease (PKD) model, the expression of IL-34 but not the dominant MCSF2 was affected by PKD, suggesting an involvement of macrophage function in this disease model. Thus IL-34 expression is sensitive to inflammatory stimuli and may regulate macrophage biology once up-regulated.