Modulatory effects of laurel-leaf cistus (Cistus laurifolius) ethanolic extract on innate immune responses and disease resistance in common carp (Cyprinus carpio).

Medicinal herbs are used for growth promotion, disease control and other health benefits in aquaculture industry. Here, we examined the effect of dietary laurel-leaf cistus (Cistus laurifolius) ethanolic extract on growth performance, digestive enzyme activity, haematological profile and nonspecific immune responses in common carp (Cyprinus carpio). In addition, resistance against Aeromonas hydrophila infection was examined. Common carp was fed diets containing 0 (Control), 0.1 (CL0.1), 0.5 (CL0.5) and 1 (CL1) g kg-1 laurel-leaf cistus extract for 45 days. After 30 days, superoxide anion production (SAP) increased in CL0.1 and CL0.5 fish groups and at the end of the study all experimental fish groups had higher SAP compared to that of the control (P ˂ 0.05). Lysozyme activity (LA) was elevated in CL0.5 and CL1 treated groups on 30th day (P < 0.05), and this increase was only observed in C0.1 fish group at the end of study compared to control (P ˂ 0.05). Myeloperoxidase activity was significantly increased in CL0.5 and CL1 fish groups at the end of study. IL-1ßgene expression was significantly increased in treated fish in a dose-depended manner. Similar results were observed for transcription of IL-6 and IL-8 (P < 0.05). Anti-inflammatory cytokines, IL-10 and TGF-ß were highly up-regulated in the intestine and head kidney of CL treated fish groups compared to control (P < 0.05). At the end of experiment, significantly higher final body weight, weight gain, and specific growth rate were obtained in CL0.1 treated fish group compared to control. However, growth was negatively affected in CL1 fish group (P < 0.05). CL1 fish group had also a significantly higher FCR. Amylase activity was significantly increased in all experimental fish groups compared to control (P ˂ 0.05). Trypsin activity was decreased in CL0.1 and CL1 fish groups (P ˂ 0.05). WBC and RBC were significantly increased (P ˂ 0.05) in CL0.5 and CL1 fish groups, whereas haemoglobin, haematocrit, mean cell, mean cell haemoglobin contents were no significantly changed among control and treatment groups. Result of challenge test with A. hydrophila exhibited that survival rate in all treatment groups was significantly higher than that of control. These findings demonstrated that laurel-leaf cistus at 0.1 g kg-1 can be a suitable candidate for growth promotion, immune system induction and infection control in 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.

Effect of acute ammonia toxicity on inflammation, oxidative stress and apoptosis in head kidney macrophage of Pelteobagrus fulvidraco and the alleviation of curcumin.

Ammonia is one of the most major pollutant and stress factors of aquaculture systems, and has seriously endangered fish health. However, few studies have been performed on mechanisms of the detrimental impact of ammonia stress and mitigation in fish. A study was carried out to investigate the response of genes involved in inflammation, antioxidation, polarization and apoptosis in head kidney macrophages to acute ammonia toxicity, and the alleviation effect of curcumin. The cells were divided into six groups, as follows: The control group composed of untreated macrophages (CON), the experimental groups, consisting of macrophages treated with 0.23 mg L-1 ammonia (AM), 45 µmol L-1 curcumin (CUR), 0.23 mg L-1 ammonia and 5 µmol L-1 curcumin (5A), 0.23 mg L-1 ammonia and 25 µmol L-1 curcumin (25A), 0.23 mg L-1 ammonia and 45 µmol L-1 curcumin (45A). The cells were pretreated with different concentrations of curcumin for 1 h and then incubated with ammonia for 24 h. The results showed that ammonia poisoning could increase ROS levels, up-regulate the expression of antioxidant enzymes (SOD and GPx), inflammatory cytokines (IL-1, IL-6 and TNF-α) and inflammatory mediators (NF-κB p65 and COX-2), decrease cell viability, down-regulate the expression of M2 marker (Arg-1) and anti-apoptosis (Bcl-2), but curcumin could alleviate the adverse effect of ammonia toxicity. Overall, these results have important implications for understanding of the mechanism of ammonia toxicity and the mitigating effect of curcumin in fish.

CCL19 variants mediate chemotactic response via CCR7 in grass carp Ctenopharyngodon idella.

CC chemokine ligand 19 (CCL19) plays a key role in the regulation of immune responses including homeostasis, inflammation, and immune tolerance. In this study, two variants of CCL19 homologues (CCL19a2 and CCL19b) and CCR7 were investigated in grass carp Ctenopharyngodon idella. The three genes were widely expressed in immune tissues and could be modulated by stimulation with LPS, PHA and poly(I:C), and infection with Flavobacterium columnare and grass carp reovirus. In an in vitro chemotaxis assay, the recombinant CCL19a2 and CCL19b were active to promote the migration of HEK293 T cells expressing CCR7 and leucocytes isolated from the gills, head kidney and spleen. Moreover, their chemotactive effects were validated in vivo. We found that the cells recruited by CCL19a2 and CCl19b are mainly monocytes/macrophages expressing high levels of IL-1ß, IFN-γ, colony stimulating factor 1 receptor (CSF1R) and MHC II. Our work suggests that CCL19a2 and CCl19b are involved in recruitment of antigen presenting cells in fish.

Functional characterization of four TIR domain-containing adaptors, MyD88, TRIF, MAL, and SARM in mandarin fish Siniperca chuatsi.

Toll/interleukin-1 receptor (TIR) domain-containing adaptors, serve as pivotal signal transduction molecules in Toll-like receptor (TLR) signalling pathway to mediate downstream signalling cascades. In this study, four TIR-domain containing adaptors, MyD88, TRIF, MAL and SARM, were identified in mandarin fish Siniperca chuatsi, and they all contain TIR domains, of which MyD88 and SARM had high sequence homology with their vertebrate homologues. The expression analysis at mRNA level indicated that these genes were ubiquitously distributed in different tissues, being high in immune- and mucosa-related tissues such as head-kidney and intestine. The transcripts of these adaptor genes were up-regulated by poly(I:C) and LPS stimulation in isolated head-kidney lymphocytes (HKLs) of mandarin fish. Fluorescence microscopy revealed that all these molecules were localized in cytoplasm, and further investigations showed that the over-expression of MyD88, TRIF and MAL activated the NF-κB, ISRE or type Ι IFN promoters and inhibited SVCV replication, whereas their antiviral effects were significantly impaired when co-transfected with SARM. It was also confirmed by co-immunoprecipitation (Co-IP) that SARM interacts separately with MyD88, TRIF and MAL, and MAL interacts with MyD88. However, the regulatory mechanisms of these adaptors involved in signalling pathways of different TLRs should be of interest for further research.

Dual function of a turbot inflammatory caspase in mediating both canonical and non-canonical inflammasome activation.

Host protective inflammatory caspase activity must be tightly regulated to prevent pathogens infection, however, the inflammatory caspase-engaged inflammasome activation in teleost fish remains largely unknown. In this study, we reveal a bifurcated evolutionary role of the inflammatory caspase in mediating both non-canonical and canonical inflammasome pathways in teleost fish. Through characterization of a unique inflammatory SmCaspase from the teleost Scophthalmus maximus (turbot), we found it can directly recognize cytosolic lipopolysaccharide (LPS) via its N-terminal CARD domain, resulting in caspase-5-like proteolytic enzyme activity-mediated pyroptosis in Turbot Muscle Fibroblasts. Interestingly, we also found that this inflammatory caspase can be recruited to SmNLRP3-SmASC to form the NLRP3 inflammasome complex, engaging the SmIL-1ß release in Head Kidney-derived Macrophages. Consequently, the SmCaspase activation can recognize and cleave the SmGSDMEb to release its N-terminal domain, mediating both pyroptosis and bactericidal activities. Furthermore, the SmCaspase-SmGSDMEb axis-gated pyroptosis governs the bacterial clearance and epithelial desquamation in fish gill filaments in vivo. To our knowledge, this study is the first to identify an inflammatory caspase acting as a central coordinator in NLRP3 inflammasome, as well as a cytosolic LPS receptor; thus uncovering a previously unrecognized function of inflammatory caspase in turbot innate immunity.

Anti-oomycetes and immunostimulatory activity of natural plant extract compounds against Saprolegnia spp.: Molecular docking and in-vitro studies.

This study aimed to investigate the effectiveness of five natural plant extract compounds Curcumin (CUR); Eugenol (EUG), Cinnamaldehyde (CIN), Stigmasterol (ST) and Morin (MOR), on two species of Saprolegnia; Saprolegnia parasitica and S. australis. Selective compounds were screened for the minimum inhibitory concentration, first for anti-oomycetes activity and then mycelium growth inhibition, spore germination inhibition and colonisation test. Nitric oxide production and myeloperoxidase activity of the compounds were tested in head kidney leukocytes of rainbow trout, Oncorhynchus mykiss to assess the immunostimulatory potential. Molecular docking of effective compounds was carried out with effector proteins of S. parasitica to investigate the target binding sites. Among all, CUR could completely inhibit zoospore production and significantly (p ≤ .05) inhibit hyphal growth at 16 mg l-1 against S. parasitica and S. australis. CIN at the concentration of 50 mg l-1 completely inhibited hyphal growth of both Saprolegnia spp., although the zoospore production of S. parasitica and S. australis was reduced at 25 mg l-1 and 10 mg l-1. In the case of EUG, significant inhibition of the hyphal growth and germination of S. parasitica zoospores was observed at 50 mg l-1. ST and MOR did not show antioomycetes activity. The molecular docking results were consistent with in vitro studies, possibly due to the binding with the vital proteins (Plasma membrane ATPase, V-type proton ATPase, TKL protein kinase, Host targeting protein 1) of S. parasitica and ultimately inhibiting their activity. CUR and CIN showed increased nitric oxide production at the highest concentration of 250 and 256 mg l-1 but the value was not significant (p ≤ .05) with control. CUR showed significantly higher peroxidase activity (p ≤ .05) at a concentration of 256 mg l-1 though values were significantly similar with concentration from 16 to 128 mg l-1. The nitric oxide and total peroxidase activity of rainbow trout leukocytes in the case of CIN showed a significant difference only at 250 mg l-1 against the control. The results conclude that CUR, CIN showed the better anti-Saprolegnia activity and could be used as phyto-additives in aquaculture. Among all, the inclusion of CUR as phyto-additives will provide additional immunostimulatory activity.

Expression of ssa-miR-155 during ISAV infection in vitro: Putative role as a modulator of the immune response in Salmo salar.

Multiple cellular components are involved in pathogen-host interaction during viral infection; in this context, the role of miRNAs have become highly relevant. We assessed the expression of selected miRNAs during an in vitro infection of a Salmo salar cell line with Infectious Salmon Anemia Virus (ISAV), the causative agent of a severe disease by the same name. Salmon orthologs for miRNAs that regulate antiviral responses were measured using RT-qPCR in an in vitro time-course assay. We observed a modulation of specific miRNAs expression, where ssa-miR-155-5p was differentially over-expressed. Using in silico analysis, we identified the putative mRNA targets for ssa-miR-155-5p, finding a high prevalence of hosts immune response-related genes; moreover, several mRNAs involved in the viral infective process were also identified as targets for this miRNA. Our results suggest a relevant role for miR-155-5p in Salmo salar during an ISAV infection as a regulator of the immune response to the virus.

Identification and expression analysis of sixteen Toll-like receptor genes, TLR1, TLR2a, TLR2b, TLR3, TLR5M, TLR5S, TLR7-9, TLR13a-c, TLR14, TLR21-23 in mandarin fish Siniperca chuatsi.

Toll-like receptors (TLRs), as a family of pattern recognition receptors (PRRs), possess specific pathogen-related molecular pattern (PAMP) recognition spectrum in inducing immune responses. In this study, sixteen TLRs were identified and characterized in mandarin fish (Siniperca chuatsi). All these TLRs consist of leucine-rich repeats (LRRs), a transmembrane domain and a Toll/interleukin-I receptor (TIR) domain, with the exception of TLR5S which lacks TIR domain, and they can be clustered into five branches, i.e. TLR1 subfamily, TLR3 subfamily, TLR5 subfamily, TLR7 subfamily and TLR11 subfamily in phylogenetic tree. These TLR genes were expressed in all tested tissues and had high expression levels in immune-related tissues such as head-kidney and spleen or mucosa-related tissues such as intestine and pyloric caecum. The transcripts of TLR2a, TLR2b, TLR3, TLR13a, TLR14, TLR22 and TLR23 were all significantly up-regulated after stimulation with poly(I:C); TLR1, TLR2a, TLR2b, TLR3, TLR5M, TLR5S, TLR13a and TLR13b transcripts were all significantly up-regulated after stimulation with PGN; and TLR2a, TLR2b, TLR5M, TLR5S, TLR7, TLR8, TLR9, TLR13c, TLR14 and TLR22 transcripts were all significantly up-regulated after stimulation with LPS in isolated head kidney lymphocytes of mandarin fish. The findings in this study may provide a valuable basis for functional study on TLR genes in mandarin fish.

Neutrophil functions can be regulated by IL-35, which is mainly expressed in IL-15Rα+ cells in grass carp (Ctenopharyngodon idella).

IL-35 plays a key role in regulatory T (Treg) and regulatory B (Breg) cell functions in mammals. CD25 has been demonstrated as one of the markers of Treg cells, and CD19+CD25hiCD71hi cells have been verified as a type of Breg cells in humans. These results indicate that there is a close relationship between IL-35 and CD25+ cells. In mammals, CD25 (alias IL-2Rα) has been identified as having high affinity and specificity for IL-2 binding, and is closely linked and structurally related to IL-15Rα, which having high affinity for IL-15 binding. In teleost, IL-15Rα can bind to both IL-2 and IL-15, with higher affinity to IL-15 than IL-2, and has been termed a CD25-like molecule in some research studies. To date, no studies of IL-35 and IL-15Rα have been documented in fish. In this work, five isoforms of IL-15Rα were cloned from grass carp, and a monoclonal antibody to the protein was developed. The results of flow cytometry and quantitative real-time PCR analyses demonstrated that grass carp IL-35 subunit genes EBI3a and IL-12p35 were mainly expressed in IL-15Rα+ cells, while the expression levels of IL-10 and TGF-ß in IL-15Rα+ and IL-15Rα- cells were insignificant. Recombinant grass carp IL-35 (rgcIL-35) could increase the proportion of IL-15Rα+ cells in leukocytes, and a certain proportion of IL-15Rα+ cells also appeared in myeloid cell subset II after stimulation with rgcIL-35. Meanwhile, the migration, phagocytic ability, and bactericidal ability of grass carp neutrophils were significantly decreased after stimulation with certain concentrations of rgcIL-35. Moreover, neutrophil apoptosis could be significantly inhibited by rgcIL-35.

Methionine and Tryptophan Play Different Modulatory Roles in the European Seabass (Dicentrarchus labrax) Innate Immune Response and Apoptosis Signaling-An In Vitro Study.

The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central roles in key pathways vital for immune support and individual AA supplementation has shown to be able to modulate fish immunity. In vitro trials are important tools to evaluate the immunomodulatory role of AA, and the present study was conceived to evaluate methionine and tryptophan roles in immune-related mechanisms aiming to understand their effects in leucocyte functioning and AA pathways. For that purpose, head-kidney leucocytes were isolated and a primary cell culture established. The effect of methionine or tryptophan surplus on cell viability was assessed. Medium L-15 10% FBS without AA addition (0.5mM of L-methionine, 0.1 mM of L-tryptophan) was used as control. To that, L-methionine or L-tryptophan were supplemented at 1 and 2 times (M1x or M2x, and T1x or T2x). Nitric oxide, ATP, total antioxidant capacity, and immune-related genes were evaluated in response to lipopolysaccharides extracted from Photobacterium damselae subsp. piscicida or UV-inactivated bacteria). Moreover, caspase 3 activity and apoptosis-related genes were evaluated in response to the apoptosis-inducing protein, AIP56. Distinct roles in leucocytes' immune response were observed, with contrasting outcomes in the modulation of individual pathways. Methionine surplus improved cell viability, polyamine production, and methionine-related genes expression in response to an inflammatory agent. Also, methionine supplementation lowered signals of apoptosis by AIP56, presenting lower caspase 3 activity and higher il1ß and nf-κb expression. Cells cultured in tryptophan supplemented medium presented signals of an attenuated inflammatory response, with decreased ATP and enhanced expression of anti-inflammatory and catabolism-related genes in macrophages. In response to AIP56, leucocytes cultured in a tryptophan-rich medium presented lower resilience to the toxin, higher caspase 3 activity and expression of caspase 8, and lower expression of several genes, including nf-κb and p65. This study showed the ability of methionine surplus to improve leucocytes' response to an inflammatory agent and to lower signals of apoptosis by AIP56 induction, while tryptophan attenuated several cellular signals of the inflammatory response to UV-inactivated bacteria and lowered leucocyte resilience to AIP56.

Characterization of TCR+ and CD8+ head kidney leucocytes in Japanese flounder (Paralichthys olivaceus) with antisera against TCRα and CD8α.

T lymphocytes play an important role in cellular and adaptive immunity in vertebrates. The mechanisms of the fish immune system are little studied because of the lack of population-specific antibodies. This study examined the expression of two T lymphocyte markers, TCRα (PoTCRα) and CD8α (PoCD8α) in the Japanese flounder (Paralichthys olivaceus). The expression of PoTCRα and PoCD8α was mainly detected in immune/mucosal tissues. Recombinant PoTCRα and PoCD8α were expressed in pET32a and pET259, respectively. Then, rabbit anti-PoTCRα serum and rat anti-PoCD8α serum were prepared. Using serum, the characteristics of TCR+ and CD8+ head kidney leucocytes (HKLs) were investigated. The results of laser scanning confocal microscopy (LSCM) demonstrated that TCRα and CD8α were transmembrane proteins localized on the cell surface. The populations of CD8α- , CD8α+ , TCRα- , and TCRα+ were sorted by flow cytometry (FCM) and analysed using qRT-PCR. The results demonstrated that all TCRα+ /TCRα- or CD8α+ /CD8α- HKLs expressed IFN-γ. The CD4-1 and IgM transcripts were detected only in TCRα- and CD8α- cells. Furthermore, HKL mitogenesis was induced with concanavalin A (ConA) stimulation. Taken together, the results from LSCM and FCM analyses showed that mammalian and P. olivaceus TCR+ and CD8+ leucocytes share basic characteristics.

Evaluation of triazophos induced immunotoxicity of spleen and head kidney in fresh water teleost, Channa punctata.

The utilization of pesticides has increased for destroying pests and protecting crops in the agriculture field. Triazophos is a commonly used organophosphorous insecticide that causes alterations in haematological and histological parameters in fish. The present study was designed to evaluate the effect of triazophos induced innate and cell mediated immunotoxicity in freshwater teleost, Channa punctata. Fishes were exposed to triazophos at concentrations 5 and 10% of LC50 value for 10 and 20 days. Splenic and head kidney macrophage phagocytosis, nitric oxide production and superoxide production were assayed to evaluate the innate immunity. Cell-mediated immunity was measured through splenic and head kidney lymphocyte proliferation in presence of T and B cell mitogens. Results of the present study revealed that macrophage phagocytosis was significantly reduced after in vivo triazophos treatment. Differential suppressive effect of triazophos was also observed where mitogen induced splenic and head kidney lymphocyte proliferations were reduced after 10 and 20 days treatment. Concentration dependent effect of triazophos was observed in in vivo studies where the production of reactive oxygen and nitrogen intermediates were suppressed. This study describes the first investigation of the effect of triazophos on immune functions and will help to determine appropriate ecotoxicity and immunotoxicity in freshwater teleosts.

Evaluation of an in vitro assay to screen for the immunotoxic potential of chemicals to fish.

A wide variety of environmental contaminants has been shown to disrupt immune functions of fish and may compromise their defense capability against pathogens. Immunotoxic effects, however, are rarely considered in ecotoxicological testing strategies. The aim of this study was to systematically evaluate the suitability of an in vitro immuno-assay using selected fish immune parameters to screen for chemicals with known immunotoxic potential and to differentiate them from non-immunotoxicants. Non-stimulated and lipopolysaccharide-stimulated head kidney leukocytes of rainbow trout (Oncorhynchus mykiss) were exposed for 3 h or 19 h to chemicals with different modes of action. As immune parameters, phagocytosis activity, oxidative burst activity and cytokine transcription (IL-1ß, TNFα, IL-10) were examined, accompanied by in silico modelling. The immunotoxicants dexamethasone, benzo(a)pyrene, ethinylestradiol and bisphenol A significantly altered the immune parameters at non-cytotoxic concentrations whereas diclofenac had only weak effects. However, the two baseline chemicals with no known immunotoxic potential, butanol and ethylene glycol, caused significant effects, too. From our results it appears that the in vitro fish leukocyte assay as performed in the present study has only a limited capacity for discriminating between immunotoxicants and non-immunotoxicants.

Cadmium induced oxystress alters Nrf2-Keap1 signaling and triggers apoptosis in piscine head kidney macrophages.

Cadmium (Cd) with no known functional role in any life-form has myriad of harmful effects. The present study was designed to elucidate the mechanism of Cd-induced oxystress generation and its impact on antioxidant and apoptosis signaling pathways in head kidney macrophage (HKM) of Channa punctatus Bloch. Fish were sampled and acclimatized with one group treated with cadmium chloride (CdCl2) (1.96 mg/L) and another as untreated control group, both kept under observation for 7 days. Exposure to Cd caused ultrastructural changes along with reduced head kidney somatic index (HKSI). Significantly increased levels of reactive oxygen species (ROS), respiratory burst activity, lipid peroxidation, DNA fragmentation and superoxide dismutase were found in the HKM from the treated group as compared to control. In contrast, antioxidant enzymes like catalase and reduced glutathione activity decreased in the Cd exposed group. The suppressed antioxidant activity was further confirmed and corroborated from the altered expression of Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) genes, the major player of antioxidant pathway. Cd induced alteration in Nrf2-Keap1 signaling pathway was also validated by the diminished levels of Nrf2 dependent expression of protein like heme oxygenase-1 (HO-1). The flow cytometry analysis supported the event of apoptosis in Cd exposed group as compared to control, which was further confirmed by the upregulated expression of caspase-3, caspase-8, caspase-9, TNF-α and p53 genes from the real-time gene expression study. In addition, altered protein level of cytochrome C validates the incidence of apoptosis. Altogether, our results demonstrate that exposure to Cd caused oxidative stress in HKM of Channa punctatus Bloch. by compromising the antioxidant enzyme activities via the down regulation of expression of genes related to antioxidant signaling pathway besides encouraging apoptosis via both mitochondrial and death receptor pathway.

A Single-Cell Transcriptome Profiling of Anterior Kidney Leukocytes From Nile Tilapia (Oreochromis niloticus).

Teleost fish anterior kidney (AK) is an important hematopoietic organ with multifarious immune cells, which have immune functions comparable to mammalian bone marrow. Myeloid and lymphoid cells locate in the AK, but the lack of useful specific gene markers and antibody-based reagents for the cell subsets makes the identification of the different cell types difficult. Single-cell transcriptome sequencing enables single-cell capture and individual library construction, making the study on the immune cell heterogeneity of teleost fish AK possible. In this study, we examined the transcriptional patterns of 11,388 AK leukocytes using 10× Genomics single-cell RNA sequencing (scRNA-seq). A total of 22 clusters corresponding to five distinct immune cell subsets were identified, which included B cells, T cells, granulocytes, macrophages, and dendritic cells (DCs). However, the subsets of myeloid cells (granulocytes, macrophages, and DCs) were not identified in more detail according to the known specific markers, even though significant differences existed among the clusters. Thereafter, we highlighted the B-cell subsets and identified them as pro/pre B cells, immature/mature B cells, activated B/plasmablasts, or plasma cells based on the different expressions of the transcription factors (TFs) and cytokines. Clustering of the differentially modulated genes by pseudo-temporal trajectory analysis of the B-cell subsets showed the distinct kinetics of the responses of TFs to cell conversion. Moreover, we classified the T cells and discovered that CD3+CD4-CD8-, CD3+CD4+CD8+, CD4+CD8-, and CD4-CD8+ T cells existed in AK, but neither CD4+CD8- nor CD4-CD8+ T cells can be further classified into subsets based on the known TFs and cytokines. Pseudotemporal analysis demonstrated that CD4+CD8- and CD4-CD8+ T cells belonged to different states with various TFs that might control their differentiation. The data obtained above provide a valuable and detailed resource for uncovering the leukocyte subsets in Nile tilapia AK, as well as more potential markers for identifying the myeloid and lymphoid cell types.

The involvement of TNF-α and TNF-ß as proinflammatory cytokines in lymphocyte-mediated adaptive immunity of Nile tilapia by initiating apoptosis.

Tumor necrosis factors (TNFs) are pleiotropic cytokines with important functions in homeostasis and disease pathogenesis. Recent advances have shown that TNFs are also involved in the regulation of adaptive immune responses. However, the knowledge about how TNF participates in and regulates adaptive immune response in early vertebrates is still limited. In present study, we identified two isoforms of TNF, TNF-α and TNF-ß, from Nile tilapia Oreochromis niloticus (On-TNF-α and ß). After analyzing the sequence characteristics, we investigated their regulatory roles in adaptive immune response of this fish species. On-TNF-α and ß are evolutionarily conserved compare with their homologs from other vertebrates. Both TNFs were distributed in a wide range of tissues in O. niloticus, and with relative higher expression level in gill. After the animals were infected by Streptococcus agalactiae, mRNA levels of On-TNF-α and TNF-ß in spleen lymphocytes were significantly upregulated during the primary response stage of adaptive immunity. Meanwhile, both TNF proteins in spleen lymphocytes were also dramatically elevated during the adaptive immune stage after bacterial infection. These results indicate the potential participation of On-TNF-α and TNF-ß in adaptive immune response of Nile tilapia. Furthermore, On-TNF-α and ß transcripts were obviously augmented, once spleen lymphocytes were activated by T cell-specific mitogen PHA. More importantly, both recombinant On-TNF-α and ß could induce the apoptosis of head-kidney leukocytes of Nile tilapia. And On-TNF-ß but not On-TNF-α promoted the apoptosis by activating caspase-8 in the target cells. Altogether, our study revealed that TNF-α and TNF-ß participated in the lymphocyte-mediated adaptive immune response of Nile tilapia by initiating the apoptosis, and thus shed novel perspective for the regulatory mechanism of adaptive immunity in teleost.

Structural insights into the co-evolution of IL-2 and its private receptor in fish.

Interleukin (IL) -2, a member of the four α-helical cytokine family, has broad regulatory roles in mediating vertebrate immune response. In mammals, IL-2 and IL-15 share a common evolutionary origin and possess overlapping but distinct functions. IL-2 and IL-15 bind to distinct private receptors for signaling. However, fish appear to possess a single IL-15Rα like gene whilst lack additional gene(s) coding for IL-2Rα. Whether the IL-2 and IL-15 interact with the same receptor in fish and how their functions and receptors have evolved are not fully understood. In this study, homologues of IL-2 and IL-2/15Rα were sequenced from a teleost species, grass carp (Ctenopharyngodon idella), and the crystal structure of IL-2 was determined. The grass carp IL-2 (termed CiIL-2) displayed a classical cytokine structure consisting of four helical bundles which shares significant similarity with human IL-15. The key amino acids involved in the interface interaction of IL-2/15 and their receptors are well conserved. The CiIL-2 has been shown to bind the IL-2/15Rα like homologue with an affinity of 2.45 nM, supporting the notion that fish IL-2 and IL-15 may share a single common private receptor for exerting functions. Syntenic analysis suggests that the IL-2Rα of tetrapods has evolved from an IL-15Rα like homologue, in which a second sushi domain (D2) in the extracellular region has been duplicated to facilitate the specific interaction with IL-2. The CiIL-2 was predominantly expressed in lymphocyte-rich tissues such as the spleen, kidney and thymus, and could be induced by PHA and IL-21. In vivo challenge with grass carp reovirus and Flavobacterium columnare also resulted in upregulation of CiIL-2 expression. The recombinant CiIL-2 was shown to activate expression of STAT5b, IL-1ß, IL-22 and IFN-γ, and to promote the proliferation of the primary cell cultures from head kidney leucocytes. Our results shed lights into the co-evolution of IL-2 and its private receptor, and the functional divergence of IL-2 and IL-15 during evolution.

Characterization of miRNAs in Extracellular Vesicles Released From Atlantic Salmon Monocyte-Like and Macrophage-Like Cells.

Cell-derived extracellular vesicles (EVs) participate in cell-cell communication via transfer of molecular cargo including genetic material like miRNAs. In mammals, it has previously been established that EV-mediated transfer of miRNAs can alter the development or function of immune cells, such as macrophages. Our previous research revealed that Atlantic salmon head kidney leukocytes (HKLs) change their morphology, phagocytic ability and miRNA profile from primarily "monocyte-like" at Day 1 to primarily "macrophage-like" at Day 5 of culture. Therefore, we aimed to characterize the miRNA cargo packaged in EVs released from these two cell populations. We successfully isolated EVs from Atlantic salmon HKL culture supernatants using the established Vn96 peptide-based pull-down. Isolation was validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. RNA-sequencing identified 19 differentially enriched (DE) miRNAs packaged in Day 1 versus Day 5 EVs. Several of the highly abundant miRNAs, including those that were DE (e.g. ssa-miR-146a, ssa-miR-155 and ssa-miR-731), were previously identified as DE in HKLs and are associated with macrophage differentiation and immune response in other species. Interestingly, the abundance relative of the miRNAs in EVs, including the most abundant miRNA (ssa-miR-125b), was different than the miRNA abundance in HKLs, indicating selective packaging of miRNAs in EVs. Further study of the miRNA cargo in EVs derived from fish immune cells will be an important next step in identifying EV biomarkers useful for evaluating immune cell function, fish health, or response to disease.

Differences in immune components of blood, spleen and head kidney between diploid and auto- and allotriploid Salmonidae.

Immune components were investigated in peripheral blood and in spleen and head kidney of autotriploid Salmo trutta f. lacustris, Salvelinus fontinalis, and Salvelinus umbla, and of allotriploid hybrids of S. trutta f. lacustris x Onchorynchus mykiss and S. fontinalis x O. mykiss in comparison to their diploid parents. In peripheral blood the number of lymphocytes was reduced in all investigated autotriploids and in the allotriploid S. trutta f. lacustris x O.mykiss, and the numbers of thrombocytes in autotriploid S. trutta f. lacustris and in both allotriploids. Alternative pathway of complement activity and immunoglobulin concentration were significantly decreased in all investigated auto- and allotriploids, lysozyme activity in autotriploid S. fontinalis and in both allotriploids. In the spleen of the 3 autotriploids the number of erythrocytes was increased, while the number of lymphoid precursor cells was decreased. In their head kidney the erythrocytes numbers were decreased and the numbers of erythropoietic precursor cells and the melanomacrophage centers were increased. Contrary, cytology of spleen and head kidney of the two allotriploid hybrids was similar to diploid controls. Caspase 1, caspase 6, lysozyme, and acid phosphatase activity and immunoglobulin concentration of spleen and head kidney showed specific changes which were related to cytological results. These data indicate alterations in immune system and in lymphoid organs of auto- and allotriploid Salmonidae.