Head Kidney Lymphocytes Research Articles

Investigating the role of the ROS/CncC-xenobiotics signaling pathway in the response to Fenpropathrin in Cyprinus carpio lymphocytes: Involving lipid peroxidation and Fe2+ metabolism imbalance

Fenpropathrin (FPT) is a synthetic pyrethroid insecticide, the persistence and accumulation in water of which could cause harmful effects on vulnerable groups like aquatic creatures, particularly posing significant risks to fish immune systems. This study aimed to investigate how environmentally relevant FPT concentrations (10–1000 μ/M) affect lipid peroxidation and Fe2+ metabolism in Cyprinus carpio head kidney lymphocytes, and its relationship with oxidative stress and immunotoxicity. Firstly, CCK-8 results demonstrated that FPT caused a significant increase in lymphocyte death. Secondly, lymphocytes exposed to FPT could lead ferroptosis in lymphocytes, accompanied by evidence of the Fe2+ transporter imbalance, lipid peroxidation, Fe2+ accumulation and ferroptosis related protein increment. Thirdly, we found that FPT esposure leads to a decrease in ATP, mitochondrial DNA and NADPH/NADP+ levels, and the mRNA associated with mitochondrial function-related genes (Fis1, Drp1, and OPA1) in lymphocytes. Additionally, FPT induced the increased the levels of inflammatory genes (TNF-α, IFN-γ, and IL-6) in head kidney lymphocytes. Importantly, exposure to FPT induced oxidative stress to produce intracellular ROS, disrupting the function of the CncC signaling pathway and expression disorder of xenobiotics detoxification (CYP 450 family) genes. Notably, Treatment with NAC (a ROS inhibitor, 5 μM) demonstrated that inhibiting ROS alleviated FPT-induced lymphocyte ferroptosis and inflammatory response via the ROS/CncC-xenobiotics signaling pathway. These findings not only introduces a novel approach to investigating the immunotoxicity of FPT but also offers critical insights into mitigating the adverse effects of FPT on aquatic animal health.

Characterization of a membrane Fcγ receptor in largemouth bass (Micropterus saloumoides) and its response to bacterial challenge.

Fc receptors (FcRs), specific to the Fc portion of immunoglobulin (Ig), are required to regulate immune responses against pathogenic infections. However, FcγR is a member of FcRs family, whose structure and function remains to be elucidated in teleost fish. In this study, the FcγRII, from largemouth bass (Micropterus saloumoides), named membrane MsFcγRII (mMsFcγRII), was cloned and identified. The opening reading frame (ORF) of mMsFcγRII was 750 bp, encoding 249 amino acids with a predicted molecular mass of 27 kDa. The mMsFcγRII contained a signal peptide, two Ig domains, a transmembrane domain, and an intracellular region, which was highly homology with FcγR from other teleost fish. The mRNA expression analysis showed that mMsFcγRII was widely distributed in all tested tissues and with the highest expression level in spleen. After bacterial challenge, the expression of mMsFcγRII was significantly upregulated in vivo (spleen and head kidney), as well as in vitro (leukocytes from head kidney). The subcellular localization assay revealed that mMsFcγRII was mostly observed on the membrane of HEK293T cells which were transfected with mMsFcγRII overexpression plasmid. Flow cytometric analysis showed that natural mMsFcγRII protein was highly expressed in head kidney lymphocytes. Moreover, indirect immunofluorescence assay and pull-down assay indicated that mMsFcγRII could bind to IgM purified from largemouth bass serum. These results suggested that mMsFcγRII was likely to play an influential role in the immune response against pathogens and provided valuable insights for studying the function of FcRs in teleost.

Micro-algal astaxanthin ameliorates polystyrene microplastics-triggered necroptosis and inflammation by mediating mitochondrial Ca2+ homeostasis in carp’s head kidney lymphocytes (Cyprinus carpio L.)

Polystyrene microplastics (PM) is a pressing global environmental concern, posing substantial risks to aquatic ecosystems. Microalgal astaxanthin (MA), a heme pigment, safeguards cells against oxidative damage induced by free radicals, which contributes to various health conditions, including aging, inflammation and chronic diseases. Herein, we investigated the potential of MA in ameliorating the immunotoxicity of PM on carp (Cyprinus carpio L.) based on head kidney lymphocytes treated with PM (250 μM) and/or MA (100 μM). Firstly, CCK8 results showed that PM resulted in excessive death of head kidney lymphocytes. Secondly, head kidney lymphocytes treated with PM had a higher proportion of necroptosis, and the levels of necroptosis-related genes in head kidney lymphocytes were increased. Thirdly, the relative red fluorescence intensity of JC-1 and MitoSox showed decreased mitochondrial membrane potential and increased mtROS in head kidney lymphocytes treated with PM. MitoTracker® Green FM fluorescence analysis revealed enhanced mitochondrial Ca2+ levels in PM-treated lymphocytes, corroborating the association between PM exposure and elevated intracellular Ca2+ dynamics. PM exposure resulted in upregulation of calcium homeostasis-related gene (Orail, CAMKIIδ and SLC8A1) in lymphocytes. Subsequent investigations revealed that PM exposure reduced miR-25-5p expression while increasing levels of MCU, MICU1, and MCUR1. Notably, these effects were counteracted by treatment with MA. Furthermore, PM led to the elevated secretion of inflammatory factors (IFN-γ, IL-1β, IL-2 and TNF-α), thereby inducing immune dysfunction in head kidney lymphocytes. Encouragingly, MA treatment effectively mitigated the immunotoxic effects induced by PM, demonstrating its potential in ameliorating necroptosis, mitochondrial dysfunction and immune impairment via regulating the miR-25-5p/MCU axis in lymphocytes. This study sheds light on safeguarding farmed fish against agrobiological threats posed by PM, highlighting the valuable applications of MA in aquaculture.

Improved innate immunity in juvenile vimba bream (Vimba vimba) fed a dry diet with an additive of hydrochloric acid (HCl)

Abstract A few reports indicate the beneficial effects of organic acids and their salts on anti-infective immunity in fish species. In the role of immunostimulants, inorganic acids may prove to be a much cheaper alternative to their organic equivalents. However, no report has described the effect of using inorganic acid as a feed additive on fish immunity. This study is the first attempt to evaluate the effect of hydrochloric acid (HCl) as a fish feed supplement on cellular immunity in the stomachless fish, cypriniform vimba bream, Vimba vimba (L.). Two groups, three replicates each, of juvenile vimba were fed a commercial dry fish diet or its variant containing a 1.5% additive of HCl for 55 days. The experiment was conducted in a recirculating aquaculture system at 25°C. Mortality, growth, condition factor, feed conversion ratio, respiratory burst activity (RBA), potential killing activity (PKA), and the proliferative response of head kidney lymphocytes were determined for both groups at the end of the experiment and compared. The PKA was higher in the group fed the diet with HCl. For all other parameters studied, there were no significant differences between the experimental groups. The results of the current study prove that the dietary additive of inorganic hydrochloric acid can substantially improve immune response to bacterial infections in juvenile vimba bream.

Immune protection and safety of Aeromonas veronii fliD deletion mutant as a live attenuated vaccine in Cyprinus carpio

Aeromonas veronii is one of the important pathogens causing motile Aeromonas septicemia (MAS) in aquaculture, leading to high fish mortality and severe economic losses. Live attenuated vaccines are considered to be an effective method to control bacterial infections and have been widely used in veterinary vaccines. In order to develop a safe and effective vaccine against MAS, the safety and protective efficacy of the fliD gene deletion strain of A. veronii as a live attenuated vaccine for Cyprinus carpio were evaluated in this study. After intraperitoneal injection vaccination, the A. veronii ΔfliD was able to colonize temporarily in the head kidney and spleen, and was finally cleared by C. carpio without causing clinical symptoms and affecting the growth performance of C. carpio. The significantly increased IgM levels in the serum indicated that both intraperitoneal injection and immersion vaccination induced a significant humoral immune response. The pro-inflammatory factors IL-1β, TNF-α and IL-6 were significantly increased at the early stage after intraperitoneal injection vaccination, indicating that A. veronii ΔfliD induced an inflammatory response in C. carpio, which facilitated bacterial clearance. In addition, a significant proliferation of head kidney lymphocytes was also observed after stimulation with A. veronii soluble antigen. The survival rates of C. carpio vaccinated by intraperitoneal injection and immersion were 83.33% and 56.67% after intraperitoneal challenge with wild-type A. veronii, compared with 13.33% and 16.67% in the control group. The histopathological examination showed that A. veronii ΔfliD could protect the liver and spleen against tissue lesion after challenge. These results indicate that attenuated strain A. veronii ΔfliD can induce an effective humoral and cellular immune response and provide protection against A. veronii for C. carpio without adverse effects, which can be developed as an effective live attenuated vaccine candidate to control the A. veronii infection.

Selection and evaluation of stable reference genes for quantitative real-time PCR in the head kidney leukocyte of Oreochromis niloticus

To investigate the antibacterial mechanisms of Nile tilapia, it is necessary to understand the responses of immune-related genes in infected lymphocytes. Usually, quantitative real-time polymerase chain reaction (qRT-PCR) is applied to investigate transcriptional profiling. However, few studies have evaluated stable reference genes for qRT-PCR in Nile tilapia head kidney lymphocytes (HKLs). In this research, we assessed the stability of seven common reference genes, which include 18 S ribosomal RNA (18 s rRNA), beta actin (β-actin), beta-2-microglobulin-like (β-MG), elongation factor 1a (EF1A), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Tubulin alpha chain-like (TUBA), and Ubiquitin-conjugating enzyme (UBE)，in both resting state and several responding states of Nile tilapia HKLs. Results showed that β-MG was the optimum reference gene in both resting state and LTA stimulation state, while GAPDH was the optimum for LPS stimulation. However, no single candidate gene could be employed as the universal reference under all situations. Next, the geNorm and RefFinder tools was applied to access the adequacy of the union of two reference genes, and the results suggested that the combination of β-MG and GAPDH was the best candidate. Finally, the expression profiles of three typical immune-related genes interleukin-1β (IL-1β), transcription factor p65(P65), and tumor necrosis factor α (TNFα) under LPS/LTA situations in HKLs were evaluated via multiple reference genes and single reference gene difference analysis. The findings revealed that TUBA and UBE were incapable of working as reference genes, and the union of two reference genes was better than any sole reference gene. The single-cell RNA-Seq (scRNA-Seq) data of Nile tilapia HKLs confirmed the above results. In conclusion, our data suggest that the combination of β-MG and GAPDH may be the best criterion for qRT-PCR calculation in tilapia HKLs.

Seasonal Immune Rhythms of head kidney and spleen cells in the freshwater Teleost, Channa punctatus

Annual rhythms in immune function are the reflection of a crucial physiological strategy to deal with environmental stressors. The fish are pivotal animal models to study the annual rhythm and to understand the evolution of the vertebrate biological system. The current research was planned to assess the annual changes in the innate immune functions of immune cells in a teleost, Channa punctatus. Head kidney and splenic macrophage phagocytosis, superoxide generation, and nitrite release were evaluated to assess innate immunity. Cell-mediated immunity was measured through head kidney and splenic lymphocyte proliferation in presence of mitogens. The superoxide anion generation by the cells of head kidney and spleen was maximum in October. A bimodal pattern in nitrite production was observed with the first peak in November and the second in March. Cosinor analysis revealed a statistically significant annual rhythm in nitrite production. Similarly, phagocytosis and lymphocyte proliferation also showed statistically significant annual rhythms. It was concluded that animals maintain an optimum immune response in seasonally changing environments. Elevated immunity during certain times of the year might assist animals deal with seasonal environmental stressors. Further research may be focused upon measuring survival rate and reproductive success after season induced elevated immunity.

CpG ODN 1668 as TLR9 agonist mediates humpback grouper (Cromileptes altivelis) antibacterial immune responses.

Cromileptes altivelis (humpback grouper) is the main farmed species in the southern coastal area of China owing to its important economic value. Toll-like receptor 9 (TLR9) belongs to the toll-like receptor (TLR) family and functions as a pattern recognition receptor, recognising unmethylated oligodeoxynucleotides containing the CpG motif (CpG ODNs) in bacterial and viral genomes, thereby activating host immune response. In this study, the C. altivelis TLR9 (CaTLR9) ligand CpG ODN 1668 was screened and found to significantly enhance the antibacterial immunity of humpback grouper in vivo and head kidney lymphocytes (HKLs) in vitro. In addition, CpG ODN 1668 also promoted the cell proliferation and immune gene expression of HKLs and strengthened the phagocytosis activity of head kidney macrophages. However, when the CaTLR9 expression was knocked down in the humpback group, the expression levels of TLR9, myeloid differentiation factor 88 (Myd88), tumour necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-1β (IL-1β), IL-6, and IL-8 were significantly reduced, and the antibacterial immune effects induced by CpG ODN 1668 were mostly abolished. Therefore, CpG ODN 1668 induced antibacterial immune responses in a CaTLR9-dependent pathway. These results enhance the knowledge of the antibacterial immunity of fish TLR signalling pathways and have important implications for exploring natural antibacterial molecules in fish.

Evolutionarily conserved IL-20 enhances the response of JAK1/STAT3 axis in Carassius cuvieri × Carassius auratus red var

The IL-20 cytokine family, which includes only IL-20, IL-22 and IL-26 in teleost, has a wide range of biological functions and is essential for epithelial integrity and innate host defense. IL-22 and IL-26 are the most intensively studied cytokine within this family; however, the immunological function of IL-20 in fish is essentially unknown. In this study, we examined the expression and function of an IL-20 homologue (WR-IL20) from White crucian carp (Carassius cuvieri, WCC, female) × Red crucian carp (Carassius auratus red var., RCC, male). WR-IL20 and its parents, WCC-IL20 and RCC-IL20, were both composed of 177 residues with high identities among them. Additionally, gene structure, functional domain, tertiary structure, multiple sequence alignment, and phylogeny analysis showed a conserved status between WR-IL20, WCC-IL20 and RCC-IL20. WR-IL20, WCC-IL20 and RCC-IL20 were mainly expressed in the head kidney and were upregulated by Aeromonas hydrophila infection in vivo. Furthermore, recombinant WR-IL20 protein (rWR-IL20) induced increased transcript levels of c-Myc, cyclin D, Bcl-2, JAK1, TYK2 and STAT3 in head kidney lymphocytes in vitro. These results provide a new perspective for understanding the origin, evolution and function of IL-20 in teleost.

CpG ODN 2102 promotes antibacterial immune responses and enhances vaccine-induced protection in golden pompano (Trachinotusovatus).

CpG oligodeoxynucleotides (ODNs) are oligodeoxynucleotides containing CpG motifs and can be recognized by toll-like receptor 9 (TLR9), activating the host's immune responses. In this study, ten different CpG ODNs were designed and synthesized to study the antibacterial immune responses of CpG ODNs in golden pompano (Trachinotus ovatus). Results showed that CpG ODN 2102 significantly improved the immunity of golden pompano against bacteria. Besides, CpG ODN 2102 promoted the proliferation of head kidney lymphocytes and activated the head kidney macrophages. When TLR9-specific small interfering RNA (siRNA) was used to interfere with TLR9 expression, the immune responses were decreased. Moreover, the expression levels of myeloid differentiation primary response 88 (Myd88), p65, tumor necrosis factor receptor-associated factor 6 (TRAF6), and tumor necrosis factor-alpha (TNF-α) in the TLR9-knockdown golden pompano kidney (GPK) cells were significantly reduced. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) promoter activity of the TLR9-knockdown GPK cells was also significantly reduced. In vivo, the antibacterial immune effects induced by CpG ODN 2102 in golden pompano were mostly abolished when TLR9 expression was knocked down. These results suggested that TLR9 was involved in the immune responses induced by CpG ODN 2102. CpG ODN 2102 also enhanced the protective effect of the Vibrio harveyi vaccine pCTssJ, where the survival rate of golden pompano was significantly improved by 20%. In addition, CpG ODN 2102 enhanced the messenger RNA (mRNA) expression levels of TLR9, Myxovirus resistance (Mx), interferon γ (IFN-γ), TNF-α, interleukin (IL)-1β, IL-8, major histocompatibility complex class (MHC) Iα, MHC IIα, Immunoglobulin D (IgD), and IgM. Therefore, TLR9 was involved in the antibacterial immune responses induced by CpG ODN 2102 and CpG ODN 2102 possessed adjuvant immune effects. These results enlarged our knowledge of the antibacterial immunity of fish TLRs signaling pathway and had important implications for exploring natural antibacterial molecules in fish and developing new vaccine adjuvants.

Effects of long-term administration of various dietary prebiotic supplements on the growth, immune cell activity and digestive tract histology of juvenile vimba (Vimba vimba).

The experiment was set to determine the effects of long-term (55-day) use of three commercial prebiotics including Saccharomyces cerevisiae-derived β-glucans and one including inulin on juvenile vimba (Vimba vimba) reared intensively under controlled conditions. Six-month-old fish were fed commercial feed (Control group, n = 90), or the same feed supplemented with 0.02% Leiber Beta-S (BS group, n = 90), 0.20% Biolex MB40 (MB group, n = 90), 0.30% CeFi (CE group, n = 90) or 1.00% inulin Orafti GR (IN group, n = 90) for 55 days. In the BS group, the final growth parameters were significantly lower than in the Control group, while the feed conversion ratio was significantly higher. No significant differences were found between any other group and the Control group in the respective parameters. The respiratory burst activity of the head-kidney phagocytes was significantly lower in all fish groups fed the prebiotic-supplemented diets compared to the Control group. The proliferative response of the head-kidney lymphocytes stimulated by concanavalin A was lower in the BS group than in the Control group, while in other groups this response was not affected. No significant differences were found in histopathological analyses of the digestive tract, liver or pancreas. The long-term supplementation of fish diets with prebiotics can negatively influence the growth, feed conversion, nonspecific cellular resistance and proliferative activity of the T lymphocytes of vimba juveniles.

Monoclonal antibody generated against Nile tilapia (Oreochromis niloticus) IgT heavy chain using a peptide-based strategy

Teleost IgT/Z plays a principal role in the defense mechanisms against infectious agents in the mucosal compartments and in systemic immunity. Previously, Nile tilapia (Oreochromis niloticus) IgT was discovered and characterized at transcription level. In this work, we generated a monoclonal antibody (mAb) that specifically recognized the Nile tilapia IgT. BALB/c mice were immunized with three synthetic peptides conjugated to KLH. The sequences of these peptides derived from the constant region of the Nile tilapia IgT heavy chain. ELISA and Western blotting confirmed the specificity of the polyclonal sera and the culture supernatant from a positive hybridoma clone. We observed immunoreactivity against a recombinant IgT fragment and native IgT in skin mucus. The anti-IgT mAb did not cross-react with purified tilapia IgM. Direct ELISA analysis allowed the quantification of skin mucus IgM and IgT concentrations. Flow cytometry analysis revealed differences in the percentage of IgT+ B cell populations between juveniles and adults in peripheral blood, head kidney and spleen lymphocytes and among the tissues analyzed. For further validation of the anti-IgT mAb utility, a recombinant vaccine candidate against sea lice (TT-P0 Ls) was injected into juvenile tilapia. Direct ELISA results revealed a differential secretion of skin mucus IgT and IgM after immunostimulation. In addition, the percentages of IgT+ B cells were determined at 7 days after booster and ex-vivo stimulation by flow cytometry. This mAb constitutes an important immunological tool to study the biological function and structural characteristics of tilapia IgT.

Insulin-like growth factor binding protein 5b of Trachinotus ovatus and its heparin-binding motif play a critical role in host antibacterial immune responses via NF-κB pathway.

Insulin-like growth factor binding protein 5 (IGFBP5) exerts an essential biological role in many processes, including apoptosis, cellular differentiation, growth, and immune responses. However, compared to mammalians, our knowledge of IGFBP5 in teleosts remains limited. In this study, TroIGFBP5b, an IGFBP5 homologue from golden pompano (Trachinotus ovatus) was identified. Quantitative real-time PCR (qRT-PCR) was used to check its mRNA expression level in healthy condition and after stimulation. In vivo overexpression and RNAi knockdown method were performed to evaluate the antibacterial profile. We constructed a mutant in which HBM was deleted to better understand the mechanism of its role in antibacterial immunity. Subcellular localization and nuclear translocation were verified by immunoblotting. Further, proliferation of head kidney lymphocytes (HKLs) and phagocytic activity of head kidney macrophages (HKMs) were detected through CCK-8 assay and flow cytometry. Immunofluorescence microscopy assay (IFA) and dual luciferase reporter (DLR) assay were used to evaluate the activity in nuclear factor-κB (NF-κβ) pathway. The TroIGFBP5b mRNA expression level was upregulated after bacterial stimulation. In vivo, TroIGFBP5b overexpression significantly improved the antibacterial immunity of fish. In contrast, TroIGFBP5b knockdown significantly decreased this ability. Subcellular localization results showed that TroIGFBP5b and TroIGFBP5b-δHBM were both present in the cytoplasm of GPS cells. After stimulation, TroIGFBP5b-δHBM lost the ability to transfer from the cytoplasm to the nucleus. In addition, rTroIGFBP5b promoted the proliferation of HKLs and phagocytosis of HKMs, whereas rTroIGFBP5b-δHBM, suppressed these facilitation effects. Moreover, the in vivo antibacterial ability of TroIGFBP5b was suppressed and the effects of promoting expression of proinflammatory cytokines in immune tissues were nearly lost after HBM deletion. Furthermore, TroIGFBP5b induced NF-κβ promoter activity and promoted nuclear translocation of p65, while these effects were inhibited when the HBM was deleted. Taken together, our results suggest that TroIGFBP5b plays an important role in golden pompano antibacterial immunity and activation of the NF-κβ signalling pathway, providing the first evidence that the HBM of TroIGFBP5b plays a critical role in these processes in teleosts.

TroTNFSF6, a tumor necrosis factor ligand superfamily member, promotes antibacterial immune response of golden pompano, Trachinotus ovatus

Tumor necrosis factor ligand superfamily member 6 (TNFSF6), also known as FasL/CD95L, is essential for maintaining the body's immune homeostasis. However, the current reports on TNFSF6 in fish are relatively scarce. In the present study, we conducted functional analyses of a TNFSF6 (TroTNFSF6) from the teleost fish golden pompano (Trachinotus ovatus). TroTNFSF6 is composed of 228 amino acids and has a low similarity with other species (9.65%–58.79%). TroTNFSF6 was expressed in the 11 tissues tested and was significantly up-regulated after Edwardsiella tarda infection. In vivo, overexpression of TroTNFSF6 effectively stimulated the AKP and ACP activities, and reduced bacterial infection in fish tissues. Correspondingly, knockdown of TroTNFSF6 expression resulted in increasing bacterial dissemination and colonization in fish tissues. In vitro, recombinant TroTNFSF6 protein promoted the proliferation of T. ovatus head kidney lymphocytes (HKLs), and promoted the apoptosis of murine liver cancer cells (Hepa1-6). The results indicated that TroTNFSF6 plays an important role in the T. ovatus antibacterial immunity. These observations will facilitate the future in-depth study of teleost TNFSF6.

Costimulatory Molecules CD80/86 Trigger Non-Specific Cytotoxic Cell of Nile tilapia (Oreochromis niloticus) to Kill CIK Cells

The teleost non-specific cytotoxic cell (NCC), as the evolutionary precursors of NK cells, is an important cytotoxic cell population in the innate immune system of teleost. We have recently realized that costimulatory CD80/86 have conservation in structural and interactional features with its ligand CD28 in Nile tilapia (Oreochromis niloticus). However, the ability of CD80/86 to regulate NCC activity has not been fully investigated. In the present study, we first obtained the recombinant fusion CD80/86 protein from O. niloticus (rOn-CD80/86). Then, NCC incubation with rOn-CD80/86 resulted in a significant production of NCC effector cytokines, including tumor necrosis factor-alpha, cellular apoptosis susceptibility and NK-lysin. Furthermore, NCC treatment with rOn-CD80/86 could significantly improve the ability to kill kidney cells of Grass carp (CIK) and up-regulate the activities of caspase-1 and caspase-3 in CIKs. The yeast, two-hybrid assay showed that On-CD80/86 cannot directly interact with non-specific cytotoxic cell receptor protein-1 of O. niloticus (On-NCCRP-1). The single-cell RNA-Seq data of Nile tilapia head kidney lymphocytes analysis found On-CD28 did not exhibit expression on NCCs subsets. The above results suggest that costimulatory molecules On-CD80/86 is independent of On-NCCRP-1 and On-CD28 receptor in modulating NCC killing activity in vitro of Nile tilapia. The results also provide more insights into the mechanism of NCC activity regulation.

Chlorpyrifos exposure induces calcium-dependent necrosis in carp (Cyprinus carpio) lymphocytes via the inhibition of T cell receptor gamma (TCR γ)

The insecticide chlorpyrifos plays an important role in agricultural production and is widely used because of its excellent insecticidal ability. However, the mechanism by which chlorpyrifos causes lymphocyte death remains unclear. In this study, transcriptomic techniques were used to analyze the head kidney tissues of carp (Cyprinus carpio) treated with chlorpyrifos. Subsequently, we screened out differentially expressed genes (DEGs) and performed the corresponding processing in the head kidney lymphocyte. Then, the intracellular calcium content and necrosis were detected by fluorescence staining, real-time fluorescence quantitative PCR, and flow cytometry. Our results showed that the expression of T cell receptor gamma (TCR γ) was significantly decreased, and TCR γ was inhibited after chlorpyrifos treatment. Also, TCR γ significantly increased the abundance of calcium channel messenger RNA (mRNA). To verify this result, we established the TCR γ overexpression group and found that the reverse results were observed in TCR γ of in the overexpression group. The results of cytoplasmic calcium concentration detection, calcium staining, and flow cytometry confirmed the conclusion of increased calcium in the cytoplasm. The function of TCR γ significantly enhanced the mRNA expression levels of necrosis-related genes, and this conclusion was evidenced by the result of necrotic flow detection. Our results showed that chlorpyrifos could inhibit TCR γ in carp lymphocytes and induce calcium-dependent necrosis.

Functional analyses of two interferon-stimulated gene 15 (ISG15) copies in large yellow croaker, Larimichthys crocea

In this study, we conducted functional analyses for two ISG15 homologues of Larimichthys crocea (LcISG15-1 and LcISG15-2). Our results of qRT-PCR showed that both LcISG15-1 and LcISG15-2 were significantly changed in head kidney and peripheral blood, after poly (I:C) stimulation. Western blot analyses with prepared polyclonal antibodies suggested that LcISG15-1 and LcISG15-2 both could be secreted by primary head kidney lymphocytes into the extracellular milieu. The purified recombinant LcISG15-1 (rLcISG15-1) and LcISG15-2 (rLcISG15-2) could both activate primary macrophages as extracellular cytokines and significantly enhance macrophage respiratory burst, NO production and bactericidal activity and induce the expression of proinflammatory cytokine genes of the cells. Moreover, rLcISG15-2 exhibited much stronger cytokine-like activities than those of rLcISG15-1, indicating the ISG15-2 gene copy evolved enhanced activity after gene duplication of ISG15 in sciaenid fishes. These results indicated important roles of LcISG15-1 and especially LcISG15-2 in immune regulation and host immune defense of large yellow croaker against viral and bacterial infection.

Novel development of cationic surfactant-based mucoadhesive nanovaccine for direct immersion vaccination against Francisella noatunensis subsp. orientalis in red tilapia (Oreochromis sp.)

Francisella noatunensis subsp. orientalis (Fno) is one of the infectious diseases that causes economic losses associated with tilapia mortality. Even though direct immersion administration of vaccines is more practicable for small fish and fry compared with oral and injection vaccination in the fields, the efficacy is still insufficient due to lower potency of antigen uptake. Herein, we accomplished the development of a mucoadhesive nanovaccine platform using cetyltrimethylammonium bromide (CTAB), a cationic surfactant, to improve the efficiency of immersion vaccination against Fno in tilapia. Cationic Fno nanovaccine (CAT-Fno-NV) was prepared though emulsification using an ultrasonic method. In our investigation, the CAT-Fno-NV increased the opportunity of Fno vaccine uptake by extending the contact time between vaccine and mucosal surface of fish gills and enhancing the protective efficacy against Fno infection. Fish were vaccinated with the CAT-Fno-NV by a direct immersion protocol. The challenge trial by Fno injection revealed that CAT-Fno-NV at the concentration 1:100 ratio (approximately 1 × 106 cfu/mL) had the highest efficacy to protect fish from Fno infection at day 30 after post challenge period according to the total number of Fno detected in head kidney, spleen and liver. A significant upregulation of IgM gene was observed in gills, skin, head kidney, serum and peripheral blood lymphocytes (PBLs) and spleen tissues treated with WC and CAT-Fno-NV (1:100) vaccines, while IgT gene was highly expressed in only gills and skin tissues for treated WC and CAT-Fno-NV (1:100) groups. We anticipate that the cationic surfactant-based nanovaccine developed in this study could become an efficient alternative for direct immersion vaccination to induce humoral immune responses against Fno in vaccinated tilapia.

Role of miR-451 in mediating cadmium induced head kidney injury in common carp via targeting cacna1ab through autophagy pathways.

Cadmium (Cd) is a common environmental pollutant, which leads to Cd residue in aquatic animals. The Cd in aquatic animals will be enriched into the human body through the food chain and seriously harm human health. The research aims to investigate the molecular mechanism of Cd poisoning in common carps. Our previous studies have confirmed that 23 differentially expressed miRNAs were potential biomarkers for Cd exposure in common carp head kidney lymphocytes. Herein, based on the prediction of the website and previous studies, miR-451 and cacna1ab were selected and their targeting relationship was verified again by dual-luciferase. Subsequently, we established the miR-451 overexpression/knockdown models and miR-451 inhibitor, cacna1ab co-knockdown models in common carp head kidney lymphocytes respectively. Immunofluorescence staining, MDC staining, calcium staining, qRT-PCR (Quantitative Real-time PCR) and western blot were used to detect the levels of autophagy. Our results demonstrated that Cd significantly decreased the expression of miR-451, miR-451 suppression thereby induced increased cacna1ab and the expression of ATG5, LC3-I, LC3-II and Beclin 1, while significantly inhibiting the expression of mTOR, P62 and Bcl-2, which indicated that autophagy was triggered. Moreover, the miR-451 knockdown group activated the expression of autophagy related factors as well as the Cd group. However, cacna1ab knockdown can reduce autophagy activation induced by miR-451 knockdown. Our results indicated that Cd induced autophagy in head kidney lymphocytes through the inhibition of miR-451 and the excitation of cacna1ab.

Macrophage migration inhibitory factor (MIF) of golden pompano (Trachinotus ovatus) is involved in the antibacterial immune response

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with a unique structure involved in immune regulation and inflammation. In the present study, we identified a MIF from Trachinotus ovatus (golden pompano) and analyzed its function. TroMIF shares high homology (58.26%–94.78%) with the other known MIF sequences of vertebrates. TroMIF is most closely related to large yellow croaker (Larimichthys crocea). The expression of TroMIF was most abundant in the liver and head kidney, and was significantly up-regulated after Edwardsiella tarda infection. The subcellular localization of TroMIF was mostly distributed in the cytoplasm. In vitro results revealed that the recombinant protein rTroMIF could inhibit the migration of head kidney lymphocytes (HKLs) and macrophages (HKMs) and enhance the phagocytic activity of HKMs. As a pro-inflammatory cytokine, rTroMIF could increase the expression levels of some pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1β), IL-6, IL-8, and interferon-gamma (IFN-γ) and decrease the expression of IL-10. The rTroMIF was proved to have enzymatic redox activity in vitro. Furthermore, overexpression of TroMIF in the head kidney cell line of golden pompano could significantly enhance its ability to resist E. tarda infection from 1 h to 4 h. The knockdown of TroMIF expression induced a significant increase in the number of bacteria after E. tarda infection at 1, 2, and 4 hpi. Our results suggest that TroMIF is an essential effector of the innate immune system and plays a pivotal role in antibacterial immunity.