Fish Immune Organs Research Articles

LC-qTOF-MS analysis of fish immune organs reveals the distribution of amino acids in response to metabolic adaptation of the survival phenotype in grouper against Vibrio infection.

The online version contains supplementary material available at 10.1007/s13205-022-03269-1.

New insights into the role of (2-Carboxyethyl) dimethylsulfonium Bromide (Br-DMPT) in enhancing the structural integrity of immune organs of on-growing grass carp (Ctenopharyngodon idella)

The immune function operation of fish immune organs depends tightly on the immune organs' structural integrity which is mainly regulated by various biological respects such as oxidative status, apoptosis and tight junctions. Previous explorations have documented that dietary (2-carboxyethyl) dimethylsulfonium Bromide (Br-DMPT) intervention boosted fish growth in association with the strengthened immune function of the head kidney, spleen and skin. Here, further exploration was performed to better decipher the primary action mechanisms by which Br-DMPT maintains the immune organs' integrity of fish. In this research, grass carp (Ctenopharyngodon idella) (216.49 ± 0.29 g) as a model were fed five graded levels of Br-DMPT diets (0-520.0 mg/kg) in triplicate for 60 days, followed by a challenge with Aeromonas hydrophila for 2 weeks. Subsequently, immune organ samples were selectively collected to investigate oxidative status, apoptosis and tight junctions associated parameters. Our data from above three immune organs manifested that compared to the control group, 260.0 mg/kg Br-DMPT diet group: (1) markedly decreased oxidative damage degree as evidenced by the obtained lowest value in all oxidative damage indicators and exhibited the highest significant value in almost all antioxidant enzymes activities and corresponding gene expressions, where this action may be in part correlated with the modulation of Nrf2 signalling (P < 0.05); (2) signally alleviated cell apoptosis by altering the expression profiles of mitochondria and death receptor pathways involved genes (such as caspase-3,8 and FasL), where this mechanism may be partly connected with p38MAPK and JNK signalling (P < 0.05); (3) dramatically elevated tight junctions by regulating the expression profiles of a series of complexes (such as occludin and claudins), where the reason may be in part correlated with myosin light chain kinase (MLCK) signalling (P < 0.05). Noticeably, no statistical distinction was examined between all Br-DMPT diet groups with respect to the gene expressions of GPx4a, GPx4b, GSTP2, Bcl-2, claudin-b and claudin-7 in above diverse immune organs (P > 0.05). Overall, for the first time, our present results confirm that the enhanced structural integrity of immune organs in Br-DMPT- supplemented fish appears to partly be associated with the increased antioxidant status and the inhibited cell apoptosis, as well as the elevated tight junctions, which provides groundbreaking insight into the undiscovered role of Br-DMPT in accelerating fish growth and increases Br-DMPT more potential to be popularly applied in future fishery production.

(2-Carboxyethyl) dimethylsulfonium bromide supplementation in non-fish meal diets for on-growing grass carp (Ctenopharyngodon idella): Beneficial effects on immune function of the immune organs via modulation of NF-κB and TOR signalling pathway

The immune function of immune organs is extremely crucial for maintaining organism health status, which ultimately affects fish growth. Our previous study has found that dietary supplementation of (2-carboxyethyl)dimethylsulfonium Bromide (Br-DMPT) in non-fish meal (NFM) diet could promote the growth of grass carp (Ctenopharyngodon idella), whereas the underlying reason or mechanism for this results is largely unclear. Herein, we further explored whether dietary supplementation of Br-DMPT promoted fish growth is connected with the enhanced immune function in the immune organs (the head kidney, spleen and skin). In this study, 540 fish (216.49 ± 0.29 g) were irregularly distributed to six groups with three replicates (30 fish replicate−1) and fed corresponding diets group containing a fish meal (FM) diet group and five different NFM diets supplemented with gradational Br-DMPT (0–520.0 mg/kg level) group for 60 days. After the 60-days feeding trial, 8 fish from each replicate were selected and then conducted a challenge test with A. hydrophila for 14 days. Our results indicated that in the NFM diets, appropriate Br-DMPT: (1) significantly decreased the morbidity of skin haemorrhage and lesion after A. hydrophila infection (P < 0.05). (2) significantly improved the innate and adaptive immune components (lysozyme, complement 3, immunoglobulin M and antibacterial peptides et al.) (P < 0.05). (3) increased the gene expressions of main anti-inflammatory cytokines partially by referring to TOR signalling pathway, and decreased the gene expressions of main pro-inflammatory cytokines partially by referring to NF-kB signalling pathway (P < 0.05). Strikingly, no statistical difference could be found in the most of above immune parameters between 260.0 mg/kg Br-DMPT diet group and FM diet group (P > 0.05). Taken together, in non-fish meal diet, appropriate supplementation of Br-DMPT could improve the disease resistance capacity, non-specific immunity and ameliorate inflammation, and simultaneously could mitigate these adverse effects induced by the non-fish meal diet in fish immune organs. Moreover, this study may be helpful to decipher the underlying mechanisms of how Br-DMPT promote fish growth by immune organs and also provide scientific theoretical evidence for the future application of Br-DMPT as a new immunopotentiator in aquaculture industry.

Diphenyl diselenide dietary supplementation protects against methylmercury-chloride-induced immunotoxicity in the head kidney and spleen of grass carp (Ctenopharyngodon idella) via regulation of purinergic signaling and the NLRP3 inflammasome

This study aimed to evaluate whether dietary supplementation with diphenyl diselenide (Ph2Se2) would prevent the impaired immune and inflammatory responses elicited by methylmercury chloride (CH3HgCl) via protective effects on purinergic signaling in fish immune organs. Tissue and lymphocytic nucleoside triphosphate diphosphohydrolase (NTPDase) activity for adenosine triphosphate (ATP) and adenosine diphosphate (ADP) was downregulated in the head kidney and spleen of grass carp (Ctenopharyngodon idella) exposed to CH3HgCl. Concomitantly, adenosine deaminase (ADA) activity was upregulated. Further, nucleotide-binding oligomerization domain-like receptor (NLRP3) inflammasome gene expression was upregulated in the spleen and head kidney of CH3HgCl-exposed grass carp. Dietary supplementation with Ph2Se2 ameliorated these CH3HgCl-mediated alterations on purinergic enzymes, and their activities returned to baseline levels (except NTPDase activity for ADP). Based on these results, purinergic signaling in immune organs and lymphocytes can be considered a pathway linked to pro-inflammatory effects during exposure to environmental CH3HgCl concentrations, which may contribute to mortality of the affected fish. Since dietary supplementation with 3 mg Ph2Se2/kg in the feed prevented the CH3HgCl-induced alterations, it can be considered a potential suitable treatment to prevent impaired immune and inflammatory responses caused by Hg.

Molecular and biological characterization of gamma-interferon-inducible lysosomal thiol reductase in silver carp (Hypophthalmichthys molitrix)

Gamma-interferon-inducible lysosomal thiol reductase (GILT) plays an important role in the processing of major histocompatibility complex (MHC) class II-restricted antigens by catalyzing disulfide bonds reduction. Herein, a GILT homolog (ScGILT) was identified from silver carp. Its open reading frame covers 771 base pairs, encoding a protein of 256 amino acids that possesses GILT signature sequence CQHGX2ECX2NX4C, active-site CXXC motif, and two potential N-linked glycosylation sites. The predicted tertiary structures of ScGILT and other GILTs were quite similar in shape and positional arrangement of the key motifs. ScGILT mRNA was constitutively expressed in all detected tissues, with high-level expression in fish immune organs, spleen and head kidney. After stimulation with lipopolysaccharide, the expression of ScGILT mRNA significantly increased in spleen and head kidney cells, and ScGILT protein translocated to late endosomes and lysosomes in HeLa cells. Recombinant ScGILT fused with a His6 tag was expressed and purified, and could reduce the interchain disulfide bonds of IgG at pH 4.5. These results suggested that ScGILT was capable of catalyzing disulfide bonds reduction, and then might play an important role in the processing of MHC class II-restricted antigens in silver carp.

Expression analysis of three immune genes Interferon-gamma, Mx and Interferon regulatory factor-1 of Japanese flounder ( Paralichthys olivaceus )

ABSTRACT IFN-γ (Interferon-gamma), Mx and IRF-1 (Interferon regulatory factor-1) are main immune-related genes and they play important roles in the innate immune system of vertebrates. In this study, the expression level of the three immune-related genes in twelve tissues of normal adult Japanese flounder was detected using semi-quantitative RT-PCR. Thirteen time points (3h, 6h, 9h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h, 120h) were selected to analyze the expression of IFN-γ, Mx and IRF-1 in spleen, head kidney, liver of Japanese flounder infected with Lipopolysaccharide (LPS). The Japanese flounder IFN-γ, Mx and IRF-1 genes were differently expressed in these tissues and had high expression levels in classical fish immune organs like spleen and head kidney. It was found that the highest expression levels of the Japanese flounder IFN-γ were detected at 24h in spleen, 36h in head kidney and 48h in liver after challenge with LPS. Interestingly, the highest expression levels of Mx in spleen and head kidney were both at 36h and IRF-1 in spleen and liver were both at 24h. The highest expression level of Mx in liver was at 48h and IRF-1 in head kidney was at 12h. The study provides a basis for further research on immune mechanism of IFN-γ, Mx, IRF-1 and the production of recombinant IFN-γ, Mx or IRF-1 used in Japanese flounder cultivation in future.

Inducible headkidney cytochrome P450 contributes to endosulfan immunotoxicity in walking catfish Clarias gariepinus

The effect of endosulfan metabolites on fish immune system is not well known. It is also not clear whether endosulfan accumulates in fish immune organs and undergoes metabolic biotransformation in situ. In the present study we investigated the role of headkidney (HK), an important fish immune organ on endosulfan metabolism and the long term effects of endosulfan metabolites on the fish immune system. C. gariepinus (walking catfish) were exposed to 2.884ppb of endosulfan (1/10th LC50) for 30d followed by their maintenance in endosulfan-free water for 30d for recovery. Endosulfan induced time-dependent reduction in the HK somatic index and histo-pathological changes in renal and hemopoietic components of the organ. At cellular level, exposure to endosulfan led to death of HK leucocytes. Gas-liquid-chromatography documented the presence of both α- and β-isomers of endosulfan along with the toxic metabolite endosulfan sulfate (ESS) in the HK of exposed fishes. We report that β-endosulfan accumulates more readily in the HK. Depuration studies suggested the persistence of ESS in the HK. Enzyme-immunoassay and qPCR results demonstrated direct relationship between cytochrome P450 1A (CYP1A) expression and ESS levels in the HK. Pre-treatment of HKL with CYP1A specific inhibitor α-Naphthoflavone (ANF) led to reduction in CYP1A mRNA, protein levels, and inhibited ESS formation together implicating the role of CYP1A on endosulfan metabolism. When the exposed fish were transferred to endosulfan-free water (⿿recovered fish⿿) it was observed that after 30d of recovery period the concentration of endosulfan and its metabolite in the HK were significantly reduced, compared to 30-d exposed fish. We also observed improvement in HK histo-architecture but no significant recovery in HKL number and viability. Collectively, our findings suggest that HK plays an important role in endosulfan metabolism. We propose that endosulfan induces the activation of CYP1A in HK which led to the generation of persistent metabolite, ESS, resulting in immunotoxicity.

Dietary myo-inositol modulates immunity through antioxidant activity and the Nrf2 and E2F4/cyclin signalling factors in the head kidney and spleen following infection of juvenile fish with Aeromonas hydrophila.

This study was conducted to investigate the effects of the dietary vitamin myo-inositol (MI), on the immunity and structural integrity of the head kidney and spleen following infection of fish with the major freshwater pathogen bacterial Aeromonas hydrophila. The results demonstrated for the first time that MI deficiency depressed the lysozyme and acid phosphatase (ACP) activities and the complement 3 (C3) and C4 contents in the head kidney and spleen compared with the optimal MI levels, indicating that MI deficiency decreased the immunity of these important fish immune organs. The depression in immunity due to MI deficiency was partially related to oxidative damage [indicated by increases in the malondialdehyde (MDA) and protein carbonyl (PC) contents] that was in turn partially due to the decreased glutathione (GSH) content and the disturbances in antioxidant enzyme activities [total superoxide dismutase (T-SOD), CuZnSOD, MnSOD, catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR)]. MI deficiency inhibited the antioxidant-related gene transcription [CuZnSOD, MnSOD, CAT, GPx1a, GR and NF-E2-related factor 2 (Nrf2)] in the head kidney and spleen following infection of the fish with A.hydrophila. The oxidative damage due to MI deficiency also resulted in the inhibition of proliferation-associated signalling (cyclin D1, cyclin A, cyclin E and E2F4). Thus, MI deficiency partially inhibited damage repair. Excessive MI exhibited negative effects that were similar to MI deficiency, whereas the optimal MI content reversed those indicators. These observations indicated that an MI deficiency or excess could cause depression of the immune system that might be partially related to oxidative damage, antioxidant disturbances, and the inhibition of the proliferation-associated signalling in the head kidney and spleen following infection of fish with A.hydrophila. Finally, the optimal MI levels were 660.7 (based on ACP) and 736.8mgkg(-1) diet (based on MDA) in the head kidney and 770.5 (based on ACP) and 766.9mgkg(-1) diet (based on MDA) in the spleen of juvenile Jian carp.

Alteration in the cytokine levels and histopathological damage in common carp induced by glyphosate

Glyphosate is one of the most frequently used herbicides, and it has been demonstrated to generate a series of toxicological problems in animals and humans. However, relatively little is known about the effects of glyphosate on the immune system of fish. In the present study, the acute toxicity of glyphosate on common carp was first determined; then, the contents of interferon-γ (IFN-γ), interleukin-1β (IL-1β), and tumor necrosis factor -α (TNF-α) and histopathological alterations in the liver, kidneys, and spleen of common carp exposed to 52.08 or 104.15mgL−1 of glyphosate for 168h were also determined and evaluated. The results of the acute toxicity tests showed that the 96h LC50 of glyphosate for common carp was 520.77mgL−1. Moreover, sub-acute exposure of glyphosate altered the contents of IFN-γ, IL-1β, and TNF-α in fish immune organs. For example, there was a remarkable increase in the IFN-γ content in the kidneys, while there was a decrease in the liver and spleen. The IL-1β content increased in liver and kidneys, but it decreased in the spleen, and TNF-α mainly increased in the fish liver, kidneys, and spleen. In addition, glyphosate-exposure also caused remarkable histopathological damage in the fish liver, kidneys, and spleen. These results suggest that glyphosate-caused cytokine alterations may result in an immune suppression or excessive activation in the treated common carp as well as may cause immune dysfunction or reduced immunity. In conclusion, glyphosate has immunotoxic effects on common carp.

Growth hormone (GH) treatment acts on the endocrine and autocrine/paracrine GH/IGF-axis and on TNF-α expression in bony fish pituitary and immune organs

There exist indications that the growth hormone (GH)/insulin-like growth factor (IGF) axis may play a role in fish immune regulation, and that interactions occur via tumour necrosis factor (TNF)-α at least in mammals, but no systematic data exist on potential changes in GH, IGF-I, IGF-II, GH receptor (GHR) and TNF-α expression after GH treatment. Thus, we investigated in the Nile tilapia the influence of GH injections by real-time qPCR at different levels of the GH/IGF-axis (brain, pituitary, peripheral organs) with special emphasis on the immune organs head kidney and spleen. Endocrine IGF-I served as positive control for GH treatment efficiency. Basal TNF-α gene expression was detected in all organs investigated with the expression being most pronounced in brain. Two consecutive intraperitoneal injections of bream GH elevated liver IGF-I mRNA and plasma IGF-I concentration. Also liver IGF-II mRNA and TNF-α were increased while the GHR was downregulated. In brain, no change occurred in the expression levels of all genes investigated. GH gene expression was exclusively detected in the pituitary where the GH injections elevated both GH and IGF-I gene expression. In the head kidney, GH upregulated IGF-I mRNA to an even higher extent than liver IGF-I while IGF-II and GHR gene expressions were not affected. Also in the spleen, no change occurred in GHR mRNA, however, IGF-I and IGF-II mRNAs were increased. In correlation, in situ hybridisation showed a markedly higher amount of IGF-I mRNA in head kidney and spleen after GH injection. In both immune tissues, TNF-α gene expression showed a trend to decrease after GH treatment. The stimulation of IGF-I and also partially of IGF-II expression in the fish immune organs by GH indicates a local role of the IGFs in immune organ regulation while the differential changes in TNF-α support the in mammals postulated interactions with the GH/IGF-axis which demand for further investigations.

Expression profiles of matrix metalloproteinase 9 in teleost fish provide evidence for its active role in initiation and resolution of inflammation

Matrix metalloproteinase 9 (MMP-9) belongs to a family of zinc-dependent endopeptidases. As a consequence of its ability to cleave structural extracellular matrix molecules, mammalian MMP-9 is associated with vital inflammatory processes such as leucocyte migration and tissue remodelling and regeneration. Interestingly, MMP-9 genes have been identified in fish, but functional data are still limited and focus on the involvement of MMP-9 in embryonic development, reproduction and post-mortem tenderization. Here, we describe the involvement of MMP-9 in the innate immunity of carp. In carp, MMP-9 was most notably expressed in classical fish immune organs and in peritoneal and peripheral blood leucocytes, indicating a role of MMP-9 in immune responses. In our well-characterized zymosan-induced peritonitis model for carp, we analysed expression of the MMP-9 gene and the gelatinolytic levels of both pro- and activated forms of MMP-9. The biphasic profile of MMP-9 mRNA expression indicated involvement during the initial phase of inflammation and during the later phase of tissue remodelling. Also, in vitro stimulation of carp phagocytes with lipopolysaccharide or concanavalin A increased MMP-9 gene expression, with a peak at 24 hr. The increase of MMP-9 mRNA correlated with the peak of MMP-9 gelatinolytic level in culture supernatants. These results provide evidence for an evolutionarily conserved and relevant role of MMP-9 in the innate immune response.