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Fish & Shellfish Immunology Apr 2024Inducible nitric oxide (NO) synthase (iNOS) is a key immune mediator for production of inflammatory mediator NO from l-arginine. Tight regulation of iNOS expression and...
Extracellular ATP- and adenosine-mediated purinergic signaling modulates inducible nitric oxide synthase (iNOS) gene expression, enzyme activity and nitric oxide production in common carp (Cyprinus carpio) head kidney macrophages.
Inducible nitric oxide (NO) synthase (iNOS) is a key immune mediator for production of inflammatory mediator NO from l-arginine. Tight regulation of iNOS expression and enzyme activity is critical for proper NO productions under inflammation and infection conditions. However, the regulatory mechanism for iNOS expression and enzyme activity in fish remains largely unknown. Here, we show that extracellular ATP treatment significantly up-regulates iNOS gene expression and enzyme activity, and consequently leads to enhanced NO production in Cyprinus carpio head kidney macrophages (HKMs). We further show that the extracellular ATP-induced iNOS enzyme activity and NO production can be attenuated by pharmacological inhibition of the ATP-gated P2X4 and P2X7 receptors with their respective specific antagonists, but enhanced by overexpression of P2X4 and P2X7 receptors in grass carp ovary cells. In contrast, adenosine administration significantly reduces iNOS gene expression, enzyme activity and NO production in carp HKMs, and these inhibitory effects can be reversed by pharmacological inhibition of adenosine receptors with the antagonist XAC. Furthermore, LPS- and poly(I:C)-induced iNOS gene expression, enzyme activity, and NO production are significantly attenuated by blockade of P2X4 and P2X7 receptors with their respective specific antagonists in carp HKMs, while overexpression of P2X and P2X7 receptors results in enhanced iNOS gene expression, enzyme activity and NO production in LPS- and poly(I:C)-treated grass carp ovary cells. Taken together, we firstly report an opposite role of extracellular ATP/adenosine-mediated purinergic signaling in modulating iNOS-NO system activity in fish.
Topics: Animals; Adenosine; Nitric Oxide Synthase Type II; Nitric Oxide; Carps; Lipopolysaccharides; Head Kidney; Macrophages; Adenosine Triphosphate; Gene Expression
PubMed: 38423488
DOI: 10.1016/j.fsi.2024.109469 -
International Journal of Molecular... Feb 2024Cisplatin is an antineoplastic agent used to treat various tumors. In mammals, it can cause nephrotoxicity, tissue damage, and inflammation. The release of inflammatory...
Cisplatin is an antineoplastic agent used to treat various tumors. In mammals, it can cause nephrotoxicity, tissue damage, and inflammation. The release of inflammatory mediators leads to the recruitment and infiltration of immune cells, particularly neutrophils, at the site of inflammation. Cisplatin is often used as an inducer of acute kidney injury (AKI) in experimental models, including zebrafish (), due to its accumulation in kidney cells. Current protocols in larval zebrafish focus on studying its effect as an AKI inducer but ignore other systematic outcomes. In this study, cisplatin was added directly to the embryonic medium to assess its toxicity and impact on systemic inflammation using locomotor activity analysis, qPCR, microscopy, and flow cytometry. Our data showed that larvae exposed to cisplatin at 7 days post-fertilization (dpf) displayed dose-dependent mortality and morphological changes, leading to a decrease in locomotion speed at 9 dpf. The expression of pro-inflammatory cytokines such as interleukin , , and increased after 48 h of cisplatin exposure. Furthermore, while a decrease in the number of neutrophils was observed in the glomerular region of the pronephros, there was an increase in neutrophils throughout the entire animal after 48 h of cisplatin exposure. We demonstrate that cisplatin can have systemic effects in zebrafish larvae, including morphological and locomotory defects, increased inflammatory cytokines, and migration of neutrophils from the hematopoietic niche to other parts of the body. Therefore, this protocol can be used to induce systemic inflammation in zebrafish larvae for studying new therapies or mechanisms of action involving neutrophils.
Topics: Animals; Cisplatin; Zebrafish; Neutrophils; Larva; Acute Kidney Injury; Inflammation; Cytokines; Mammals
PubMed: 38397041
DOI: 10.3390/ijms25042363 -
Marine Biotechnology (New York, N.Y.) Apr 2024The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation, is studied. First, gilthead seabream (Sparus aurata L.)...
The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation, is studied. First, gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs) were incubated with λ-carrageenin to study the expression of hepcidin and iron metabolism-related genes. While the expression of most of the genes studied was upregulated, the expression of ferroportin gene (slc40a) was downregulated. In the second part of the study, seabream specimens were injected intramuscularly with λ-carrageenin or buffer (control). The expression of the same genes was evaluated in the head kidney, liver, and skin at different time points after injection. The expression of Hamp1m, ferritin b, and ferroportin genes (hamp1, fthb, and slc40a) was upregulated in the head kidney of fish from the λ-carrageenin-injected group, while the expression of Hamp2C and Hamp2E genes (hamp2.3 and hamp2.7) was downregulated. In the liver, the expression of hamp1, ferritin a (ftha), slc40a, Hamp2J, and Hamp2D (hamp2.5/6) genes was downregulated in the λ-carrageenin-injected group. In the skin, the expression of hamp1 and (Hamp2A Hamp2C) hamp2.1/3/4 genes was upregulated in the λ-carrageenin-injected group. A bioinformatic analysis was performed to predict the presence of transcription factor binding sites in the promoter region of hepcidins. The primary sequence of hepcidin was conserved among the different mature peptides, although changes in specific amino acid residues were identified. These changes affected the charge, hydrophobicity, and probability of hepcidins being antimicrobial peptides. This study sheds light on the poorly understood roles of hepcidins in fish. The results provide insight into the regulatory mechanisms of inflammation in fish and could contribute to the development of new strategies for treat inflammation in farm animals.
Topics: Animals; Sea Bream; Hepcidins; Fish Proteins; Inflammation; Liver; Fish Diseases; Head Kidney; Iron; Gene Expression Regulation; Leukocytes; Skin; Cation Transport Proteins; Ferritins; Promoter Regions, Genetic
PubMed: 38353762
DOI: 10.1007/s10126-024-10293-0 -
The Science of the Total Environment Mar 2024Reactive oxygen species (ROS) over-production and oxidative stress resulted from climate change and environmental pollution seriously endangered global fish populations...
Reactive oxygen species (ROS) over-production and oxidative stress resulted from climate change and environmental pollution seriously endangered global fish populations and healthy development of marine aquaculture. Peroxiredoxins (Prxs), a highly conserved family of thiol-specific antioxidants, can mitigate ROS and protect cells from oxidative stress. We previously demonstrated that large yellow croaker PrxIV (LcPrxIV) could not only regulate the pro-inflammatory responses, but also scavenge ROS. However, the underlying mechanism how LcPrxIV regulated immune response and redox homeostasis remains unknown. MicroRNAs (miRNAs) are non-coding RNAs that play important roles in the regulation of various biological processes. In this study, mRNA and miRNA expression profiles from LYCK-pcDNA3.1 and LYCK-PrxIV cells, with or without oxidative stress stimulated by HO were evaluated using high-throughput sequencing. A series of differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs), as well as DEM-DEG pairs were identified from each two-group comparison, respectively. GO and KEGG functional analyses indicated that most significant DEGs were associated with signaling pathways related to oxidative stress and immune response. Subsequent DEM-DEG interaction analysis revealed that miR-731 and miR-1388 may be involved in both redox regulation and immune response via synergistic effect with LcPrxIV. Interestingly, miR-731 could regulate the expression of different down-stream DEGs under different stimulations of LcPrxIV over-expression, HO, or both. Moreover, miR-731 could cause the DEG, γ-glutamyl hydrolase (GGH), to be expressed in opposite ways under different stimulations. On the other hand, the expression of miR-1388 could be negatively or positively regulated under the stimulation of LcPrxIV over-expression with or without oxidative stress, thus regulating gene expression of different mRNAs. Based on these results, we speculate that LcPrxIV may participate in immune response or redox regulation by regulating the expression of different down-stream genes through controlling the expression level of a certain miRNA or by regulating the varieties of expressed miRNAs.
Topics: Animals; MicroRNAs; Reactive Oxygen Species; RNA, Messenger; Head Kidney; Hydrogen Peroxide; Perciformes; Oxidation-Reduction; Gene Expression Profiling
PubMed: 38301776
DOI: 10.1016/j.scitotenv.2024.170503 -
Fish & Shellfish Immunology Mar 2024Single-cell transcriptomics is the current gold standard for global gene expression profiling, not only in mammals and model species, but also in non-model fish species....
Single-cell transcriptomics is the current gold standard for global gene expression profiling, not only in mammals and model species, but also in non-model fish species. This is a rapidly expanding field, creating a deeper understanding of tissue heterogeneity and the distinct functions of individual cells, making it possible to explore the complexities of immunology and gene expression on a highly resolved level. In this study, we compared two single cell transcriptomic approaches to investigate cellular heterogeneity within the head kidney of healthy farmed Atlantic salmon (Salmo salar). We compared 14,149 cell transcriptomes assayed by single cell RNA-seq (scRNA-seq) with 18,067 nuclei transcriptomes captured by single nucleus RNA-Seq (snRNA-seq). Both approaches detected eight major cell populations in common: granulocytes, heamatopoietic stem cells, erythrocytes, mononuclear phagocytes, thrombocytes, B cells, NK-like cells, and T cells. Four additional cell types, endothelial, epithelial, interrenal, and mesenchymal cells, were detected in the snRNA-seq dataset, but appeared to be lost during preparation of the single cell suspension submitted for scRNA-seq library generation. We identified additional heterogeneity and subpopulations within the B cells, T cells, and endothelial cells, and revealed developmental trajectories of heamatopoietic stem cells into differentiated granulocyte and mononuclear phagocyte populations. Gene expression profiles of B cell subtypes revealed distinct IgM and IgT-skewed resting B cell lineages and provided insights into the regulation of B cell lymphopoiesis. The analysis revealed eleven T cell sub-populations, displaying a level of T cell heterogeneity in salmon head kidney comparable to that observed in mammals, including distinct subsets of cd4/cd8-negative T cells, such as tcrγ positive, progenitor-like, and cytotoxic cells. Although snRNA-seq and scRNA-seq were both useful to resolve cell type-specific expression in the Atlantic salmon head kidney, the snRNA-seq pipeline was overall more robust in identifying several cell types and subpopulations. While scRNA-seq displayed higher levels of ribosomal and mitochondrial genes, snRNA-seq captured more transcription factor genes. However, only scRNA-seq-generated data was useful for cell trajectory inference within the myeloid lineage. In conclusion, this study systematically outlines the relative merits of scRNA-seq and snRNA-seq in Atlantic salmon, enhances understanding of teleost immune cell lineages, and provides a comprehensive list of markers for identifying major cell populations in the head kidney with significant immune relevance.
Topics: Animals; Salmo salar; Gene Expression Regulation; Head Kidney; Endothelial Cells; Gene Expression Profiling; Transcriptome; RNA, Small Nuclear; Mammals
PubMed: 38181891
DOI: 10.1016/j.fsi.2024.109357 -
Doklady Biological Sciences :... Oct 2023The erythroid lineage was studied in the flounder-gloss (Platichthys flesus Linnaeus, 1758) during the annual cycle. The erythrocyte count in the blood was determined...
The erythroid lineage was studied in the flounder-gloss (Platichthys flesus Linnaeus, 1758) during the annual cycle. The erythrocyte count in the blood was determined along with the contents of immature erythroid forms (basophilic and polychromatophilic normoblasts) in the head kidney (pronephros) and the blood. Cell proliferative activity was inferred from the [H]thymidine inclusion in circulating immature erythrocytes. Irregularity was observed in erythropoiesis occurring in flounder-gloss hematopoietic tissue. Intense production of erythroid mass was mainly associated with a post-spawning period. This was evident from an increase in the contents of immature erythroid forms in the pronephros and circulating blood and an increase in their proliferative activity. The changes were associated with peculiarities of the erythroid system organization, which precludes regular production of erythropoietin in the kidney in teleost fish.
Topics: Animals; Flounder
PubMed: 38087018
DOI: 10.1134/S0012496623700564 -
Development & Reproduction Sep 2023The Ruvb-like AAA ATPase1 (Ruvbl1; also known as Pontin) is an evolutionary conserved protein belonging to the adenosine triphosphates associated with diverse cellular...
The Ruvb-like AAA ATPase1 (Ruvbl1; also known as Pontin) is an evolutionary conserved protein belonging to the adenosine triphosphates associated with diverse cellular activities (AAA+) superfamily of ATPases. Ruvbl1 is a component of various protein supercomplexes and is involved in a variety of cellular activities, including chromatin remodeling, DNA damage repair, and mitotic spindle assembly however, the developmental significance of this protein is unknown and needs detailed investigation. We investigated the developmental significance of Ruvbl1 in multiciliated cells of the epidermis since is expressed in the multiciliated cells and pronephros during embryogenesis. The knockdown of significantly impaired cilia-driven fluid flow and basal body polarity in the epidermis compared to control embryos, but did not affect cilia morphology. Our results suggest that Ruvbl1 plays a significant role in embryonic development by regulating ciliary beating; however, further investigation is needed to determine the mechanisms involved.
PubMed: 38074458
DOI: 10.12717/DR.2023.27.3.159 -
Scientific Reports Nov 2023Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the...
Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS kidney cells and injection into ctns zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns larvae, and restoration of the zebrafish pronephros function.
Topics: Animals; Cystinosis; Cystine; Zebrafish; RNA, Messenger; Models, Theoretical; Dietary Supplements; Amino Acid Transport Systems, Neutral
PubMed: 38016974
DOI: 10.1038/s41598-023-47085-w -
Fish & Shellfish Immunology Dec 2023As one of short-chain fatty acids, butyrate is an important metabolite of dietary fiber by the fermentation of gut commensals. Our recent study uncovered that butyrate...
As one of short-chain fatty acids, butyrate is an important metabolite of dietary fiber by the fermentation of gut commensals. Our recent study uncovered that butyrate promoted IL-22 production in fish macrophages to augment the host defense. In the current study, we further explored the underlying signaling pathways in butyrate-induced IL-22 production in fish macrophages. Our results showed that butyrate augmented the IL-22 expression in head kidney macrophages (HKMs) of turbot through binding to G-protein receptor 41 (GPR41) and GPR43. Moreover, histone deacetylase 3 (HDAC3) inhibition apparently up-regulated the butyrate-enhanced IL-22 generation, indicating HDACs were engaged in butyrate-regulated IL-22 secretion. In addition, butyrate triggered the STAT3/HIF-1α signaling to elevate the IL-22 expression in HKMs. Importantly, the evidence in vitro and in vivo was provided that butyrate activated autophagy in fish macrophages via IL-22 signaling, which contributing to the elimination of invading bacteria. In conclusion, we clarified in the current study that butyrate induced STAT3/HIF-1α/IL-22 signaling pathway via GPCR binding and HDAC3 inhibition in fish macrophages to activate autophagy that was involved in pathogen clearance in fish macrophages.
Topics: Animals; Butyrates; Flatfishes; Head Kidney; Macrophages; Signal Transduction; Autophagy; Interleukin-22
PubMed: 37977544
DOI: 10.1016/j.fsi.2023.109214 -
The Science of the Total Environment Jan 2024Edema represents a notable outcome in fishes exposed to aquatic pollutants, yet the underlying etiology remains inadequately understood. This investigation delves into...
Edema represents a notable outcome in fishes exposed to aquatic pollutants, yet the underlying etiology remains inadequately understood. This investigation delves into the etiological factors of edema formation in 7 days post fertilization (dpf) zebrafish larvae following their exposure to InP/ZnS quantum dots (QDs), which was chosen as a prototypical edema inducer. Given the fundamental role of the kidney in osmoregulation, we used transgenic zebrafish lines featuring fluorescent protein labeling of the glomerulus, renal tubule, and blood vessels, in conjunction with histopathological scrutiny. We identified the pronounced morphological and structural aberrations within the pronephros. By means of tissue mass spectrometry imaging and hyperspectral microscopy, we discerned the accumulation of InP/ZnS QDs in the pronephros. Moreover, InP/ZnS QDs impeded the renal clearance capacity of the pronephros, as substantiated by diminished uptake of FITC-dextran. InP/ZnS QDs also disturbed the expression levels of marker genes associated with kidney development and osmoregulatory function at the earlier time points, which preceded the onset of edema. These results suggest that impaired fluid clearance most likely resulting from pronephros injury contributes to the emergence of zebrafish edema. Briefly, our study provides a perspective: the kidney developmental injury induced by exogenous substances may regulate edema in a zebrafish model.
Topics: Animals; Zebrafish; Quantum Dots; Larva; Kidney Glomerulus
PubMed: 37963533
DOI: 10.1016/j.scitotenv.2023.168420