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Cells Apr 2022The anterior-posterior (AP) axis in chordates is regulated by a conserved set of genes and signaling pathways, including genes and retinoic acid (RA), which play...
The anterior-posterior (AP) axis in chordates is regulated by a conserved set of genes and signaling pathways, including genes and retinoic acid (RA), which play well-characterized roles in the organization of the chordate body plan. The intermediate mesoderm (IM), which gives rise to all vertebrate kidneys, is an example of a tissue that differentiates sequentially along this axis. Yet, the conservation of the spatiotemporal regulation of the IM across vertebrates remains poorly understood. In this study, we used a comparative developmental approach focusing on non-conventional model organisms, a chondrichthyan (catshark), a cyclostome (lamprey), and a cephalochordate (amphioxus), to assess the involvement of RA in the regulation of chordate and vertebrate pronephros formation. We report that the anterior expression boundary of early pronephric markers ( and ), positioned at the level of somite 6 in amniotes, is conserved in the catshark and the lamprey. Furthermore, RA, driving the expression of genes like in amniotes, regulates the anterior pronephros boundary in the catshark. We find no evidence for the involvement of this regulatory hierarchy in the AP positioning of the lamprey pronephros and the amphioxus pronephros homolog, Hatschek's nephridium. This suggests that despite the conservation of and expressions in chordate pronephros homologs, the responsiveness of the IM, and hence of pronephric genes, to RA- and -dependent regulation is a gnathostome novelty.
Topics: Animals; Chordata; Genes, Homeobox; Lampreys; Pronephros; Tretinoin; Vertebrates
PubMed: 35455988
DOI: 10.3390/cells11081304 -
Fish & Shellfish Immunology Apr 2022Cantharidin is a toxic vesicant terpene used in folk and traditional medicine due to its various therapeutic effects. Since there are no previous data on the effect of...
Cantharidin is a toxic vesicant terpene used in folk and traditional medicine due to its various therapeutic effects. Since there are no previous data on the effect of cantharidin in fish, this study aimed to investigate the in vitro related-inflammatory effects of cantharidin in gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs). In the first experiment, the HKLs were incubated with 0, 5 and 10 μg mL of cantharidin for 24 h to delimit its possible toxic effects. In a second experiment, leucocytes were incubated with ranging concentrations from 0 to 10 μg mL for 3, 6, or 12 h. Cell viability was higher in acidophilic granulocytes than in monocytes/macrophages and lymphocytes. Cantharidin caused apoptosis as was evidenced by transmission electron microscopy. In addition, cantharidin produced a time- and dose-dependent decrease of respiratory burst and phagocytic activities in HKLs, while their peroxidase activity was increased at 24 h of incubation with 5 and 10 μg mL of cantharidin. Different changes in the gene expression were observed after incubation with cantharidin. While the gene expression of tnfa, il1b and crel was up-regulated in HKLs, the nfkb1 and igmh genes were down-regulated in comparison to the expression found in control HKLs. Present results offer a first view of the possible effects and action mechanisms of cantharidin in HKLs, as well as its implication in the inflammatory process, which could be of interest not only for basic research but also in the aquaculture sector.
Topics: Animals; Cantharidin; Head Kidney; Kidney; Leukocytes; Sea Bream
PubMed: 35218974
DOI: 10.1016/j.fsi.2022.02.045 -
Toxins Feb 2022subsp. () is a Gram-negative bacterium that infects a large number of marine fish species in Europe, Asia, and America, both in aquacultures and in the natural...
subsp. () is a Gram-negative bacterium that infects a large number of marine fish species in Europe, Asia, and America, both in aquacultures and in the natural environment. Among the affected hosts are economically important cultured fish, such as sea bream (), sea bass (), yellowtail (), and cobia (). The best characterized virulence factor of is the Apoptosis-Inducing Protein of 56 kDa (AIP56), a secreted AB-type toxin that has been shown to induce apoptosis of sea bass phagocytes during infection. AIP56 has an A subunit that displays metalloprotease activity against NF-kB p65 and a B subunit that mediates binding and internalization of the A subunit in susceptible cells. Despite the fact that the gene is highly prevalent in isolates from different fish species, the toxicity of AIP56 has only been studied in sea bass. In the present study, the toxicity of AIP56 for sea bream was evaluated. Ex vivo assays showed that sea bream phagocytes are resistant to AIP56 cytotoxicity and that resistance was associated with an inefficient internalization of the toxin by those cells. Accordingly, in vivo intoxication assays revealed that sea bream is much more resistant to AIP56-induced lethality than sea bass. These findings, showing that the effect of AIP56 is different in sea bass and sea bream, set the basis for future studies to characterize the effects of AIP56 and to fully elucidate its virulence role in different susceptible hosts.
Topics: Animals; Apoptosis Regulatory Proteins; Bacterial Proteins; Bacterial Toxins; Bass; Head Kidney; Leukocytes; Liver; Photobacterium; Sea Bream; Spleen; Transcription Factor RelA
PubMed: 35202146
DOI: 10.3390/toxins14020119 -
Pathogens & Immunity 2022Uropathogenic (UPEC) infections are common and when they disseminate can be of high morbidity.
BACKGROUND
Uropathogenic (UPEC) infections are common and when they disseminate can be of high morbidity.
METHODS
We studied the effects of UPEC infection using single cell RNA sequencing (scRNAseq) in zebrafish. Bulk RNA sequencing has historically been used to evaluate gene expression patterns, but scRNAseq allows gene expression to be evaluated at the single cell level and is optimal for evaluating heterogeneity within cell types and rare cell types. Zebrafish cohorts were injected with either saline or UPEC, and scRNAseq and canonical pathway analyses were performed.
RESULTS
Canonical pathway analysis of scRNAseq data provided key information regarding innate immune pathways in the cells determined to be thymus cells, ionocytes, macrophages/monocytes, and pronephros cells. Pathways activated in thymus cells included interleukin 6 (IL-6) signaling and production of reactive oxygen species. Fc receptor-mediated phagocytosis was a leading canonical pathway in the pronephros and macrophages. Genes that were downregulated in UPEC vs saline exposed embryos involved the cellular response to the Gram-negative endotoxin lipopolysaccharide (LPS) and included Forkhead Box O1a , Tribbles Pseudokinase 3 (, Arginase 2 ( and Polo Like Kinase 3 (
CONCLUSIONS
Because 4-day post fertilization zebrafish embryos only have innate immune systems, the scRNAseq provides insights into pathways and genes that cell types utilize in the bacterial response. Based on our analysis, we have identified genes and pathways that might serve as genetic targets for treatment and further investigation in UPEC infections at the single cell level.
PubMed: 35178490
DOI: 10.20411/pai.v7i1.479 -
International Journal of Molecular... Jan 2022Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies...
Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream () after subsp. () infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 µL of PBS (placebo) or 100 µL of exponentially growing (1 × 10 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.
Topics: Animals; Blood Cells; Fish Proteins; Gene Expression Regulation; Head Kidney; Immunity; Immunity, Humoral; Immunity, Innate; Oxidative Stress; Photobacterium; Sea Bream
PubMed: 35163486
DOI: 10.3390/ijms23031561 -
Frontiers in Immunology 2021Teleost fish anterior kidney (AK) is an important hematopoietic organ with multifarious immune cells, which have immune functions comparable to mammalian bone marrow....
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 CD3CD4CD8, CD3CD4CD8, CD4CD8, and CD4CD8 T cells existed in AK, but neither CD4CD8 nor CD4CD8 T cells can be further classified into subsets based on the known TFs and cytokines. Pseudotemporal analysis demonstrated that CD4CD8 and CD4CD8 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.
Topics: Animals; Cichlids; Head Kidney; Immunophenotyping; Leukocytes; RNA-Seq; Single-Cell Analysis
PubMed: 35027916
DOI: 10.3389/fimmu.2021.783196 -
Frontiers in Immunology 2021The intestine has many types of cells that are present mostly in the epithelium and lamina propria. The importance of the intestinal cells for the mammalian mucosal... (Comparative Study)
Comparative Study
The intestine has many types of cells that are present mostly in the epithelium and lamina propria. The importance of the intestinal cells for the mammalian mucosal immune system is well-established. However, there is no in-depth information about many of the intestinal cells in teleosts. In our previous study, we reported that adherent intestinal cells (AIC) predominantly express macrophage-related genes. To gather further evidence that AIC include macrophage-like cells, we compared their phagocytic activity and morphology with those of adherent head kidney cells (AKC), previously characterized as macrophage-like cells. We also compared equally abundant as well as differentially expressed mRNAs and miRNAs between AIC and AKC. AIC had lower phagocytic activity and were larger and more circular than macrophage-like AKC. RNA-Seq data revealed that there were 18309 mRNAs, with 59 miRNAs that were equally abundant between AIC and AKC. Integrative analysis of the mRNA and miRNA transcriptomes revealed macrophage heterogeneity in both AIC and AKC. In addition, analysis of AIC and AKC transcriptomes revealed functional characteristics of mucosal and systemic macrophages. Five pairs with significant negative correlations between miRNA and mRNAs were linked to macrophages and epithelial cells and their interaction could be pointing to macrophage activation and differentiation. The potential macrophage markers suggested in this study should be investigated under different immune conditions to understand the exact macrophage phenotypes.
Topics: Animals; Biodiversity; Cell Adhesion; Cell Communication; Cell Differentiation; Head Kidney; Intestines; Macrophage Activation; Macrophages; MicroRNAs; Phagocytosis; RNA, Messenger; Salmon; Sequence Analysis, RNA; Transcriptome
PubMed: 35003123
DOI: 10.3389/fimmu.2021.798156 -
Frontiers in Immunology 2021The mechanisms underlying -induced mycobacteriosis remain unexplored. Using head kidney macrophages (HKM) from catfish (), we report that Ca surge across...
The mechanisms underlying -induced mycobacteriosis remain unexplored. Using head kidney macrophages (HKM) from catfish (), we report that Ca surge across mitochondrial-Ca uniporter (MICU), and consequent mitochondrial ROS (mtROS) production, is imperative for mycobactericidal activity. Inhibition of mtROS alleviated HKM apoptosis and enhanced bacterial survival. Based on RNA interference (RNAi) and inhibitor studies, we demonstrate that the Toll-like receptor (TLR)-2-endoplasmic reticulum (ER) stress-store-operated calcium entry (SOCE) axis is instrumental for activating the mt-Ca/mtROS cascade in -infected HKM. Additionally, pharmacological inhibition of mtROS attenuated the expression of , , and , which suggests a positive feedback loop between ER-stress-induced SOCE and mtROS production. Elevated tumor necrosis factor alpha (TNF-α) levels and caspase-8 activity were observed in HKM consequent to infection, and our results implicate that mtROS is crucial in activating the TNF-mediated caspase-8 activation. Our results for the first time demonstrate mitochondria as an innate immune signaling center regulating mycobacteriosis in fish. We conclude that induced persistent SOCE signaling leads to mtROS production, which in turn activates the TNF-α/caspase-8 axis culminating in HKM apoptosis and bacterial clearance.
Topics: Animals; Calcium; Catfishes; Head Kidney; Macrophages; Mitochondria; Mycobacterium Infections, Nontuberculous; Mycobacterium fortuitum; Reactive Oxygen Species; Signal Transduction; Toll-Like Receptor 2
PubMed: 34987503
DOI: 10.3389/fimmu.2021.748758 -
Molecular Immunology Feb 2022Fungal diseases of fish are a significant economic problem in aquaculture. Using high-throughput expression analysis, we identified potential transcript markers in...
Fungal diseases of fish are a significant economic problem in aquaculture. Using high-throughput expression analysis, we identified potential transcript markers in primary head kidney and secondary embryonic cells from salmonid fish after stimulation with the inactivated fungi Mucor hiemalis and Fusarium aveneacium and with purified fungal molecular patterns. The transcript levels of most of the 45 selected genes were altered in head-kidney cells after 24 h of stimulation with fungal antigens. Stimulation with the inactivated fungus M. hiemalis induced the most pronounced transcriptional changes, including the pathogen receptor-encoding genes CLEC18A and TLR22, the cytokine-encoding genes IL6 and TNF, and the gene encoding the antimicrobial peptide LEAP2. In parallel, we analyzed the total GlcNAcylation status of embryonic salmonid cells with or without stimulation with inactivated fungi. O-GlcNAcylation modulates gene expression, intracellular protein, and signal activity, but we detected no significant differences after a 3-h stimulation. A pathway analysis tool identified the "apoptosis of leukocytes" based on the expression profile 24 h after fungal stimulation. Fluorescence microscopy combined with flow cytometry revealed apoptosis in 50 % of head-kidney leukocytes after 3 h stimulation with M. hiemalis, but this level decreased by > 5% after 24 h of stimulation. The number of apoptotic cells significantly increased in all blood cells after a 3-h stimulation with fungal molecular patterns compared to unstimulated controls. This in vitro approach identified transcript-based parameters that were strongly modulated by fungal infections of salmonid fish.
Topics: Acetylglucosamine; Animals; Antimicrobial Cationic Peptides; Apoptosis; Fish Diseases; Fusarium; Gene Expression Regulation, Developmental; Head Kidney; Interleukin-6; Lectins, C-Type; Mucor; Mycoses; Oncorhynchus mykiss; Protein Processing, Post-Translational; Salmon; Toll-Like Receptor 3; Tumor Necrosis Factor-alpha
PubMed: 34979452
DOI: 10.1016/j.molimm.2021.12.019 -
Frontiers in Immunology 2021β-Glucans (BG) are glucose polymers which are produced in bacteria and fungi but not in vertebrate organisms. Being recognized by phagocytic leukocytes including...
β-Glucans (BG) are glucose polymers which are produced in bacteria and fungi but not in vertebrate organisms. Being recognized by phagocytic leukocytes including macrophages and neutrophils through receptors such as dectin-1 and Complement receptor 3 (CR3), the BG are perceived by the innate immune system of vertebrates as foreign substances known as Pathogen Associated Molecular Patterns (PAMPs). The yeast-derived BG has been recognized for its potent biological activity and it is used as an immunomodulator in human and veterinary medicine. The goal of the current study was to characterize the immunostimulatory activity of soluble yeast BG in primary cultures of Atlantic salmon () head kidney leukocytes (HKLs) in which phagocytic cell types including neutrophils and mononuclear phagocytes predominate. The effect of BG on the secretome of HKL cultures, including secretion of extracellular vesicles (EVs) and soluble protein55s was characterized through western blotting and mass spectrometry. The results demonstrate that, along with upregulation of proinflammatory genes, BG induces secretion of ubiquitinated proteins (UbP), MHCII-containing EVs from professional antigen presenting cells as well as proteins derived from granules of polymorphonuclear granulocytes (PMN). Among the most abundant proteins identified in BG-induced EVs were beta-2 integrin subunits, including CD18 and CD11 homologs, which highlights the role of salmon granulocytes and mononuclear phagocytes in the response to soluble BG. Overall, the current work advances the knowledge about the immunostimulatory activity of yeast BG on the salmon immune system by shedding light on the effect of this PAMP on the secretome of salmon leukocytes.
Topics: Animals; Extracellular Vesicles; Gene Expression Profiling; Head Kidney; Immunity, Innate; Leukocytes; Phagocytes; Salmo salar; Secretome; beta-Glucans
PubMed: 34917074
DOI: 10.3389/fimmu.2021.736964