Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish.

Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.

Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.

Tissue damage and infection are deemed likewise triggers of innate immune responses. But whereas neutrophil responses to microbes are generally protective, neutrophil recruitment into damaged tissues without infection is deleterious. Why neutrophils respond to tissue damage and not just to microbes is unknown. Is it a flaw of the innate immune system that persists because evolution did not select against it, or does it provide a selective advantage? Here we dissect the contribution of tissue damage signaling to antimicrobial immune responses in a live vertebrate. By intravital imaging of zebrafish larvae, a powerful model for innate immunity, we show that prevention of tissue damage signaling upon microbial ear infection abrogates leukocyte chemotaxis and reduces animal survival, at least in part, through suppression of cytosolic phospholipase A2 (cPla2), which integrates tissue damage- and microbe-derived cues. Thus, microbial cues are insufficient, and damage signaling is essential for antimicrobial neutrophil responses in zebrafish.

Host genotype-specific therapies can optimize the inflammatory response to mycobacterial infections.

Susceptibility to tuberculosis is historically ascribed to an inadequate immune response that fails to control infecting mycobacteria. In zebrafish, we find that susceptibility to Mycobacterium marinum can result from either inadequate or excessive acute inflammation. Modulation of the leukotriene A(4) hydrolase (LTA4H) locus, which controls the balance of pro- and anti-inflammatory eicosanoids, reveals two distinct molecular routes to mycobacterial susceptibility converging on dysregulated TNF levels: inadequate inflammation caused by excess lipoxins and hyperinflammation driven by excess leukotriene B(4). We identify therapies that specifically target each of these extremes. In humans, we identify a single nucleotide polymorphism in the LTA4H promoter that regulates its transcriptional activity. In tuberculous meningitis, the polymorphism is associated with inflammatory cell recruitment, patient survival and response to adjunctive anti-inflammatory therapy. Together, our findings suggest that host-directed therapies tailored to patient LTA4H genotypes may counter detrimental effects of either extreme of inflammation.

Macrophages provide a transient muscle stem cell niche via NAMPT secretion.

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.

Dynamic Changes in Lymphocyte Populations Establish Zebrafish as a Thymic Involution Model.

The thymus is the site of T lymphocyte development and T cell education to recognize foreign, but not self, Ags. B cells also reside and develop in the thymus, although their functions are less clear. During "thymic involution," a process of lymphoid atrophy and adipose replacement linked to sexual maturation, thymocytes decline. However, thymic B cells decrease far less than T cells, such that B cells comprise ∼1% of human neonatal thymocytes but up to ∼10% in adults. All jawed vertebrates possess a thymus, and we and others have shown zebrafish (Danio rerio) also have thymic B cells. In this article, we investigated the precise identities of zebrafish thymic T and B cells and how they change with involution. We assessed the timing and specific details of zebrafish thymic involution using multiple lymphocyte-specific, fluorophore-labeled transgenic lines, quantifying the changes in thymic T- and B-lymphocytes pre- versus postinvolution. Our results prove that, as in humans, zebrafish thymic B cells increase relative to T cells postinvolution. We also performed RNA sequencing on D. rerio thymic and marrow lymphocytes of four novel double-transgenic lines, identifying distinct populations of immature T and B cells. Collectively, this is, to our knowledge, the first comprehensive analysis of zebrafish thymic involution, demonstrating its similarity to human involution and establishing the highly genetically manipulatable zebrafish model as a template for involution studies.

Single-cell Transcriptional Landscape of Temporal Neutrophil Response to Burn Wound in Larval Zebrafish.

Neutrophils accumulate early in tissue injury. However, the cellular and functional heterogeneity of neutrophils during homeostasis and in response to tissue damage remains unclear. In this study, we use larval zebrafish to understand neutrophil responses to thermal injury. Single-cell transcriptional mapping of myeloid cells during a 3-d time course in burn and control larvae revealed distinct neutrophil subsets and their cell-cell interactions with macrophages across time and conditions. The trajectory formed by three zebrafish neutrophil subsets resembles human neutrophil maturation, with varying transition patterns between conditions. Through ligand-receptor cell-cell interaction analysis, we found that neutrophils communicate more in burns in a pathway and temporal manner. Finally, we identified the correlation between zebrafish myeloid signatures and human burn severity, establishing GPR84+ neutrophils as a potential marker of early innate immune response in burns. This work builds a comparative single-cell transcriptomic framework to identify neutrophil markers of tissue damage using model organisms.

Crystal Structures of DNA-bound Fish IRF10 and IRF11 Reveal the Determinants of IFN Regulation.

IFN regulatory factors (IRFs) are transcription factors that mediate homeostatic mechanisms of host defense against pathogens. In addition to IRF1-9, which are conserved across vertebrates, teleost fishes have two other IRFs, IRF10 and IRF11. In zebrafish (Danio rerio), IRF10 represses the expression of IFNφ1 and IFNφ3, whereas IRF11 exerts the opposite effect. In this study, we found IRF10 could significantly inhibit the expression of IFNφ1 and IFNφ3 induced by IFN11 to synergistically regulate type I IFN expression. To clarify the synergistically regulatory mechanism of IRF10 and IRF11 in type I IFN expression, we determined and analyzed the crystal structures of the DNA-binding domains (DBDs) of zebrafish IRF10 and IRF11 bound to DNA, as well as IRF11 DBD in apo form. The interactions of IRF10-DBD and IRF11-DBD with DNA backbone were elaborated in detail. Further analysis showed that IRF10 and IRF11 have the same binding patterns and comparable affinities with the IFN-sensitive response elements of IFNφ1 and IFNφ3 promoters. Therefore, IRF10 could function as a controlling factor for IRF11 by competitive binding of the IFN-sensitive response elements to coregulate the host IFN response. Accordingly, similar to IRF1 and IRF2 in mammals, IRF10 and IRF11 act as another pair of negative and positive regulators to balance the antiviral responses in fish.

LGP2 Facilitates Bacterial Escape through Binding Peptidoglycan via EEK Motif and Suppressing NOD2-RIP2 Axis in Cyprinidae and Xenocyprididae Families.

RIG-I-like receptors and NOD-like receptors play pivotal roles in recognizing microbe-associated molecular patterns and initiating immune responses. The LGP2 and NOD2 proteins are important members of the RIG-I-like receptor and NOD-like receptor families, recognizing viral RNA and bacterial peptidoglycan (PGN), respectively. However, in some instances bacterial infections can induce LPG2 expression via a mechanism that remains largely unknown. In the current study, we found that LGP2 can compete with NOD2 for PGN binding and inhibit antibacterial immunity by suppressing the NOD2-RIP2 axis. Recombinant CiLGP2 (Ctenopharyngodon idella LGP2) produced using either prokaryotic or eukaryotic expression platform can bind PGN and bacteria in pull-down and ELISA assays. Comparative protein structure models and intermolecular interaction prediction calculations as well as pull-down and colocalization experiments indicated that CiLGP2 binds PGN via its EEK motif with species and structural specificity. EEK deletion abolished PGN binding of CiLGP2, but insertion of the CiLGP2 EEK motif into zebrafish and mouse LGP2 did not confer PGN binding activity. CiLGP2 also facilitates bacterial replication by interacting with CiNOD2 to suppress expression of NOD2-RIP2 pathway genes. Sequence analysis and experimental verification demonstrated that LGP2 having EEK motif that can negatively regulate antibacterial immune function is present in Cyprinidae and Xenocyprididae families. These results show that LGP2 containing EEK motif competes with NOD2 for PGN binding and suppresses antibacterial immunity by inhibiting the NOD2-RIP2 axis, indicating that LGP2 plays a crucial negative role in antibacterial response beyond its classical regulatory function in antiviral immunity.

HIF-1 Transcriptionally Regulates Basal Expression of STING to Maintain Cellular Innate Immunity.

Stimulator of IFN genes (STING) is a critical component of the innate immune system, playing an essential role in defending against DNA virus infections. However, the mechanisms governing basal STING regulation remain poorly understood. In this study, we demonstrate that the basal level of STING is critically maintained by hypoxia-inducible factor 1 (HIF-1)α through transcription. Under normal conditions, HIF-1α binds constitutively to the promoter region of STING, actively promoting its transcription. Knocking down HIF-1α results in a decrease in STING expression in multiple cell lines and zebrafish, which in turn reduces cellular responses to synthetic dsDNAs, including cell signaling and IFN production. Moreover, this decrease in STING levels leads to an increase in cellular susceptibility to DNA viruses HSV-1 and pseudorabies virus. These findings unveil a (to our knowledge) novel role of HIF-1α in maintaining basal STING levels and provide valuable insights into STING-mediated antiviral activities and associated diseases.

Glucocorticoids Suppress NF-κB-Mediated Neutrophil Control of Aspergillus fumigatus Hyphal Growth.

Glucocorticoids are a major class of therapeutic anti-inflammatory and immunosuppressive drugs prescribed to patients with inflammatory diseases, to avoid transplant rejection, and as part of cancer chemotherapy. However, exposure to these drugs increases the risk of opportunistic infections such as with the fungus Aspergillus fumigatus, which causes mortality in >50% of infected patients. The mechanisms by which glucocorticoids increase susceptibility to A. fumigatus are poorly understood. In this article, we used a zebrafish larva Aspergillus infection model to identify innate immune mechanisms altered by glucocorticoid treatment. Infected larvae exposed to dexamethasone succumb to infection at a significantly higher rate than control larvae. However, both macrophages and neutrophils are still recruited to the site of infection, and dexamethasone treatment does not significantly affect fungal spore killing. Instead, the primary effect of dexamethasone manifests later in infection with treated larvae exhibiting increased invasive hyphal growth. In line with this, dexamethasone predominantly inhibits neutrophil function rather than macrophage function. Dexamethasone-induced mortality also depends on the glucocorticoid receptor. Dexamethasone partially suppresses NF-κB activation at the infection site by inducing the transcription of IκB via the glucocorticoid receptor. Independent CRISPR/Cas9 targeting of IKKγ to prevent NF-κB activation also increases invasive A. fumigatus growth and larval mortality. However, dexamethasone treatment of IKKγ crispant larvae further increases invasive hyphal growth and host mortality, suggesting that dexamethasone may suppress other pathways in addition to NF-κB to promote host susceptibility. Collectively, we find that dexamethasone acts through the glucocorticoid receptor to suppress NF-κB-mediated neutrophil control of A. fumigatus hyphae in zebrafish larvae.

Retinal ganglion cell survival after severe optic nerve injury is modulated by crosstalk between Jak/Stat signaling and innate immune responses in the zebrafish retina.

Visual information is transmitted from the eye to the brain along the optic nerve, a structure composed of retinal ganglion cell (RGC) axons. The optic nerve is highly vulnerable to damage in neurodegenerative diseases, such as glaucoma, and there are currently no FDA-approved drugs or therapies to protect RGCs from death. Zebrafish possess remarkable neuroprotective and regenerative abilities. Here, utilizing an optic nerve transection (ONT) injury and an RNA-seq-based approach, we identify genes and pathways active in RGCs that may modulate their survival. Through pharmacological perturbation, we demonstrate that Jak/Stat pathway activity is required for RGC survival after ONT. Furthermore, we show that immune responses directly contribute to RGC death after ONT; macrophages/microglia are recruited to the retina and blocking neuroinflammation or depleting these cells after ONT rescues survival of RGCs. Taken together, these data support a model in which crosstalk between macrophages/microglia and RGCs, mediated by Jak/Stat pathway activity, regulates RGC survival after optic nerve injury.

A zebrafish NLRX1 isoform downregulates fish IFN responses by targeting the adaptor STING.

In mammals, NLRX1 is a unique member of the nucleotide-binding domain and leucine-rich repeat (NLR) family showing an ability to negatively regulate IFN antiviral immunity. Intron-containing genes, including NLRX1, have more than one transcript due to alternative splicing; however, little is known about the function of its splicing variants. Here, we identified a transcript variant of NLRX1 in zebrafish (Danio rerio), termed NLRX1-tv4, as a negative regulator of fish IFN response. Zebrafish NLRX1-tv4 was slightly induced by viral infection, with an expression pattern similar to the full-length NLRX1. Despite the lack of an N-terminal domain that exists in the full-length NLRX1, overexpression of NLRX1-tv4 still impaired fish IFN antiviral response and promoted viral replication in fish cells, similar to the full-length NLRX1. Mechanistically, NLRX1-tv4 targeted STING for proteasome-dependent protein degradation by recruiting an E3 ubiquitin ligase RNF5 to drive the K48-linked ubiquitination, eventually downregulating the IFN antiviral response. Mapping of NLRX1-tv4 domains showed that its N-terminal and C-terminal regions exhibited a similar potential to inhibit STING-mediated IFN antiviral response. Our findings reveal that like the full-length NLRX1, zebrafish NLRX-tv4 functions as an inhibitor to shape fish IFN antiviral response.IMPORTANCEIn this study, we demonstrate that a transcript variant of zebrafish NLRX1, termed NLRX1-tv4, downregulates fish IFN response and promotes virus replication by targeting STING for protein degradation and impairing the interaction of STING and TBK1 and that its N- and C-terminus exhibit a similar inhibitory potential. Our results are helpful in clarifying the current contradictory understanding of structure and function of vertebrate NLRX1s.

Zebrafish phd1 enhances mavs-mediated antiviral responses in a hydroxylation-independent manner.

PHD1 is a member of the prolyl hydroxylase domain protein (PHD1-4) family, which plays a prominent role in the post-translational modification of its target proteins by hydroxylating proline residues. The best-characterized targets of PHD1 are hypoxia-inducible factor α (HIF-1α and HIF-2α), two master regulators of the hypoxia signaling pathway. In this study, we show that zebrafish phd1 positively regulates mavs-mediated antiviral innate immunity. Overexpression of phd1 enhances the cellular antiviral response. Consistently, zebrafish lacking phd1 are more susceptible to spring viremia of carp virus infection. Further assays indicate that phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, zebrafish phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. However, the enzymatic activity of phd1 is not required for phd1 to activate mavs. In conclusion, this study reveals a novel function of phd1 in the regulation of antiviral innate immunity.IMPORTANCEPHD1 is a key regulator of the hypoxia signaling pathway, but its role in antiviral innate immunity is largely unknown. In this study, we found that zebrafish phd1 enhances cellular antiviral responses in a hydroxylation-independent manner. Phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. Zebrafish lacking phd1 are more susceptible to spring viremia of carp virus infection. These findings reveal a novel role for phd1 in the regulation of mavs-mediated antiviral innate immunity.

Macrophages Mediate the Repair of Brain Vascular Rupture through Direct Physical Adhesion and Mechanical Traction.

Hemorrhagic stroke and brain microbleeds are caused by cerebrovascular ruptures. Fast repair of such ruptures is the most promising therapeutic approach. Due to a lack of high-resolution in vivo real-time studies, the dynamic cellular events involved in cerebrovascular repair remain unknown. Here, we have developed a cerebrovascular rupture system in zebrafish by using multi-photon laser, which generates a lesion with two endothelial ends. In vivo time-lapse imaging showed that a macrophage arrived at the lesion and extended filopodia or lamellipodia to physically adhere to both endothelial ends. This macrophage generated mechanical traction forces to pull the endothelial ends and facilitate their ligation, thus mediating the repair of the rupture. Both depolymerization of microfilaments and inhibition of phosphatidylinositide 3-kinase or Rac1 activity disrupted macrophage-endothelial adhesion and impaired cerebrovascular repair. Our study reveals a hitherto unexpected role for macrophages in mediating repair of cerebrovascular ruptures through direct physical adhesion and mechanical traction.

Zebrafish TMEM47 is an effective blocker of IFN production during RNA and DNA virus infection.

IMPORTANCE: Mitochondrial antiviral signaling protein (MAVS) and stimulator of interferon (IFN) genes (STING) are key adaptor proteins required for innate immune responses to RNA and DNA virus infection. Here, we show that zebrafish transmembrane protein 47 (TMEM47) plays a critical role in regulating MAVS- and STING-triggered IFN production in a negative feedback manner. TMEM47 interacted with MAVS and STING for autophagic degradation, and ATG5 was essential for this process. These findings suggest the inhibitory function of TMEM47 on MAVS- and STING-mediated signaling responses during RNA and DNA virus infection.

Zebrafish MAP2K7 Simultaneously Enhances Host IRF7 Stability and Degrades Spring Viremia of Carp Virus P Protein via Ubiquitination Pathway.

During viral infection, host defensive proteins either enhance the host immune response or antagonize viral components directly. In this study, we report on the following two mechanisms employed by zebrafish mitogen-activated protein kinase kinase 7 (MAP2K7) to protect the host during spring viremia of carp virus (SVCV) infection: stabilization of host IRF7 and degradation of SVCV P protein. In vivo, map2k7+/- (map2k7-/- is a lethal mutation) zebrafish showed a higher lethality, more pronounced tissue damage, and more viral proteins in major immune organs than the controls. At the cellular level, overexpression of map2k7 significantly enhanced host cell antiviral capacity, and viral replication and proliferation were significantly suppressed. Additionally, MAP2K7 interacted with the C terminus of IRF7 and stabilized IRF7 by increasing K63-linked polyubiquitination. On the other hand, during MAP2K7 overexpression, SVCV P proteins were significantly decreased. Further analysis demonstrated that SVCV P protein was degraded by the ubiquitin-proteasome pathway, as the attenuation of K63-linked polyubiquitination was mediated by MAP2K7. Furthermore, the deubiquitinase USP7 was indispensable in P protein degradation. These results confirm the dual functions of MAP2K7 during viral infection. IMPORTANCE Normally, during viral infection, host antiviral factors individually modulate the host immune response or antagonize viral components to defense infection. In the present study, we report that zebrafish MAP2K7 plays a crucial positive role in the host antiviral process. According to the weaker antiviral capacity of map2k7+/- zebrafish than that of the control, we find that MAP2K7 reduces host lethality through two pathways, as follows: enhancing K63-linked polyubiquitination to promote host IRF7 stability and attenuating K63-mediated polyubiquitination to degrade the SVCV P protein. These two mechanisms of MAP2K7 reveal a special antiviral response in lower vertebrates.

Evaluation of the synbiotic effects of Saccharomyces cerevisiae and mushroom extract on the growth performance, digestive enzyme activity, and immune status of zebrafish danio rerio.

BACKGROUND: The quest for candidate probiotics and prebiotics to develop novel synbiotics for sustainable and profitable fish farming remains a major focus for various stakeholders. In this study, we examined the effects of combining two fungal probiotics, Saccharomyces cerevisiae and Aspergillus niger with extracts of Jerusalem artichoke and white button mushroom to develop a synbiotic formulation to improve the growth and health status of zebrafish (Danio rerio). An initial in vitro study determined the most effective synbiotic combination, which was then tested in a 60-day in vivo nutritional trial using zebrafish (80 ± 1.0 mg) as a model animal. Four experimental diets were prepared: a control diet (basal diet), a prebiotic diet with 100% selected mushroom extract, a probiotic diet with 107 CFU of S. cerevisiae/g of diet, and a synbiotic diet with 107 CFU of S. cerevisiae/g of diet and 100% mushroom extract. As readouts, growth performance, survival, digestive enzyme activity and innate immune responses were evaluated. RESULTS: In vitro results showed that the S. cerevisiae cultured in a medium containing 100% mushroom extract exhibited the maximum specific growth rate and shortest doubling time. In the in vivo test with zebrafish, feeding them with a synbiotic diet, developed with S. cerevisiae and mushroom extract, led to a significant improvement in the growth performance of zebrafish (P < 0.05). The group of zebrafish fed with the synbiotic diet showed significantly higher levels of digestive enzyme activity and immune responses compared to the control group (P < 0.05). CONCLUSION: Taken together, these results indicated that the combination of S. cerevisiae and mushroom extract forms an effective synbiotic, capable of enhancing growth performance and immune response in zebrafish.

Effects of Lactobacillus acidophilus AC on the growth, intestinal flora and metabolism of zebrafish (Danio rerio).

The intensive aquaculture model has resulted in a heightened prevalence of diseases among farmed animals. It is imperative to identify healthy and efficacious alternatives to antibiotics for the sustainable progression of aquaculture. In this investigation, a strain of Lactobacillus acidophilus AC was introduced into the cultural water at varying concentrations (105 CFU/mL, 106 CFU/mL, 107 CFU/mL) to nourish zebrafish (Danio rerio). The findings revealed that L. acidophilus AC effectively increased the growth performance of zebrafish, improved the ion exchange capacity of gills, and enhanced hepatic antioxidant and immune-enzyme activities. Furthermore, L. acidophilus AC notably enhanced the intestinal morphology and augmented the activity of digestive enzymes within the intestinal tract. Analysis of intestinal flora revealed that L. acidophilus AC exerted a significant impact on the intestinal flora community, manifested by a reduction in the relative abundance of Burkholderiales, Candidatus_Saccharibacteria_bacterium, and Sutterellaceae, coupled with an increase in the relative abundance of Cetobacterium. Metabolomics analysis demonstrated that L. acidophilus AC significantly affected intestinal metabolism of zebrafish. PG (i-19:0/PGE2) and 12-Hydroxy-13-O-d-glucuronoside-octadec-9Z-enoate were the metabolites with the most significant up- and down-regulation folds, respectively. Finally, L. acidophilus AC increased the resistance of zebrafish to Aeromonas hydrophila. In conclusion, L. acidophilus AC was effective in enhancing the health and immunity of zebrafish. Thus, our findings suggested that L. acidophilus AC had potential applications and offered a reference for its use in aquaculture.

Leukolectins support lifelong innate immunity in lower vertebrates, and reveal dichotomies of several leukocytic lineages.

Innate immunity is vital for animal homeostasis and survival. First-line immuno-defense for fish larvae involves mucus enriched with leukolectin (LL) secreted by dermal lectocytes. Later during the critical transition from yolk-nutrition to feeding, additional larval immuno-protection in zebrafish (zF) is provided by macrophages containing LL (lectophages). This work investigated new LL-expression in embryos and in blood, structures of fish leukocytic LL and LL-genes, and LL-presence in chicken leukocytes. In zF-embryos, lectophages appear ∼10 hpf, while later, cells co-expressing myeloperoxidase- and LL-mRNA were detected (∼19 hpf). Furthermore, protein-extracts of Atlantic salmon (Ssal) leukocytes contained LL-proteins, compartmentalized in the cytosol. Cloning and sequencing revealed 94 % nt-sequence identity between variants of Ssal-leukolectins. Highly conserved LLs allowed production of epitope-specific anti-LL IgGs. Immuno-fluorescence-analysis demonstrated that most Ssal-bloodcells were LL-negative, but both some large cells with protrusions and some small, rounded cells did express LL. Immunoperoxidase-staining method confirmed LL-expression in some Ssal-leukocytes, identified as macrophages, PMN-leukocytes, thrombocytes and dendritic cells. However, closer examination revealed a dichotomy of these cell-categories into either LL-positive, or LL-negative variants. In situ hybridization demonstrated profuse LL-expression in Ssal head kidney interstitial tissue, while LL-transcripts were absent in large kidney tubules. Both hematopoietic (non-pigmented) marrow cells and melano-macrophages expressed LL-mRNA, implying that leukolectins provide lifelong innate immuno-protection. PCR-amplification using Ssal-leukocytic DNA as template, and direct sequencing yielded a leukocytic ll-gene. Some cells in salmon, cod, halibut, oikopleura and zebrafish embryos express LL-proteins and/or LL-mRNA, and LL-mRNA is detected in salmon, cod and chicken leukocytes. However, current genomes for these species lack recognizable LL-loci except the Ssal_v3.1 Genome-assembly. The data demonstrate an unexpected dichotomy of some leukocyte lineages into LL-positive or LL-negative cell-variants. Such dichotomies suggest exploring differential impacts from the duplicated leukocyte-lineages in health and disease.

Isolation, characterization, and immunomodulatory effects of extracellular vesicles isolated from fish pathogenic Aeromonas hydrophila.

Bacterial extracellular vesicles (BEVs) are natural nanocarriers that have shown great potential for biomedical applications such as biomarkers, cancer therapy, immunomodulators, vaccines, wound healing, tissue engineering, and drug carriers. In the present study, BEVs were isolated from the gram-negative bacterium, Aeromonas hydrophila using the ultracentrifugation method and denoted as AhEVs. Using transmission electron microscopy imaging, we confirmed the ultrastructure and spherical shape morphology of AhEVs. Nanoparticle-tracking analysis results showed a mean particle size of 105.5 ± 2.0 nm for AhEVs. Moreover, the particle concentration of AhEVs was 2.34 ± 0.12 × 1011 particles/mL of bacterial supernatant. AhEV-treated fathead minnow (FHM) cells did not show cytotoxicity effects up to 50 µg/mL with no significant decrease in cells. Moreover, no mortality was observed in larval zebrafish up to 50 µg/mL which indicates that the AhEVs are biocompatible at this concentration. Furthermore, fluorescent-labeled AhEVs were internalized into FHM cells. Results of qRT-PCR analysis in FHM cells revealed that cellular pro-inflammatory cytokines such as nuclear factor (NF)-κB, interferon (Ifn), Irf7, interleukin (Il) 8, and Il11 were upregulated while downregulating the expression of anti-inflammatory Il10 in a concentration-dependent manner. AhEV-treated adult zebrafish (5 µg/fish) induced toll-like receptor (tlr) 2 and tlr4; tumor necrosis factor-alpha (tnfα); heat shock protein (hsp) 70; and il10, il6, and il1ß in kidney. Protein expression of NF-κB p65 and Tnfα presented amplified levels in the spleen of AhEVs-treated zebrafish. Based on the collective findings, we conclude that AhEVs exhibited morphological and physicochemical characteristics to known EVs of gram (-)ve bacteria. At biocompatible concentrations, the immunomodulatory activity of AhEVs was demonstrated by inducing different immune response genes in FHM cells and zebrafish. Hence, we suggest that AhEVs could be a novel vaccine candidate in fish medicine due to their ability to elicit strong immune responses.