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Poly(I:C) is known as an agonist of the TLR3 receptor which could prime inflammation and elicit the host immune response, which is widely applied as adjuvant or antivirus treatment. However, the negative effects of poly(I:C) on regulating immune response to protect the host from inflammatory diseases remain largely unknown. Here, we establish an in vivo model to pre-treat zebrafish larvae with poly(I:C) at 2 dpf, then challenge them with LPS at 6 dpf, and find that poly(I:C) training could significantly alleviate the LPS challenge-induced septic shock and inflammatory phenotypes. Moreover, the poly(I:C)-trained larvae exhibit decreased number of macrophages, but not neutrophils, after secondary LPS challenge. Furthermore, training the larvae with poly(I:C) could elevate the transcripts of mTOR signaling and heighten the H3K4me3-mediated epigenetic modifications. And interestingly, we find that inhibiting the H3K4me3 modification, rather than mTOR signaling, could recover the number of macrophages in poly(I:C)-trained larvae, which is consistent with the observations of inflammatory phenotypes. Taken together, these results suggest that poly(I:C) training could induce epigenetic rewiring to mediate the anti-inflammatory response against secondary LPS challenge-induced septic shock through decreasing macrophages' number in vivo, which might expand our understanding of poly(I:C) in regulating fish immune response., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

T-helper 17 lymphocytes (Th17) are the most common inflammatory cells identified in mammals. However, the identification of Th17 cells and the clarification of their function in teleost fish remain largely unknown. In this study, we took advantage of the single-cell RNA sequencing-based immune cell atlas that was identified in turbot (Scophthalmus maximus), and revealed two chemokine-related genes, ccl20a.3 and ccl20l, that were specifically expressed in Th17 cells. Moreover, through immuno-fluorescence analysis, we found that CCL20a.3 or CCL20l was co-expressed with the classical makers in Th17 cells, including IL17a/f1 and IL22. Furthermore, through a Th17 lineage-specific transcription factor RORc inhibitor GSK805 treatment, we found that the expression of ccl20a.3 and ccl20l was significantly impaired, compared with other T cell markers. Besides, we also found that ccl20a.3 and ccl20l exhibited the same dynamic response with the classical markers that were identified in Th17 cells during bacterial infection. Taken together, these results provide potential gene markers for better understanding of the dynamic immune responses of Th17 cells in teleost fish., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Bacterial disease is one of the important factors leading to economic losses in the turbot (Scophthalmus maximus) cultivation industry. T lymphocytes are major components of cellular immunity, whereas B lymphocytes produce immunoglobulins (Ig) that are key elements of humoral immune responses against infection. However, the genomic organization of genes encoding T-cell receptors (TCR) and immunoglobulin heavy chains (IgHs) in turbot remains largely unknown. In this study, abundant full-length transcripts of TCRs and IgHs were sequenced by Isoform-sequencing (Iso-seq), and we investigated and annotated the V, D, J and C gene loci of TCRα, TCRβ, IgT, IgM and IgD in turbot. Furthermore, through single-cell RNA sequencing (scRNA-seq) of blood leukocytes, we confirmed that these identified TCRs and IgHs were highly expressed in T/B cell clusters, respectively. Meanwhile, we also identified the IgM + IgD + B and IgT + B cells with differential gene expression profiles and potential functions. Taken together, our results provide a comprehensive understanding of TCRs and IgHs loci in turbot, which will contribute to evolutionary and functional characterization of T and B lymphocytes in teleost., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Neutrophils can capture and kill pathogens by releasing neutrophils extracellular traps (NETs), which play critical roles in anti-microbial infection in mammals; however, the mechanisms involved in NETs formation and its role in anti-bacterial infection in teleost fish remains largely unknown. In this study, to explore the function of NETs in turbot, we established an in vitro bacterial infection model in head kidney derived neutrophils, and found that the haemolysin over-expressed Edwardsiella piscicida (ethA + ) could induce a robust phenotype of NETs, compared with that in wild type or ethA mutant (ethA +  -ΔethA) strains. Besides, the NETosis was mediated by ethA +  -induced pyroptosis, and arms the ability of bacterial killing in neutrophils of turbot. Moreover, we found that neutrophils elastase (NE) might involves in this pyroptotic signaling, rather than inflammatory Smcaspase. Taken together, this study reveals the important role of pyroptosis in NETs formation in turbot neutrophils, suggesting that NETs formation is a critical immune response during bacterial infection in teleost fish., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Propolis is non-hazardous resinous substance mixture containing bioactive ingredients such as polyphenols, flavonoids and organic acid. It has been widely used as food supplement and immune adjuvant due to its benefits in anti-microbial and immunomodulation. Edwardsiella piscicida is a kind of threatening pathogen which could cause high mortality in turbot. However, whether propolis could enhance the innate immune response against E. piscicida infection in turbot remains unknown. In this study, we found dietary propolis addition could improve the expression of anti-oxidative stress related enzymes, e.g., SOD, CAT and GPT, and relieved the histopathological changes of juvenile turbot after E. piscicida infection. Moreover, propolis addition increased the expression of cytokines such as il-1β, il-6 and tnf-α in different organs of juvenile turbot. Importantly, rescued survival and decreased bacterial loads were observed in propolis feeding group. Taken together, these findings suggest that the important roles of propolis in protecting juvenile turbot from E. piscicida infection, indicating propolis might be applied as a promising immunopotentiator candidate in aquaculture., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

NLRP3 inflammasome is one of the most well-known inflammasomes in mammals, which plays critical roles in innate immunity. However, knowledge about this inflammasome in non-mammalian species, especially in teleost fish, remains rarely known. Herein, we established an Edwardsiella piscicida-head-kidney macrophages (HKMs) infection model in Japanese flounder, and found a robust caspase-1 activation and IL-1β maturation. To characterize the upstream receptor, we established a bioinformatic screening analysis, and found an NLRP3 homolog (JfNLRP3) from Japanese flounder, which shares an overall conservative structure architecture to human NLRP3. Moreover, the JfNLRP3 can assemble JfASC through PYD-PYD domain interaction and trigger JfCaspase-1 activation and JfIL-1β maturation. Meanwhile, the classical inflammasome activation stimulators, including nigericin, ATP or MSU, can trigger the JfCaspase-1 activation and JfIL-1β maturation in Japanese flounder HKMs. During intraperitoneal infection of E. piscicida in Japanese flounder, we found a dynamic up-regulated transcription of JfNLRP3 and JfCaspase-1 in vivo. Furthermore, knockdown of either JfNLRP3 or JfCaspase-1 reduces the serum JfIL-1β level, and promotes the bacterial colonization in systemic immune organs at 2 day-post infection, while overexpression of JfNLRP3 or JfCaspase-1 hampers the bacterial colonization in these organs of Japanese flounder. Taken together, our results identified the NLRP3 inflammasome paradigm in Japanese flounder, which not only providing new insight into the molecular mechanisms of teleost NLRP3 inflammasome and revealing its role in restricting bacterial infection in vivo, but also shedding light on the evolutionary of NLRP3 inflammasome in teleost., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Apoptosis-associated speck-like protein containing a C-terminal caspase recruit domain (ASC) is an important adapter protein in the inflammasome complex that mediates inflammatory caspase activation and host innate immunity in mammals. However, the function of inflammasome components in lower vertebrate remains poorly understood. In this study, full length of SmASC was cloned from turbot (Scophthalmus maximus). Through bioinformatic analysis, we found that SmASC shares relatively high identity with ASC in bony fish. Furthermore, we found that the intact SmASC can form an oligomeric speck-like structure, while the PYD segment of SmASC can form the filamentous structure. Moreover, expression of SmASC was induced after intraperitoneal injection of Edwardsiella piscicida (E. piscicida) in vivo. To further explore the role of SmASC during infection, we constructed SmASC knockdown and overexpression models by administration of siRNA and overexpression plasmids in vivo, respectively. Expression of SmASC decreased the propagation of E. piscicida in different immune organs. In summary, our results characterize the function of SmASC in S. maximus, suggesting that the SmASC plays a critical role in turbot immune responses., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Interleukine-1β (IL-1β) is the first identified pro-inflammatory cytokine, which is cleaved by caspase-1 following the inflammasomes activation, playing critical roles in innate immunity. However, few studies have been performed to characterize the IL-1β in lower vertebrates. Herein, we distinguished the Scophthalmus maximus IL-1β (SmIL-1β) from three IL-1β like sequences and found that SmIL-1β was cleaved by S. maximus caspase at a non-conserved Asp 86 , then targeted to the plasma membrane. Moreover, during the immersion infection of Edwardsiella piscicida, we found that E. piscicida were mainly colonized in gills at early time points and invaded to systemic sites after 5 days post infection, which was consistent with the dynamic up-regulated transcription of SmIL-1β. Furthermore, knockdown of SmIL-1β promotes the bacterial colonization in gills at early time points and result into systemic colonization, while overexpression of SmIL-1β hampers the bacterial colonization in both spleen and kidney. Taken together, these data provide new insights into the molecular mechanisms of SmIL-1β and reveal its role in limiting bacterial infection in vivo, which will support the idea for better understanding the evolutionary of IL-1β functions in teleost., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Edwardsiella piscicida is an important pathogen that infects a wide range of hosts, from fish to human. Its infection leads to extensive losses in a diverse array of commercially important fish, like Japanese flounder, turbot, and tilapia. During the infection, type III secretion system (T3SS) and type VI secretion system (T6SS) of E. piscicida play significant roles, but how T3SS and T6SS cooperatively contribute to its virulence is still unknown. In this study, we first examined the roles of T3SS and T6SS in different processes during E. piscicida infection of host cells, and revealed that T3SS of E. piscicida is responsible for promoting bacterial invasion, the following intracellular replication and inducing cell death in host cells, while T6SS restrains E. piscicida intracellular replication and cell death in J774A.1 cells, which suggested that T3SS and T6SS antagonistically concert E. piscicida infection. Furthermore, we found an significant decrease in transcription level of IL-1β in zebrafish kidney infected with T3SS mutant and an drastically increase in transcription level of TNF- α infected with T6SS mutant when compared with the wild-type. Interestingly, both T3SS and T6SS mutants showed significant attenuated virulence in the zebrafish infection model when compared with the wild-type. Finally, considering the cooperative role of T3SS and T6SS, we generated a mutant strain WEDΔT6SS based on the existing live attenuated vaccine (LAV) WED which showed improved vaccine safety and comparable immune protection. Therefore, WEDΔT6SS could be used as an optimized LAV in the future. Taken together, this work suggested a bilateral role of T3SS and T6SS which respectively act as spear and shield during E. piscicida infection, together contribute to E. piscicida virulence., (Copyright © 2019. Published by Elsevier Ltd.)

Innate immunity is regulated by phagocytic cells and is critical for host control of bacterial infection. In many bacteria, the type VI secretion system (T6SS) can affect bacterial virulence in certain environments, but little is known about the mechanisms underlying T6SS regulation of innate immune responses during infection in vivo. Here, we developed an infection model by microinjecting bacteria into the tail vein muscle of 3-day-post-fertilized zebrafish larvae, and found that both macrophages and neutrophils are essential for bacterial clearance. Further study revealed that EvpP plays a critical role in promoting the pathogenesis of Edwardsiella piscicida (E. piscicida) via inhibiting the phosphorylation of Jnk signaling to reduce the expression of chemokine (CXC motif) ligand 8 (cxcl8a), matrix metallopeptidase 13 (mmp13) and interleukin-1β (IL-1β) in vivo. Subsequently, by utilizing Tg (mpo:eGFP +/+ ) zebrafish larvae for E. piscicida infection, we found that the EvpP-inhibited Jnk-caspy (caspase-1 homolog) inflammasome signaling axis significantly suppressed the recruitment of neutrophils to infection sites, and the caspy- or IL-1β-morpholino (MO) knockdown larvae were more susceptible to infection and failed to restrict bacterial colonization in vivo. taken together, this interaction improves our understanding about the complex and contextual role of a bacterial T6SS effector in modulating the action of neutrophils during infection, and offers new insights into the warfare between bacterial weapons and host immunological surveillance., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Edwardsiella piscicida is a facultative intracellular pathogen that causes hemorrhagic septicemia and haemolytic ascites disease in aquaculture fish. During bacterial infection, macrophages and neutrophils are the first line of host innate immune system. However, the role of neutrophils in response to E. piscicida infection in vivo remains poorly understood. Here, through developing an immersion infection model in the 5 day-post fertilization (dpf) zebrafish larvae, we found that E. piscicida was mainly colonized in intestine, and resulted into significant pathological changes in paraffin sections. Moreover, a dynamic up-regulation of inflammatory cytokines (TNF-α, IL-1β, GCSFb, CXCL8 and MMP9) was detected in zebrafish larvae during E. piscicida infection. Furthermore, a significant recruitment of neutrophils was observed during the E. piscicida infection in Tg(mpx:eGFP) zebrafish larvae. Thus, we utilized the CRISPR/Cas9 system to generate the neutrophil-knockdown (gcsfr -/- crispants) larvae, and found a comparative higher mortality and bacterial colonization in gcsfr -/- crispants, which reveals the critical role of fish neutrophils in bacterial clearance. Taken together, our results developed an effective E. piscicida immersion challenge model in zebrafish larvae to clarify the dynamic of bacterial infection in vivo, which would provide a better understanding of the action about innate immune cells during infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)