Essential role of an ERV-derived Env38 protein in adaptive humoral immunity against an exogenous SVCV infection in a zebrafish model.

Endogenous retroviruses (ERVs) are the relics of ancient retroviruses occupying a substantial fraction of vertebrate genomes. However, knowledge about the functional association of ERVs with cellular activities remains limited. Recently, we have identified approximately 3,315 ERVs from zebrafish at genome-wide level, among which 421 ERVs were actively expressed in response to the infection of Spring viraemia of carp virus (SVCV). These findings demonstrated the previously unrecognized activity of ERVs in zebrafish immunity, thereby making zebrafish an attractive model organism for deciphering the interplay among ERVs, exogenous invading viruses, and host immunity. In the present study, we investigated the functional role of an envelope protein (Env38) derived from an ERV-E5.1.38-DanRer element in zebrafish adaptive immunity against SVCV in view of its strong responsiveness to SVCV infection. This Env38 is a glycosylated membrane protein mainly distributed on MHC-II+ antigen-presenting cells (APCs). By performing blockade and knockdown/knockout assays, we found that the deficiency of Env38 markedly impaired the activation of SVCV-induced CD4+ T cells and thereby led to the inhibition of IgM+/IgZ+ B cell proliferation, IgM/IgZ Ab production, and zebrafish defense against SVCV challenge. Mechanistically, Env38 activates CD4+ T cells by promoting the formation of pMHC-TCR-CD4 complex via cross-linking MHC-II and CD4 molecules between APCs and CD4+ T cells, wherein the surface subunit (SU) of Env38 associates with the second immunoglobin domain of CD4 (CD4-D2) and the first α1 domain of MHC-IIα (MHC-IIα1). Notably, the expression and functionality of Env38 was strongly induced by zebrafish IFNφ1, indicating that env38 acts as an IFN-stimulating gene (ISG) regulated by IFN signaling. To the best of our knowledge, this study is the first to identify the involvement of an Env protein in host immune defense against an exogenous invading virus by promoting the initial activation of adaptive humoral immunity. It improved the current understanding of the cooperation between ERVs and host adaptive immunity.

PD-L1/BTLA Checkpoint Axis Exploited for Bacterial Immune Escape by Restraining CD8+ T Cell-Initiated Adaptive Immunity in Zebrafish.

Programmed death-ligand 1/programmed cell death 1 (PD-L1/PD-1) is one of the most important immune checkpoints in humans and other mammalian species. However, the occurrence of the PD-L1/PD-1 checkpoint in evolutionarily ancient vertebrates remains elusive because of the absence of a PD-1 homolog before its appearance in tetrapods. In this article, we identified, to our knowledge, a novel PD-L1/B and T lymphocyte attenuator (BTLA) checkpoint in zebrafish by using an Edwardsiella tarda-induced bacterial infection model. Results showed that zebrafish (Danio rerio) PD-L1 (DrPD-L1) and BTLA (DrBTLA) were differentially upregulated on MHC class II+ macrophages (Mϕs) and CD8+ T cells in response to E. tarda infection. DrPD-L1 has a strong ability to interact with DrBTLA, as shown by the high affinity (KD = 5.68 nM) between DrPD-L1/DrBTLA proteins. Functionally, the breakdown of DrPD-L1/DrBTLA interaction significantly increased the cytotoxicity of CD8+BTLA+ T cells to E. tarda-infected PD-L1+ Mϕ cells and reduced the immune escape of E. tarda from the target Mϕ cells, thereby enhancing the antibacterial immunity of zebrafish against E. tarda infection. Similarly, the engagement of DrPD-L1 by soluble DrBTLA protein diminished the tolerization of CD8+ T cells to E. tarda infection. By contrast, DrBTLA engagement by a soluble DrPD-L1 protein drives aberrant CD8+ T cell responses. These results were finally corroborated in a DrPD-L1-deficient (PD-L1-/-) zebrafish model. This study highlighted a primordial PD-L1/BTLA coinhibitory axis that regulates CD8+ T cell activation in teleost fish and may act as an alternative to the PD-L1/PD-1 axis in mammals. It also revealed a previously unrecognized strategy for E. tarda immune evasion by inducing CD8+ T cell tolerance to target Mϕ cells through eliciting the PD-L1/BTLA checkpoint pathway.

Single-cell transcriptome profiling reveals diverse immune cell populations and their responses to viral infection in the spleen of zebrafish.

Teleost fish are indispensable model organisms for comparative immunology research that should lead to an improved understanding of the general principles of vertebrate immune system design. Although numerous studies on fish immunology have been conducted, knowledge about the cell types that orchestrate piscine immune systems remains limited. Here, we generated a comprehensive atlas of immune cell types in zebrafish spleen on the basis of single-cell transcriptome profiling. We identified 11 major categories from splenic leukocyte preparations, including neutrophils, natural killer cells, macrophages/myeloid cells, T cells, B cells, hematopoietic stem and progenitor cells, mast cells, remnants of endothelial cells, erythroid cells, erythroid progenitors, and a new type of serpin-secreting cells. Notably, we derived 54 potential subsets from these 11 categories. These subsets showed differential responses to spring viremia of carp virus (SVCV) infection, implying that they have diverse roles in antiviral immunity. Additionally, we landscaped the populations with the induced expression of interferons and other virus-responsive genes. We found that trained immunity can be effectively induced in the neutrophil and M1-macrophage subsets by vaccinating zebrafish with inactivated SVCV. Our findings illustrated the complexity and heterogeneity of the fish immune system, which will help establish a new paradigm for the improved understanding of fish immunology.

Zbtb46 Controls Dendritic Cell Activation by Reprogramming Epigenetic Regulation of cd80/86 and cd40 Costimulatory Signals in a Zebrafish Model.

The establishment of an appropriate costimulatory phenotype is crucial for dendritic cells (DCs) to maintain a homeostatic state with optimal immune surveillance and immunogenic activities. The upregulation of CD80/86 and CD40 is a hallmark costimulatory phenotypic switch of DCs from a steady state to an activated one for T cell activation. However, knowledge of the regulatory mechanisms underlying this process remains limited. In this study, we identified a Zbtb46 homolog from a zebrafish model. Zbtb46 deficiency resulted in upregulated cd80/86 and cd40 expression in kidney marrow-derived DCs (KMDCs) of zebrafish, which was accompanied with a remarkable expansion of CD4+/CD8+ T cells and accumulation of KMDCs in spleen of naive fish. Zbtb46 -/- splenic KMDCs exhibited strong stimulatory activity for CD4+ T cell activation. Chromatin immunoprecipitation-quantitative PCR and mass spectrometry assays showed that Zbtb46 was associated with promoters of cd80/86 and cd40 genes by binding to a 5'-TGACGT-3' motif in resting KMDCs, wherein it helped establish a repressive histone epigenetic modification pattern (H3K4me0/H3K9me3/H3K27me3) by organizing Mdb3/organizing nucleosome remodeling and deacetylase and Hdac3/nuclear receptor corepressor 1 corepressor complexes through the recruitment of Hdac1/2 and Hdac3. On stimulation with infection signs, Zbtb46 disassociated from the promoters via E3 ubiquitin ligase Cullin1/Fbxw11-mediated degradation, and this reaction can be triggered by the TLR9 signaling pathway. Thereafter, cd80/86 and cd40 promoters underwent epigenetic reprogramming from the repressed histone modification pattern to an activated pattern (H3K4me3/H3K9ac/H3K27ac), leading to cd80/86 and cd40 expression and DC activation. These findings revealed the essential role of Zbtb46 in maintaining DC homeostasis by suppressing cd80/86 and cd40 expression through epigenetic mechanisms.

Negative Regulatory Role of the Spring Viremia of Carp Virus Matrix Protein in the Host Interferon Response by Targeting the MAVS/TRAF3 Signaling Axis.

Spring viremia of carp virus (SVCV) is a severe infectious pathogen that causes high rates of mortality in cyprinids and other fish species. Despite numerous investigations of SVCV infection, the underlying molecular mechanisms remain poorly understood. In this study, we found that the SVCV matrix protein (SVCV-M) played an inhibitory role in the host interferon (IFN) response by targeting the MAVS/TRAF3 signaling axis, thereby uncovering a previously unrecognized mechanism of SVCV escape from host innate antiviral immunity. Mechanistically, SVCV-M was located at the mitochondria independent of MAVS, which allowed SVCV-M to build an arena for competition with the MAVS platform. A microscale thermophoresis assay showed that SVCV-M had a high affinity for TRAF3, as indicated by a lower equilibrium dissociation constant (KD) value than that of MAVS with TRAF3. Therefore, the association of MAVS with TRAF3 was competitively impaired by SVCV-M in a dose-dependent manner. Accordingly, SVCV-M showed a potent ability to inhibit the K63-linked polyubiquitination of TRAF3. This inhibition was accompanied by the impairment of the IFN response, as shown by the marked decline in IFN-φ1-promoter (pro) luciferase reporter activity. By constructing truncated TRAF3 and SVCV-M proteins, the RING finger, zinc finger, and coiled-coil domains of TRAF3 and the hydrophobic-pocket-like structure formed by the α2-, α3-, and α4-helices of SVCV-M may be the major target and antagonistic modules responsible for the protein-protein interaction between the TRAF3 and SVCV-M proteins. These findings highlighted the intervention of SVCV-M in host innate immunity, thereby providing new insights into the extensive participation of viral matrix proteins in multiple biological activities. IMPORTANCE The matrix protein of SVCV (SVCV-M) is an indispensable structural element for nucleocapsid condensation and virion formation during viral morphogenesis, and it connects the core nucleocapsid particle to the outer membrane within the mature virus. Previous studies have emphasized the architectural role of SVCV-M in viral construction; however, the potential nonstructural functions of SVCV-M in viral replication and virus-host interactions remain poorly understood. In this study, we identified the inhibitory role of the SVCV-M protein in host IFN production by competitively recruiting TRAF3 from the MAVS signaling complex and impairing TRAF3 activation via inhibition of K63-linked polyubiquitination. This finding provided new insights into the regulatory role of SVCV-M in host innate immunity, which highlighted the broader functionality of rhabdovirus matrix protein apart from being a structural protein. This study also revealed a previously unrecognized mechanism underlying SVCV immune evasion by inhibiting the IFN response by targeting the MAVS/TRAF3 signaling axis.

New Insights into IgZ as a Maternal Transfer Ig Contributing to the Early Defense of Fish against Pathogen Infection.

IgZ or its equivalent IgT is a newly discovered teleost specific Ig class that is highly specialized in mucosal immunity. However, whether this IgZ/IgT class participates in other biological processes remains unclear. In this study, we unexpectedly discovered that IgZ is highly expressed in zebrafish ovary, accumulates in unfertilized eggs, and is transmitted to offspring from eggs to zygotes. Maternally transferred IgZ in zygotes is found at the outer and inner layers of chorion, perivitelline space, periphery of embryo body, and yolk, providing different lines of defense against pathogen infection. A considerable number of IgZ+ B cells are found in ovarian connective tissues distributed between eggs. Moreover, pIgR, the transporter of IgZ, is also expressed in the ovary and colocalizes with IgZ in the zona radiata of eggs. Thus, IgZ is possibly secreted by ovarian IgZ+ B cells and transported to eggs through association with pIgR in a paracrine manner. Maternal IgZ in zygotes showed a broad bacteriostatic activity to different microbes examined, and this reactivity can be manipulated by orchestrating desired bacteria in water where parent fish live or immunizing the parent fish through vaccination. These observations suggest that maternal IgZ may represent a group of polyclonal Abs, providing protection against various environmental microbes encountered by a parent fish that were potentially high risk to offspring. To our knowledge, our findings provide novel insights into a previously unrecognized functional role of IgZ/IgT Ig in the maternal transfer of immunity in fish, greatly enriching current knowledge about this ancient Ig class.

The zebrafish NLRP3 inflammasome has functional roles in ASC-dependent interleukin-1ß maturation and gasdermin E-mediated pyroptosis.

The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) model. We found that DrNLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase (DrCaspase-A/-B) activation and interleukin 1ß (DrIL-1ß) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which DrNLRP3 organizes DrASC into a filament that recruits DrCaspase-A/-B by homotypic pyrin domain (PYD)-PYD interactions. DrCaspase-A/-B activation in the DrNLRP3 inflammasome occurred in two steps, with DrCaspase-A being activated first and DrCaspase-B second. DrNLRP3 also directly activated full-length DrCaspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by DrNLRP3 to ensure efficient IL-1ß secretion for the initiation of host innate immunity. By knocking down DrNLRP3 in zebrafish embryos and generating a DrASC-knockout (DrASC-/-) fish clone, we characterized the function of the DrNLRP3 inflammasome in anti-bacterial immunity in vivo The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis.

Differential immune responses of immunoglobulin Z subclass members in antibacterial immunity in a zebrafish model.

Immunoglobulin Z (IgZ) or its equivalent immunoglobulin T (IgT) is a newly identified immunoglobulin (Ig) class from teleost fish. This Ig class is characterized by its involvement in mucosa-associated lymphoid tissues (MALTs) for mucosal defence against pathogen infection. Recently, several subclass members of IgZ/IgT, such as IgZ, IgZ2, Igτ1, Igτ2 and Igτ3, have been further identified from zebrafish, common carp and rainbow trout. However, the functional diversity and correlation among these subclasses remain uncertain. Here, we explored the differential immune reactions of the IgZ and IgZ2 subclasses in antibacterial immunity in a zebrafish model. IgZ was extensively distributed in the peripheral serum and skin/gill MALTs and showed a rapid induction upon bacterial infection. IgZ2 was specialized in skin/gill MALTs and showed a strong induction following IgZ production. Correspondingly, the IgZ+ B cells had a wider distribution in the systemic primary/secondary lymphoid tissues and MALTs than the IgZ2+ B cells, which were predominant in MALTs. IgZ and IgZ2 exhibited a complementary effect in antibacterial immunity by possessing differential abilities. That is, IgZ is preferentially involved in bactericidal reaction that is in part C1q-dependent, and IgZ2 participates in neutralization action through bacteria-coating activity. The production of IgZ largely depended on the αß T/CD4+ T cells, whereas that of IgZ2 did not, suggesting the different dependencies of IgZ and IgZ2 on systemic immunity. Our findings demonstrate that the functional behaviour and mechanism of the IgZ/IgT family are more diverse than previously recognized and thus improve the current knowledge about this ancient Ig class.

Characterization of cGAS homologs in innate and adaptive mucosal immunities in zebrafish gives evolutionary insights into cGAS-STING pathway.

Cyclic GMP-AMP synthase (cGAS) is one of the most-characterized cytoplasmic DNA sensors in humans and other mammals. However, knowledge about cGAS homologs in nonmammalian species remains limited. In this study, we report the molecular and functional identification of two cGAS homologs, namely, DrcGASa and DrcGASb, from a zebrafish (Danio rerio) model. DrcGASa and DrcGASb share the same overall conservative structural architectures and functional domains/residues to mammalian cGASs. Both homologs synthesized a 2'3'-cGAMP isomer but not a 3'3'-cGAMP isomer via oligomerization in response to DNA stimulation. Overexpression of DrcGASa/b in HEK293T cells and zebrafish embryos significantly activated NF-κB and IFN-I signaling pathways in a STING-dependent manner. Knockdown of DrcGASa or DrSTING impaired such activations, thereby reducing the host innate immunity against bacterial and viral infections. DrcGASa, but not DrcGASb, was involved in immunoglobulin Z-mediated mucosal immunity in gill-associated lymphoid tissue, suggesting differential functions between the two DrcGASs. This reaction was associated with the DrcGAS-DrSTING-IFNφ1 signaling axis in GALT's γδ T cells. Our findings provide experimental evidence that a modern cGAS-STING pathway that mainly participates in IFN-mediated immunity originated from teleost fish based on the functional constraint of cGAS and STING proteins during vertebrate evolution.

BTLA-HVEM Checkpoint Axis Regulates Hepatic Homeostasis and Inflammation in a ConA-Induced Hepatitis Model in Zebrafish.

The BTLA-HVEM checkpoint axis plays extensive roles in immunomodulation and diseases, including cancer and autoimmune disorders. However, the functions of this checkpoint axis in hepatitis remain limited. In this study, we explored the regulatory role of the Btla-Hvem axis in a ConA-induced hepatitis model in zebrafish. Results showed that Btla and Hvem were differentially expressed on intrahepatic Cd8+ T cells and hepatocytes. Knockdown of Btla or Hvem significantly promoted hepatic inflammation. Btla was highly expressed in Cd8+ T cells in healthy liver but was downregulated in inflamed liver, as evidenced by a disparate proportion of Cd8+Btla+ and Cd8+Btla- T cells in individuals without or with ConA stimulation. Cd8+Btla+ T cells showed minimal cytotoxicity to hepatocytes, whereas Cd8+Btla- T cells were strongly reactive. The depletion of Cd8+Btla- T cells reduced hepatitis, whereas their transfer enhanced hepatic inflammation. These observations indicate that Btla endowed Cd8+Btla+ T cells with self-tolerance, thereby preventing them from attacking hepatocytes. Btla downregulation deprived this tolerization. Mechanistically, Btla-Hvem interaction contributed to Cd8+Btla+ T cell tolerization, which was impaired by Hvem knockdown but rescued by soluble Hvem protein administration. Notably, Light was markedly upregulated on Cd8+Btla- T cells, accompanied by the transition of Cd8+Btla+Light- to Cd8+Btla-Light+ T cells during hepatitis, which could be modulated by Cd4+ T cells. Light blockade attenuated hepatitis, thereby suggesting the positive role of Light in hepatic inflammation. These findings provide insights into a previously unrecognized Btla-Hvem-Light regulatory network in hepatic homeostasis and inflammation, thus adding a new potential therapeutic intervention for hepatitis.

Characterization of an NLRP1 Inflammasome from Zebrafish Reveals a Unique Sequential Activation Mechanism Underlying Inflammatory Caspases in Ancient Vertebrates.

NLRP1 inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, the existence of this inflammasome in nonmammalian species remains poorly understood. In this study, we report the molecular and functional identification of an NLRP1 homolog, Danio rerio NLRP1 (DrNLRP1) from a zebrafish (D. rerio) model. This DrNLRP1 possesses similar structural architecture to mammalian NLRP1s. It can trigger the formation of a classical inflammasome for the activation of zebrafish inflammatory caspases (D. rerio Caspase [DrCaspase]-A and DrCaspase-B) and maturation of D. rerio IL-1ß in a D. rerio ASC (DrASC)-dependent manner. In this process, DrNLRP1 promotes the aggregation of DrASC into a filament with DrASCCARD core and DrASCPYD cluster. The assembly of DrNLRP1 inflammasome depends on the CARD-CARD homotypic interaction between DrNLRP1 and DrASCCARD core, and PYD-PYD interaction between DrCaspase-A/B and DrASCPYD cluster. The FIIND domain in DrNLRP1 is necessary for inflammasome assembly. To understand the mechanism of how the two DrCaspases are coordinated in DrNLRP1 inflammasome, we propose a two-step sequential activation model. In this model, the recruitment and activation of DrCaspase-A/B in the inflammasome is shown in an alternate manner, with a preference for DrCaspase-A followed by a subsequent selection for DrCaspase-B. By using morpholino oligonucleotide-based knockdown assays, the DrNLRP1 inflammasome was verified to play important functional roles in antibacterial innate immunity in vivo. These observations demonstrate that the NLRP1 inflammasome originated as early as in teleost fish. This finding not only gives insights into the evolutionary history of inflammasomes but also provides a favorable animal model for the study of NLRP1 inflammasome-mediated immunology and diseases.

B cells in teleost fish act as pivotal initiating APCs in priming adaptive immunity: an evolutionary perspective on the origin of the B-1 cell subset and B7 molecules.

The long-held paradigm that B cells cannot uptake nonspecific particulate Ags for the initiation of primary adaptive immunity has been challenged by the recent discovery that teleost B cells have potent phagocytic and microbicidal abilities. This discovery provides preliminary clues that primitive B cells might act as initiating APCs in priming adaptive immunity. In this study, zebrafish B cells clearly showed a potent Ag-presenting ability to both soluble Ags and bacterial particles to prime naive CD4(+) T cell activation. This finding demonstrates the innate-like nature of teleost B cells in the interface of innate and adaptive immunity, indicating that they might consist of a major population of initiating APCs whose performance is similar to that of dendritic cells. Given the functional similarities between teleost B cells and the mammalian B-1 subset, we hypothesize that B-1 lineage and teleost B cells might originate from a common ancestor with potent phagocytic and initiating APC capacities. In addition, CD80/86 and CD83 costimulatory signals were identified as being essential for B cell-initiated adaptive immunity. This result suggests that the costimulatory mechanism originated as early as the origin of adaptive immunity and is conserved throughout vertebrate evolution. In fish, only a single CD80/86 copy exists, which is similar to mammalian CD86 rather than to CD80. Thus, CD86 might be a more primordial B7 family member that originated from fish. This study provides valuable insights into the evolutionary history of professional APCs, B cell lineages, and the costimulatory mechanism underlying adaptive immunity as a whole.

Essential Role of RIG-I in Hematopoietic Precursor Emergence in Primitive Hematopoiesis during Zebrafish Development.

Retinoic acid-inducible gene I (RIG-I) is an important cytosolic pattern recognition receptor crucial for sensing RNA virus infection and initiating innate immune responses. However, the participation of RIG-I in cellular development under physiological conditions remains limited. In this study, the regulatory role of RIG-I in embryonic hematopoiesis was explored in a zebrafish model. Results showed that rig-I was ubiquitously expressed during embryogenesis at 24 h postfertilization (hpf). A defect in RIG-I remarkably disrupted the emergence of primitive hematopoietic precursors and subsequent myeloid and erythroid lineages. In contrast, RIG-I deficiency did not have an influence on the generation of endothelial precursors and angiogenesis and the development of mesoderm and adjacent tissues. The alteration in these phenotypes was confirmed by whole-mount in situ hybridization with lineage-specific markers. In addition, immunostaining and TUNEL assays excluded the abnormal proliferation and apoptosis of hematopoietic precursors in RIG-I-deficient embryos. Mechanistically, RIG-I regulates primitive hematopoiesis through downstream IFN signaling pathways, as shown by the decline in ifnφ2 and ifnφ3 expression, along with rig-I knockdown, and rescue of the defects of hematopoietic precursors in RIG-I-defective embryos after administration with ifnφ2 and ifnφ3 mRNAs. Additionally, the defects of hematopoietic precursors in RIG-I morphants could be efficiently rescued by the wild-type RIG-I but could not be restored by the RNA-binding-defective RIG-I with site mutations at the RNA-binding pocket, which are essential for association with RNAs. This finding suggested that endogenous RNAs may serve as agonists to activate RIG-I-modulated primitive hematopoiesis. This study revealed the functional diversity of RIG-I under physiological conditions far beyond that previously known.

Extensive involvement of CD40 and CD154 costimulators in multiple T cell-mediated responses in a perciform fish Larimichthys crocea.

CD40 and CD154 are well-characterized costimulatory molecules involved in adaptive humoral immunity in humans and other mammals. These two costimulatory molecules were found to be originated from teleost fish during vertebrate evolution. However, the functionality of fish CD40 and CD154 remains to be explored. In this study, we identified the CD40 and CD154 homologs (LcCD40 and LcCD154) from large yellow croaker (Larimichthys crocea), a marine species of the perciform fish family. The LcCD40 and LcCD154 share conserved structural features to their mammalian counterparts, and are widely expressed in immune-relevant tissues and leukocytes at different transcriptional levels. Immunofluorescence staining and FCM analysis showed that LcCD40 and LcCD154 proteins are distributed on MHC-II+ APCs and CD4-2+ T cells, and are significantly upregulated in response to antigen stimulation. Co-IP assay exhibited strong association between LcCD40 and LcCD154 proteins. Blockade of LcCD154 with anti-LcCD154 antibody (Ab) or recombinant soluble LcCD40-Ig fusion protein remarkably decreased the MHC-II+ APC-initiated CD4+ T cell response upon Aeromonas hydrophila stimulation, and alloreactive T cell activation as examined by mixed lymphocyte reaction (MLR). These findings highlight the costimulatory role of LcCD40 and LcCD154 in T cell activities in Larimichthys crocea. Thus, the CD40 and CD154 costimulators may extensively participate in the regulation of multiple T cell-mediated immune responses in teleost fish. It is anticipated that this study would provide a cross-species understanding of the evolutionary history of CD40 and CD154 costimulatory signals from fish to mammals.

Regulatory role of BTLA and HVEM checkpoint inhibitors in T cell activation in a perciform fish Larimichthys crocea.

The BTLA and HVEM are two well-characterized immune checkpoint inhibitors in humans and other mammalian species. However, the occurrence and functionality of these two molecules in non-mammalian species remain poorly understood. In the present study, we identified the BTLA and HVEM homologs from large yellow croaker (Larimichthys crocea), an economically important marine species of the perciform fish family. The Larimichthys crocea BTLA and HVEM (LcBTLA and LcHVEM) share conserved structural features to their mammalian counterparts, and they were expressed in various tissues and cells examined at different transcriptional levels, with particular abundance in immune-relevant tissues and splenic leukocytes. Immunofluorescence staining and flow cytometry analysis showed that LcHVEM and LcBTLA proteins were distributed on MHC-II+ APCs and CD4-2+ T cells, and a strong interaction between LcBTLA and LcHVEM was detected in splenic leukocytes in the mixed lymphocyte reaction (MLR). By blockade assays using anti-LcBTLA and anti-LcHVEM Abs as well as recombinant soluble LcBTLA and LcHVEM proteins in different combinations, it was found that LcBTLA-LcHVEM interactions play an important inhibitory role in the activation of alloreactive T cells using MLR as a model, and APC-initiated antigen-specific CD4-2+ T cells in response to A. hydrophila (A. h) stimulation. These observations highlight the extensive functional roles of LcBTLA and LcHVEM immune-checkpoint inhibitors in allogeneic T cell reactions, and CD4-2+ T cell-mediated adaptive immune responses in Larimichthys crocea. Thus, the BTLA-HVEM checkpoint may represent an ancient coinhibitory pathway, which was originated in fish and was conserved from fish to mammals throughout the vertebrate evolution.

The DC-SIGN of zebrafish: insights into the existence of a CD209 homologue in a lower vertebrate and its involvement in adaptive immunity.

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN/CD209) has become hot topic in recent studies because of its important roles in immune responses and immune escape. CD209 has been well characterized in humans and several other mammals, but little documentation exists about it in lower vertebrates. This is the first report on the identification and functional characterization of a fish DC-SIGN/CD209 molecule. The zebrafish DC-SIGN/CD209 cDNA translates into 343 aa organized into three domains structurally conserved among vertebrates. An EPN motif essential for interacting with Ca(2+) and for recognizing mannose-containing motifs has been identified. Several conserved motifs crucial for internalization and signal transduction are also present within the cytoplasmic tail. Phylogenetic analysis supports the hypothesis that CD209 family members diverged from a common ancestor. The expression of DC-SIGN/CD209 in immune-related tissues can be significantly up-regulated by exogenous Ags and IL-4. This molecule associates with various APCs, including macrophages, B lymphocytes, and a possible dendritic cell-like (CD83(+)/CD80(+)CD209(+)) population. Functionally, T cell activation, Ab (IgM) production, and bacterial vaccination-elicited immunoprotection can be dramatically inhibited by a CD209 blockade after stimulation with keyhole limpet hemocyanin (KLH) in vivo or challenged with Aeromonas hydrophila, suggesting that DC-SIGN/CD209 in zebrafish is crucial for the initiation and development of adaptive immunity. Phagocytosis analysis showed that DC-SIGN/CD209 does not participate in the uptake of KLH Ag, suggesting that other mechanisms might exist that underlie DC-SIGN/CD209 involvement. We hope that the present study will contribute to a better cross-species understanding of the evolutionary history of the DC-SIGN/CD209 family.

Genome-Wide Characterization of Zebrafish Endogenous Retroviruses Reveals Unexpected Diversity in Genetic Organizations and Functional Potentials.

Endogenous retroviruses (ERVs) occupy a substantial fraction of mammalian genomes. However, whether ERVs extensively exist in ancient vertebrates remains unexplored. Here, we performed a genome-wide characterization of ERVs in a zebrafish (Danio rerio) model. Approximately 3,315 ERV-like elements (DrERVs) were identified as Gypsy, Copia, Bel, and class I-III groups. DrERVs accounted for approximately 2.3% of zebrafish genome and were distributed in all 25 chromosomes, with a remarkable bias on chromosome 4. Gypsy and class I are the two most abundant groups with earlier insertion times. The vast majority of the DrERVs have varied structural defects. A total of 509 gag and 71 env genes with coding potentials were detected. The env-coding elements were well-characterized and classified into four subgroups. A ERV-E4.8.43-DanRer element shows high similarity with HERV9NC-int in humans and analogous sequences were detected in species spanning from fish to mammals. RNA-seq data showed that hundreds of DrERVs were expressed in embryos and tissues under physiological conditions, and most of them exhibited stage and tissue specificity. Additionally, 421 DrERVs showed strong responsiveness to virus infection. A unique group of DrERVs with immune-relevant genes, such as fga, ddx41, ftr35, igl1c3, and tbk1, instead of intrinsic viral genes were identified. These DrERVs are regulated by transcriptional factors binding at the long terminal repeats. This study provided a survey of the composition, phylogeny, and potential functions of ERVs in a fish model, which benefits the understanding of the evolutionary history of ERVs from fish to mammals. IMPORTANCE Endogenous retroviruses (ERVs) are relics of past infection that constitute up to 8% of the human genome. Understanding the genetic evolution of the ERV family and the interplay of ERVs and encoded RNAs and proteins with host function has become a new frontier in biology. Fish, as the most primitive vertebrate host for retroviruses, is an indispensable integral part for such investigations. In the present study, we report the genome-wide characterization of ERVs in zebrafish, an attractive model organism of ancient vertebrates from multiple perspectives, including composition, genomic organization, chromosome distribution, classification, phylogeny, insertion time, characterization of gag and env genes, and expression profiles in embryos and tissues. The result helps uncover the evolutionarily conserved and fish-specific ERVs, as well as the immune-relevant ERVs in response to virus infection. This study demonstrates the previously unrecognized abundance, diversification, and extensive activity of ERVs at the early stage of ERV evolution.

Mesenchymal stem cells attenuate liver fibrosis by targeting Ly6Chi/lo macrophages through activating the cytokine-paracrine and apoptotic pathways.

Mesenchymal stem cell (MSC) therapy has become a promising treatment for liver fibrosis due to its predominant immunomodulatory performance in hepatic stellate cell inhibition and fibrosis resolution. However, the cellular and molecular mechanisms underlying these processes remain limited. In the present study, we provide insights into the functional role of bone marrow-derived MSCs (BM-MSCs) in alleviating liver fibrosis by targeting intrahepatic Ly6Chi and Ly6Clo macrophage subsets in a mouse model. Upon chronic injury, the Ly6Chi subset was significantly increased in the inflamed liver. Transplantation of BM-MSCs markedly promoted a phenotypic switch from pro-fibrotic Ly6Chi subset to restorative Ly6Clo subpopulation by secreting paracrine cytokines IL-4 and IL-10 from the BM-MSCs. The Ly6Chi/Ly6Clo subset switch significantly blocked the source of fibrogenic TGF-ß, PDGF, TNF-α, and IL-1ß cytokines from Ly6Chi macrophages. Unexpectedly, BM-MSCs experienced severe apoptosis and produced substantial apoptotic bodies in the fibrotic liver during the 72 h period of transplantation. Most apoptotic bodies were engulfed by Ly6Clo macrophages, and this engulfment robustly triggered MMP12 expression for fibrosis resolution through the PtdSer-MerTK-ERK signaling pathway. This paper is the first to show previously unrecognized dual regulatory functions of BM-MSCs in attenuating hepatic fibrosis by promoting Ly6Chi/Ly6Clo subset conversion and Ly6Clo macrophage restoration through secreting antifibrogenic-cytokines and activating the apoptotic pathway.

Functional role of CD40 and CD154 costimulatory signals in IgZ-mediated immunity against bacterial infection.

CD40 and CD154 are one of the best-characterized costimulatory molecules essential for adaptive immunity, which extensively involved in T and B cell activation, IgM Ab production, isotype class switching, germinal center formation and affinity maturation. However, the functionality of CD40 and CD154 in IgZ-mediated immunity remains limited. In this study, we explored the regulatory role of Cd40-Cd154 interaction in IgZ-mediated antibacterial immunity in zebrafish. The results showed that the IgZ-mediated antibacterial response can be significantly induced in response to A. hydrophila infection. The percentage of Cd40+IgZ+ B cells and the production of IgZ Ab were substantially increased upon A. hydrophila stimulation, but these reactions were markedly declined in Cd154 blockade fish by administering anti-Cd154 Ab or recombinant sCd40-Ig protein, accompanied with the impairment of the vaccine-initiated IgZ-mediated immunoprotection of fish against A. hydrophila infection. These observations suggested the essential role of Cd40-Cd154 interaction in IgZ-mediated bacterial immunity. Notably, the Cd40 and Cd154 costimulatory signals are required for a TD antigen-induced IgZ immunity, but are not indispensable for a TI antigen-induced IgZ immune response. These findings indicated the differential role of Cd40-Cd154 interaction in bacterial TD and TI antigen-induced IgZ immunity, which suggested the existence of diverse regulatory mechanisms underlying IgZ-mediated antibacterial immune reactions. To our knowledge, this is the first report to show the functional role of Cd40-Cd154 costimulatory signaling pathway in IgZ-mediated immune defense against bacterial infection. We hope this study will improve the current understanding of the coevolution between the IgZ/IgT immunoglobins and CD40/CD154 costimulatory molecules.

Inhibitory Role of an Aeromonas hydrophila TIR Domain Effector in Antibacterial Immunity by Targeting TLR Signaling Complexes in Zebrafish.

The Toll/interleukin-1 receptor (TIR) domain is a structural unit responsible for the assembly of signal protein complexes in Toll-like receptor (TLR) and interleukin-1 receptor signaling pathways. TIR domain homologs are found in a considerable number of bacteria and enhance bacterial infection and survival in host organisms. However, whether TIR domain homologs exist in Aeromonas hydrophila, a ubiquitous waterborne bacterium in aquatic environments, remains poorly understood. In this study, a TIR domain protein (TcpAh) was identified from A. hydrophila JBN2301. TIR domain of TcpAh is highly homologous to the counterpart domains in TLRs and myeloid differentiation factor 88 (MyD88). The zebrafish infected with mutant A. hydrophila with tcpAh deletion had a remarkably lower mortality than those infected with the wild-type strain. This result suggests that TcpAh is a crucial virulence factor for A. hydrophila infection. TcpAh exhibited a strong ability to associate with MyD88, tumor necrosis factor receptor-associated factor 3 (TRAF3) and TRAF-associated NF-κB activator-binding kinase 1 (TBK1) in TIR-TIR, TIR-Death domain (DD), and other alternative interactions. This finding suggests that TcpAh extensively interferes with MyD88 and TIR domain-containing adapter inducing interferon (IFN)-ß (TRIF) signaling pathways downstream of TLRs. Consequently, CD80/86 expression was suppressed by TcpAh via attenuating TLR-stimulated NF-κB activation, which ultimately led to the impairment of the major costimulatory signal essential for the initiation of adaptive humoral immunity against A. hydrophila infection. We believe that this study is the first to show a previously unrecognized mechanism underlying A. hydrophila evades from host antibacterial defense by intervening CD80/86 signal, which bridges innate and adaptive immunity. The mechanism will benefit the development of therapeutic interventions for A. hydrophila infection and septicemia by targeting TcpAh homologs.