Negative regulation of interferon (IFN) responses is crucial for preventing IFN-induced cellular damage and restoring immune homeostasis after viral clearance. However, whether such regulation occurs at the receptor level has not been explored in lower vertebrates. Here we characterized a decoy receptor (CRFB15) in zebrafish, belonging to the cytokine receptor family B, that functions as a decoy receptor to antagonize IFN-mediated signaling. CRFB15 was shown to be upregulated upon viral infection. It bound to IFN ligands and suppressed STAT1 phosphorylation and downstream antiviral effector gene expression, ultimately promoting viral replication. It also interacted with IFN receptors, including CRFB1, CRFB2, and CRFB5, selectively promoting degradation of CRFB2 and CRFB5 and attenuating IFN-induced antiviral response. Furthermore, crfb15−/− zebrafish exhibited enhanced resistance to viral infection, confirming its role as a negative regulator of IFN signaling. This study establishes CRFB15 as a key negative regulator of teleost type I IFN signaling. Upon viral infection, CRFB15 is upregulated and suppresses IFN signaling through two distinct pathways: by functioning as a decoy receptor that competitively binds type I IFNs and their functional receptors, and/or by promoting the degradation of the key receptor subunits CRFB2 and CRFB5.

Cell-mediated cytotoxicity is a defense mechanism in which host immune cells kill infected cells. Cell-mediated cytotoxicity refers to the Major Histocompatibility Complex Class (MHC) restricted killing of infected cells with CD8+ T cells serving as key players. Intracellularly processed antigenic peptides are displayed by MHC-I molecules at the cell's surface for surveillance of immune cells. Naïve CD8+ T cells patrolling the lymphoid and peripheral tissues recognize the MHC-I loaded cognate peptide (pMHC-I) via their specialized T cell receptor (TCR). This results in TCR/pMHC-I binding and initiation of a cascade of events to facilitate T cell activation and subsequent differentiation into effector/cytotoxic CD8+ T cells (CTLs). Effector CTLs typically produce effector molecules and cytokines including granzymes, perforins and IFN-γ to eliminate infected cells. Fish cell-mediated cytotoxic response is complex and is far from understood. This review provides a general overview of the current knowledge of CD8+ cell mediated cytotoxic responses in fish and highlights the research gaps that need to be filled.

Integrins are important transmembrane receptors involved in cell adhesion, migration, and immune regulation. However, their functional roles remain poorly understood in fish. In this study, the itgb1b and itgb2 genes were cloned from grass carp (Ctenopharyngodon idella). Phylogenetic and synteny analyses showed that teleost itgb1 underwent gene duplication, and diverged into itgb1a and itgb1b, whereas itgb2 remained as a single-copy gene. Polyclonal antibodies against Itgb1b and Itgb2 were generated and characterized. Immunofluorescence staining demonstrated abundant populations of Itgb1b+ and Itgb2+ cells in the head kidney and spleen of healthy fish. Among head kidney leukocytes, Itgb1b+ and Itgb2+ cells accounted for 6.75% and 7.68%, respectively, whereas their proportions in the spleen were 9.25% and 3.99%. Notably, Itgb1b+/Mcsfr+ cells accounted for 4.64% and 5.21% of total live leukocytes in the head kidney and spleen, respectively, whereas Itgb2+/Mcsfr+ cells accounted for 5.05% and 3.05%, respectively, indicating that Itgb1b and Itgb2 are expressed by subsets of Mcsfr+ monocytes/macrophages. Following Aeromonas hydrophila infection, both the mRNA expression of itgb1b and itgb2 and the abundance of Itgb1b+ and Itgb2+ cells were significantly increased in the head kidney and spleen. Moreover, bacterial challenge promoted the proliferation of Itgb1b+ and Itgb2+ cells in these immune tissues. These findings indicate that Itgb1b+ and Itgb2+ leukocytes, including a macrophage subpopulation, participate in the immune response to bacterial infection in fish.

Cullin-RING ligases (CRLs) constitute the most diverse E3 ligase family in the ubiquitin-proteasome system, yet their antiviral roles in bony fish remain poorly understood. Here, we identify the catalytic cores of CRLs, the RING-box proteins RBX1 and RBX2, as key modulators of antiviral signaling in cyprinid fish. Phylogenetic and structural analyses revealed that Rbx1 and Rbx2 are highly conserved and widely expressed, with preferential enrichment in primordial germ and immune-related cells. Upon infection with grass carp reovirus (GCRV) or spring viremia of carp virus (SVCV) in cyprinid fish, Rbxs were rapidly induced in vivo and in vitro. RBX1 and RBX2 synergistically promote viral replication by directly interacting with interferon regulatory factors (IRF) 3 and IRF7 and facilitating their ubiquitin-mediated regulation. Together, our findings uncover RBX1 and RBX2 as evolutionarily conserved negative regulators of fish innate immunity and provide mechanistic insight into the ubiquitin-mediated control of interferon homeostasis across vertebrates.

Fish cell lines suffer from extremely low transfection efficiency (most <10%), severely limiting functional genomics and genetic engineering in aquaculture. Current strategies focus on optimizing delivery vehicles, while intracellular mechanisms restricting exogenous nucleic acid expression remain unexplored in fish. In mammals, TRIM25 is a key sensor mediating degradation of endocytosed exogenous RNA. Here, we confirmed structural conservation of TRIM25 across 18 teleost species and demonstrated that siRNA-mediated trim25 knockdown dramatically enhanced transfection efficiency: in zebrafish PAC2 cells, mRNA transfection increased from 19.27% to 74.63% (3.87-fold); in Chinese tongue sole CST cells, 3.47-fold. These results suggest TRIM25 is a conserved barrier to exogenous nucleic acid expression in teleosts, offering a simple, low-cost strategy to overcome the transfection bottleneck in fish cells.

Proteasome β (PSMB) subunits are essential components of the proteasome complex and play important roles in antigen processing and immune regulation. In this study, we identified 14 Psmb genes in grass carp (Ctenopharyngodon idella), including seven constitutive Psmbs (Psmb1-7), three immunoproteasome genes (Psmb8-10), two thymoproteasome-related paralogs (Psmb11a and Psmb11b), and two telelost-specific members (Psmb12 and Psmb13). Comparative genomic analyses showed that grass carp Psmb genes are highly conserved in genomic organization, gene synteny, and predicted β-subunit-like protein structures, supporting the evolutionary conservation of the proteasome β-subunit family in fish. Phylogenetic and syntenic analyses further revealed lineage-specific expansion of immunoproteasome-related Psmb genes in teleost fish, with Psmb12 and Psmb13 likely derived from duplications of Psmb9 and Psmb10, respectively. Tissue expression analysis suggested functional divergence among duplicated Psmb members, as constitutive Psmbs were relatively enriched in the brain, whereas immunoproteasome-related and teleost-specific Psmbs were highly expressed in immune- and mucosa-associated tissues. Moreover, GCRV-I infection rapidly induced Psmb8-10 and Psmb11b expression in CIK cells. IFN-γ induced a broader set of Psmb genes than IFNa, whereas IL-10 selectively suppressed several Psmbs. Together, these findings highlight the evolutionary conservation, expansion, and immune-related diversification of the Psmb family in teleost fish.

The interleukin-1 (IL-1) family is a central regulator of vertebrate inflammation. While eleven IL-1 family members have been identified in mammals, only three members, IL-1β, IL-18, and a teleost-specific novel IL-1 family member (nIL-1F), have been reported in fish to date. Here, we report a previously uncharacterized IL-1 family cytokine (termed nIL-1Fb) in cyprinid fish. The gene encoding nIL-1Fb is tandemly linked with the nIl-1f gene and is phylogenetically related to the nIl-1f, suggesting their common ancestral origin. We characterized the expression and function of nIL-1Fb in grass carp (Ctenopharyngodon idella), a major aquaculture species in Asia. Following infection with grass carp reovirus type II (GCRV-II), the nIl-1fb expression was markedly upregulated in the gills and hindgut. Immunofluorescence analyses revealed that nIL-1Fb was produced by virus infected cells and upregulated in infected tissues. Furthermore, nIL-1Fb co-localized with macrophage colony-stimulating factor receptor (MCSFR), indicating that monocytes/macrophages (Mo/Mφ) are the major source of nIL-1Fb. Functionally, nIL-1Fb not only induced the expression of pro-inflammatory genes (Tnf-α3, Il-8, and inos) but also enhanced the phagocytic activity of Mo/Mφ. However, nIL-1Fb exhibited a much weaker pro-inflammatory activity than IL-1β. Taken together, our findings identify nIL-1Fb as a cyprinid-specific pro-inflammatory cytokine, offering novel insights into the evolution and functional diversification of the IL-1 family in teleost fish.

Type I interferons (IFNs) are central antiviral cytokines in vertebrates, yet the mechanisms underlying their functional diversification in early vertebrates remain unclear. Teleost fish, whose IFN repertoires expanded through whole-genome duplication, provide a powerful model to address this question. Here, we systematically characterize grass carp (Ctenopharyngodon idella) IFNd to elucidate the mechanisms underlying its functional divergence from IFNa. IFNd used the same receptors of IFNa to activate JAK-STAT pathway, albeit with significantly lower antiviral potency than IFNa. Structural analysis revealed significant differences in F helix length and key receptor-binding residues between IFNa and IFNd. Furthermore, single-cell RNA sequencing demonstrated high heterogeneity among immune cell subpopulations responding to IFNa and IFNd, Notably, the signaling pathways enriched by differentially expressed genes in monocytes and macrophages were distinct for the 2 cytokines. Together, our findings link ligand structural conformation and receptor-binding composition to IFN signaling strength and immune cell specificity, providing mechanistic insight into the functional diversification of type I IFNs in lower vertebrates.

Meteorin-like (Metrnl) is a cytokine known to play multifunctional roles in mammalian immunity, yet its immunoregulatory role in teleost remains largely unexplored. In the present study, we systematically investigate the effects of Metrnl on primary monocytes/macrophages (Mo/Mφ) in grass carp (Ctenopharyngodon idella). In vitro, Metrnl significantly increased the viability of primary Mo/Mφ, with increases of 12.7% on day 3 and 7.9% on day 6, as determined by flow cytometric quantification of MCSFR+ cells across multiple biological replicates. Furthermore, Metrnl stimulation promoted Mo/Mφ proliferation and phagocytic activity, resulting in absolute increases of 12.43% and 16.86%, respectively, calculated as the proportion of EdU+MCSFR+ or Beads+MCSFR+ cells within the total MCSFR+ population. Consistent with these findings, in vivo experiments using a Metrnl-expressing plasmid demonstrated enhanced Mo/Mφ proliferation and phagocytosis. Moreover, Metrnl also significantly upregulated the mRNA levels of the chemokines Cxcl8 and Cxcl11.1b, indicating its ability to recruit Mo/Mφ to target sites by activating chemokine-mediated chemotactic signaling. Importantly, neutralization of Metrnl with a specific antibody markedly impaired the enhanced Mo/Mφ recruitment, proliferation and phagocytosis, confirming that the effect is specifically mediated by Metrnl. Our results demonstrate that Metrnl acts as an important regulatory factor that activates and enhances Mo/Mφ-mediated innate immune response.

Abstract Why microbes invest energy in producing structurally complex natural products at extremely low concentrations within hosts remains an important topic of interest in microbial ecology. This is because such compounds with potential “biomolecular activities” can reflect molecule-target interactions, thereby providing conceptual and mechanistic insights that inform the rational design of essential clinical agents. Here, we uncover the in situ ecological function of the dithiolopyrrolone antibiotic holomycin produced by the fish pathogen Yersinia ruckeri . We show that holomycin’s biosynthesis is strictly gated by low temperature and that trace amounts of holomycin accumulate in vivo during infection in rainbow trout. In addition, the holomycin-deficient mutant leads to markedly reduced virulence. Integrated multiomics, phenotypic analyses, and in vivo bioassay test reveal that holomycin is not a direct lethal factor but instead remodels host-associated microbiota and enhances Y. ruckeri biofilm formation. These coordinated effects promote pathogen colonization and infection progression. Our findings establish holomycin as a host-associated effector and illustrate how its existence and ecological function are mechanistically intertwined in the evolution of pathogenic bacterial chemical arsenals.

Arrestin domain-containing 3 (ARRDC3), a member of the α-arrestins family, plays an important regulatory role in host anti-tumor and cellular metabolism by modulating the degradation and transportation of G protein-coupled receptors (GPCRs). However, the function of ARRDC3 in fish remains largely unexplored, particularly in host antiviral response. Here, we identified a paralog of ARRDC3 in teleost, zebrafish ARRDC3a, which was found to be induced by spring viremia of carp virus (SVCV) in vivo and in vitro. Overexpression of ARRDC3a significantly attenuated IFN expression and facilitated SVCV replication, whereas its knockdown inhibited viral proliferation. Furthermore, ARRDC3a overexpression markedly suppressed the IFN response triggered by retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) signaling pathway, and reduced the protein stability of TBK1, MITA, and IRF3. In summary, our findings suggest that zebrafish ARRDC3a acts as a negative regulator of type I IFN production by targeting RLR-associated signaling molecules, thereby promoting SVCV replication in the host. Our study provides novel insights into the biological function of ARRDC3a in fish.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of 34–120 kDa nuclear proteins that have recently been reported to participate in virus replication. The hnRNP family contains approximately 20 members, including hnRNP A1, hnRNP A2, hnRNP A2B1, hnRNPC, hnRNPD and hnRNPK. HnRNPC plays important roles in RNA biology, including expression, stability, mRNA splicing, nonspecific sequence export and 3’-end processing; however, the mechanisms underlying hnRNPC regulatory roles are not fully understood. Here, we found that zebrafish hnRNPC promoted spring viraemia of carp virus (SVCV) replication by increasing the stability of SVCV phosphoprotein while inhibiting the K48-linked ubiquitination of virus phosphoprotein, thereby suppressing the type I interferon (IFN) response. Mechanistically, hnRNPC could interact with the mediator of IFN regulatory factor 3 activation (MITA) to activate K48-linked ubiquitination for MITA degradation through the C-terminal domain of hnRNPC. We also showed that human hnRNPC could interact with MITA and that the overexpression of human hnRNPC decreased MITA protein in HEK293 cells, suggesting that the negative regulatory effects of hnRNPC on the type I IFN response are evolutionarily conserved. Collectively, our data indicate that hnRNPC promotes virus replication by suppressing IFN production activated by MITA and increasing the availability of viral proteins. Our work reveals an evolutionarily conserved mechanism that controls the IFN-mediated antiviral response by a member of the hnRNP family in vertebrates.

While T cells are well established as key players in mammalian adaptive immunity, their functional roles in teleosts remain poorly understood. Here, a monoclonal antibody against grass carp (Ctenopharyngodon idella) CD3ε was generated, and characterized by Western blotting, flow cytometry, and immunofluorescence. In healthy fish, CD3ε+ T cells accounted for 5.1 %, 4.0 %, and 2.6 % of splenic, head kidney, and peripheral blood lymphocytes, respectively. Upon Aeromonas hydrophila infection, CD3ε+ T cell populations elevated significantly in immune tissues such as spleen, head kidney, and posterior intestine, accompanied by upregulated transcription of T cell markers (Cd3ε, Tcr-β, and Lck). Strikingly, immunofluorescence analysis revealed that CD3ε+ T cells and MCSFR+ macrophages were located in the same region in the spleen tissue of A. hydrophila-infected fish, suggesting functional crosstalk between T cells and macrophages. Our findings provide the first evidence of T cell-macrophage interaction in teleost antibacterial defense, offering insights into the adaptive immune responses in fish.

A balanced IFN response, tightly regulated at multiple levels, is essential for host defense against viral infection. Tripartite motif-containing (TRIM) proteins are a large group of E3 ubiquitin ligases, and have been shown to be involved in the regulation of IFN response. However, the regulatory functions of individual TRIM proteins remain controversial. Here, we show that a virus-inducible TRIM2 homolog acts as a negative regulator for IFN production in zebrafish. Zebrafish Trim2a was upregulated in response to spring viremia of carp virus (SVCV) infection, and knockout of Trim2a significantly increased the expression of antiviral genes, leading to enhanced resistance to SVCV. Overexpression of Trim2a resulted in pronounced ubiquitination of IFN regulatory factor 3 (IRF3) via K11, K27, K29, and K48, promoting IRF3 degradation and stability of SVCV phosphoprotein to favor viral replication. Moreover, TRIM2a induced ubiquitination of autophagic cargo receptor p62, which then interacted with IRF3, instigating IRF3 degradation. Further, the inhibitory effects of TRIM2a on IFN production were also observed in human HEK293 cells, suggesting that the regulatory functions of TRIM2 are likely to be conserved during evolution. Collectively, our findings demonstrate that TRIM2a is a negative regulator of IFN production, and could serve as a potential target to dampen exacerbated IFN response triggered by aberrant activation of retinoic acid-inducible gene 1 (RIG-I)-like receptors. Our study provides insights into a previously uncharacterized role of TRIM2 in the regulation of IFN signaling.

Anaplastic large cell lymphoma (ALCL), an aggressive T-cell malignancy, is marked by elevated expression of CD30 and the immune checkpoint molecule PD-L1. While CD30-directed chimeric antigen receptor (CAR) therapies have demonstrated clinical promise, therapeutic resistance remains a major hurdle. Here, we conducted integrated genome-wide CRISPR-Cas9 loss-of-function screens using CD30-specific CAR-engineered natural killer (CAR-NK) cells, alongside a complementary PD-L1 regulator screen, and uncovered a critical role for interleukin-1 receptor (IL-1R) signaling in modulating CAR therapy efficacy in both ALK⁺ and ALK⁻ ALCL. Mechanistically, IL-1R signaling drives an NFKBIZ – IL-17F – MAPK axis that sustains PD-L1 expression via an autocrine loop, while simultaneously inducing proinflammatory cytokines and chemokines that reinforce immune evasion and shape an immunosuppressive tumor microenvironment. Notably, NFKBIZ (IκBζ) emerges as a central transcriptional regulator orchestrating this immune suppression program upstream of IL-17F. Importantly, pharmacologic inhibition of IL-1R signaling significantly enhances the antitumor activity of CD30-specific CAR therapies both in vitro and in ALCL xenograft models. Collectively, our findings uncover a novel mechanism of immune resistance and nominate IL-1R blockade as a promising combinatorial strategy to improve CAR-based immunotherapy in ALCL.

Interleukin-26 (IL-26) belongs to the IL-10 cytokine family and exerts diverse biological functions in regulating immune responses in vertebrates. Although IL-26 has been extensively studied in mammals, the functions of IL-26 remain largely unexplored in lower vertebrates. In this study, we determined the tissue and cell sources of IL-26 using a monoclonal antibody (mAb) generated against grass carp (Ctenopharyngodon idella, Ci) IL-26, and investigated the responses of IL-26 producing cells to bacterial infection. We showed that the CiIL-26 mAb specifically recognized the recombinant CiIL-26 proteins expressed in the Escherichia coli and HEK293 cells. Flow cytometry analysis revealed that the CiIL-26 mAb could detect the intracellular CiIL-26 expressed in the HEK293 cells and CIK cells stimulated with inactivated Aeromonas hydrophila (A. hydrophila). Using confocal microscopy, we analyzed IL-26+ cells in various tissues of grass carp following infection with A. hydrophila. It was shown that the IL-26+ cells were significantly increased in the gills, head kidney, posterior intestine and spleen. Remarkably, for the first time, we observed that most IL-26+ cells were CD3γ/δ+ T cells and MCSFR+ monocytes/macrophages, which could be induced by A. hydrophila. Our findings highlight the essential roles of CD3γ/δ+/IL-26+ T cells and MCSFR+/IL-26+ macrophages in the immune defense against bacterial infections in fish.

Interferons (IFNs) are a group of secreted cytokines that play a crucial role in antiviral immunity. Type I IFNs display functional disparities. In teleosts, type I IFNs are categorized into two subgroups containing one or two pairs of disulfide bonds. However, their functional differences have not been fully elucidated. In this study, we comparatively characterized the antiviral activities of zebrafish IFNI:p1 and IFNI:p4 belonging to the group I type I IFNs. It was found that ifn phi 1 and ifn phi 4 were differentially modulated during viral infection. Although both IFNI:p1 and IFNI:p4 activated JAK-STAT signaling pathway via CRFB1/CRFB5 receptor complex, IFNI:p4 was less potent in inducing phosphorylation of STAT1a, STAT1b and STAT2 and the expression of antiviral genes than IFNI:p1, thereby conferring weaker antiviral resistance of target cells. Taken together, our results provide insights into the functional divergence of type I IFNs in lower vertebrates.

Grass carp, one of the major freshwater aquaculture species in China, is susceptible to grass carp reovirus (GCRV). GCRV is a non-enveloped RNA virus and has a double-layered capsid, causing hemorrhagic disease and high mortalities in infected fish. However, the tropism of GCRV infection has not been investigated. In this study, monoclonal antibodies against recombinant VP35 protein were generated in mice and characterized. The antibodies exhibited specific binding to the N terminal region (1-155 aa) of the recombinant VP35 protein expressed in the HEK293 cells, and native VP35 protein in the GCRV-II infected CIK cells. Immunofluorescent staining revealed that viruses aggregated in the cytoplasm of infected cells. In vivo challenge experiments showed that high levels of GCRV-II viruses were present in the gills, intestine, spleen and liver, indicating that they are the major sites for virus infection. Our study showed that the VP35 antibodies generated in this study exhibited high specificity, and are valuable for the development of diagnostic tools for GCRV-II infection.

Macrophage colony-stimulating factor receptor (MCSFR) is a cell lineage marker for monocytes/macrophages and has been applied for cell typing in mammals. However, lack of high-quality monoclonal antibodies hampers isolation and functional characterization of monocytes/macrophages in fish. A monoclonal antibody (mAb) against the extracellular region of grass carp (Ctenopharyngodon idella, Ci) MCSFR using the recombinant protein produced in the E. coli cells was generated in this study. Western blotting showed the mAb could recognize the native CiMCSFR protein derived from tissues and the recombinant CiMCSFR protein expressed in the E. coli cells and HEK293 cells. The antibody did not cross-react with the MCSFR homolog in zebrafish, indicating that it has good species specificity. Immunofluorescence analysis showed MCSFR+ monocytes/macrophages distributed in the gills, hindgut, head kidney and spleen of healthy grass carp. Furthermore, Aeromonas hydrophila infection resulted in increased MCSFR+ monocytes/macrophages in the lamina propria and epithelium of hindgut mucosa. In addition, Flavobacterium columnare infection led to the infiltration of MCSFR+ monocytes/macrophages in the gills, and interestingly, the MCSFR+ monocytes/macrophages in the gill lamellae but not filaments are activated to produce IL-4/13A and IL-4/13B, while MCSFR+ monocytes/macrophages in the hindgut did not produce IL-4/13A and IL-4/13B. Our results indicate that monocytes/macrophages are participated in mediating mucosal immune responses to bacterial infections and will help understand host immune defense to bacterial diseases.