TLR5M and TLR5S Synergistically Sense Flagellin in Early Endosome in Lamprey Petromyzon marinus, Switched by the N-Glycosylation Site N239.

In mammals, TLR5 functions as a homodimer to recognize bacterial flagellin on the cytomembrane. The current investigations reveal the existence of two types of TLR5, a membrane-bound PmTLR5M, and a soluble variant PmTLR5S, in lamprey (Petromyzon marinus). Although both PmTLR5M and PmTLR5S can bind flagellin, only PmTLR5M is capable of eliciting a proinflammatory response, whereas PmTLR5S can detect the flagellin and facilitate the role of PmTLR5M in early endosomes. The trafficking chaperone UNC93B1 enhances the ligand-induced signaling via PmTLR5M or the combination of PmTLR5M and PmTLR5S. PmTLR5M recruits MyD88 as an adaptor. Furthermore, chimeric receptor studies demonstrate the indispensability of the intradomain of PmTLR5M in effective activation of the proinflammatory pathway upon flagellin stimulation, and the combination of PmTLR5S with a singular intradomain in both homodimer and heterodimer ectodomain arrangements can very significantly augment the immune response. Furthermore, the flagellin binding sites between PmTLR5M and PmTLR5S are conserved, which are essential for ligand binding and signal transduction. Moreover, investigations on N-linked glycosylation modifications reveal that the N239 site in PmTLR5M and PmTLR5S plays a switch role in both flagellin binding and immune responses. In addition, PmTLR5M exhibits the high-mannose-type and complex-type N-glycosylation modifications; however, PmTLR5S shows exclusive complex-type N-glycosylation modification. The key N239 site demonstrates complex-type N-glycosylation modification. The findings address the function and mechanism of TLR5 in ligand recognition, subcellular localization, and signaling pathway in lowest vertebrate and immune system transition species, highlight the regulatory role of N-glycosylation modification in TLRs, and augment immune evolutionary research on the TLR signaling pathway.

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.

GCRV-II invades monocytes/macrophages and induces macrophage polarization and apoptosis in tissues to facilitate viral replication and dissemination.

Grass carp reovirus (GCRV), particularly the highly prevalent type II GCRV (GCRV-II), causes huge losses in the aquaculture industry. However, little is known about the mechanisms by which GCRV-II invades grass carp and further disseminates among tissues. In the present study, monocytes/macrophages (Mo/Mφs) were isolated from the peripheral blood of grass carp and infected with GCRV-II. The results of indirect immunofluorescent microscopy, transmission electron microscopy, real-time quantitative RT-PCR (qRT-PCR), western blot (WB), and flow cytometry analysis collectively demonstrated that GCRV-II invaded Mo/Mφs and replicated in them. Additionally, we observed that GCRV-II induced different types (M1 and M2) of polarization of Mo/Mφs in multiple tissues, especially in the brain, head kidney, and intestine. To assess the impact of different types of polarization on GCRV-II replication, we recombinantly expressed and purified the intact cytokines CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B and successfully induced M1 and M2 type polarization of macrophages using these cytokines through in vitro experiments. qRT-PCR, WB, and flow cytometry analyses showed that M2 macrophages had higher susceptibility to GCRV-II infection than other types of Mo/Mφs. In addition, we found GCRV-II induced apoptosis of Mo/Mφs to facilitate virus replication and dissemination and also detected the presence of GCRV-II virus in plasma. Collectively, our findings indicated that GCRV-II could invade immune cells Mo/Mφs and induce apoptosis and polarization of Mo/Mφs for efficient infection and dissemination, emphasizing the crucial role of Mo/Mφs as a vector for GCRV-II infection.IMPORTANCEType II grass carp reovirus (GCRV) is a prevalent viral strain and causes huge losses in aquaculture. However, the related dissemination pathway and mechanism remain largely unclear. Here, our study focused on phagocytic immune cells, monocytes/macrophages (Mo/Mφs) in blood and tissues, and explored whether GCRV-II can invade Mo/Mφs and replicate and disseminate via Mo/Mφs with their differentiated type M1 and M2 macrophages. Our findings demonstrated that GCRV-II infected Mo/Mφs and replicated in them. Furthermore, GCRV-II infection induces an increased number of M1 and M2 macrophages in grass carp tissues and a higher viral load in M2 macrophages. Furthermore, GCRV-II induced Mo/Mφs apoptosis to release viruses, eventually infecting more cells. Our study identified Mo/Mφs as crucial components in the pathway of GCRV-II dissemination and provides a solid foundation for the development of treatment strategies for GCRV-II infection.

IFN1 Enhances Thrombocyte Phagocytosis through IFN Receptor Complex-JAK/STAT-Complement C3.3-CR1 Pathway and Facilitates Antibacterial Immune Regulation in Teleost.

Type I IFNs with strong positive charges exhibit robust bactericidal activity and a protective effect against bacterial infections. However, the antibacterial mechanism in vivo remains unknown. In this study, Ab blockade of IFN1, a member of type I IFNs in grass carp (Ctenopharyngodon idella), resulted in high mortality, tissue bacterial loads, and low expression of immune factors after bacterial challenge, which indicates that the antibacterial activity of IFN1 has physiological significance. Meanwhile, we injected grass carp with the recombinant and purified intact IFN1 protein after bacterial injection, and the result demonstrated a remarkable therapeutic effect. Furthermore, we found that IFN1 expression was remarkably induced in blood cells after bacterial challenge, and prophagocytosis via IFN1 mostly increased in thrombocytes. Then, we isolated peripheral blood thrombocytes by polyclonal Ab of CD41 and stimulated thrombocytes with recombinant IFN1, and the results indicated that immune factors and complement components (especially C3.3) were induced. Unexpectedly, complements demonstrated not only bacteriolysis but also bacterial aggregation. Furthermore, Ab blockades of the three subunits (CRFB1/CRFB2/CRFB5) of the IFN1 receptor or inhibition of STAT1 almost abolished the prophagocytosis via IFN1 and reduced C3.3 and immune factor expression in thrombocytes. Meanwhile, Ab blockade of the complement receptor CR1 greatly attenuated the prophagocytosis of IFN1. In contrast, mouse IFN-ß did not show the promotion of antibacterial activity. These results clarify the prophagocytosis and immune regulation pathways of IFN1 in antibacterial immunity in teleosts. This study reveals the antibacterial mechanisms of type I IFNs in vivo and inspires functional studies of IFN in bacterial infections.

Cyprinid-specific duplicated membrane TLR5 senses dsRNA as functional homodimeric receptors.

Membrane-embedded Toll-like receptor 5 (TLR5) functions as a homodimer to detect bacterial flagellin. Cyprinid grass carp (Ctenopharyngodon idella) encodes two TLR5 genes, CiTLR5a and CiTLR5b. Here, we show that cyprinid TLR5a and TLR5b homodimers unexpectedly bind the dsRNA analog poly(I:C) and regulate interferon (IFN) response in early endosomes and lysosomes. Although TLR5 homodimers also bind flagellin, an immune response to flagellin is only triggered by TLR5a/b heterodimer. Moreover, we demonstrate that two TLR5 paralogs have opposite effects on antiviral response: CiTLR5a slightly promotes and powerfully maintains, whereas CiTLR5b remarkably inhibits virus replication. We show that the ectodomain of CiTLR5 is required for dsRNA-induced IFN signaling, and we map the key poly(I:C) binding sites to G240 for CiTLR5a and to N547 for CiTLR5b. Furthermore, we reveal that differential N-glycosylation of CiTLR5a/b affects dsRNA-IFN signaling but has no role in flagellin-mediated NF-κB induction, with paralog-specific roles for CiTLR5a-T101 and corresponding CiTLR5b-I99. Moreover, we provide evidence that the ability to sense dsRNA represents a neofunctionalization specific for membrane-bound TLR5 in cyprinid, bridging viral and bacterial immune responses.

Antioxidant anthocyanin synergistic immune enhancer nanopeptide C-I20 remarkably enhances the protective effect of largemouth bass against largemouth bass ranavirus.

Largemouth bass ranavirus (LMBV) infection results in huge economic losses in largemouth bass (Micropterus salmoides) industry. Nanopeptide C-I20 and anthocyanins have a positive effect on promoting immune responses and antioxidant mechanisms in several aquatic organisms, and are therefore used to inhibit LMBV infection. In this study, we developed an LMBV immersion challenge model using three different viral concentrations (1 × 104 copies/mL, 1 × 105 copies/mL, and 1 × 106 copies/mL) to infect largemouth bass, and LMBV-MCP mRNA expression was detected in infected fish. Following infection, the fish exhibited severe external ulceration, redness swelling, and darkening of the skin. Histopathological examination revealed significant necrosis and inflammation in muscle tissue, epithelial cell shedding in renal tubules, macrophage aggregation centers and cellular vacuolization in spleen and head kidney, and cellular hypertrophy in liver. To mitigate LMBV infection, we explored the protective effects of a combined treatment strategy involving C-I20 and anthocyanin. Overall, the combination of anthocyanin and C-I20 demonstrated the highest protective efficacy, significantly reducing viral loads in muscle, liver, spleen, and head kidney. Moreover, this treatment regimen enhanced antioxidant enzyme activities (T-AOC, TSOD, GSH-Px, CAT) and modulated important immune genes (IL-1, IL-8, TNF-α, IL-10, Mx, and IgM) expression. In conclusion, the synergistic application of anthocyanin and C-I20 demonstrates significant efficacy in mitigating LMBV infection. This research introduces a novel and promising approach to managing infectious diseases in aquaculture settings.

Lactobacillus casei displaying MCP2α and FlaC delivered by PLA microspheres effectively enhances the immune protection of largemouth bass (Micropterus salmoides) against LMBV infection.

Largemouth bass ranavirus (LMBV) seriously affects the development of largemouth bass (Micropterus salmoides) industry and causes huge economic losses. Oral vaccine can be a promising method for viral disease precaution. In this study, MCP2α was identified as a valuable epitope region superior to MCP and MCP2 of LMBV by neutralizing antibody experiments. Then, recombinant Lactobacillus casei expressing the fusion protein MCP2αC (MCP2α as antigen, C represents flagellin C from Aeromonas hydrophila as adjuvant) on surface was constructed and verified. Further, PLA microsphere vaccine loading recombinant MCP2αC L. casei was prepared. The PLA microspheres vaccine were observed by scanning electron microscopy and showed a smooth, regular spherical surface with a particle size distribution between 100 and 200 µm. Furthermore, we evaluated the tolerance of PLA-MCP2αC vaccine in simulated gastric fluid and simulated intestinal fluid, and the results showed that PLA-MCP2αC can effectively resist the gastrointestinal environment. Moreover, the protective effect of PLA-MCP2αC against LMBV was evaluated after oral immunization and LMBV challenge. The results showed that PLA-MCP2αC effectively up-regulated the activity of serum biochemical enzymes (T-SOD, T-AOC, LZM, complement C3) and induced the mRNA expression of representative immune genes (IL-1ß, TNF-α, IFN-γ, MHC-IIα, Mx, IgM) in spleen and head kidney tissues. The survival rate of largemouth bass vaccinated with PLA-MCP2αC increased from 24 % to 68 %. Meanwhile, PLA-MCP2αC inhibited the LMBV burden in spleen, head kidney and liver tissues and attenuated tissue damage in spleen. These results suggested that PLA-MCP2αC can be used as a candidate oral vaccine against LMBV infection in aquaculture.

Selenomethionine alleviates Aeromonas hydrophila-induced oxidative stress and ferroptosis via the Nrf2/HO1/GPX4 pathway in grass carp.

Aeromonas hydrophila infection is a severe, acute, and life-threatening disease affecting grass carp (Ctenopharyngodon idella) in aquaculture. Ferroptosis is a novel form of cell death characterized by the accumulation of free iron and harmful lipid peroxides within cells. While selenomethionine (Se-Met) is known to effectively inhibit ferroptosis and alleviate cell damage, its ability to counteract oxidative stress and ferroptosis induced by A. hydrophila remains unclear. The objective of this study is to reveal the possible mechanism behind the ferroptosis phenomenon during A. hydrophila infection. We established a macrophage model of A. hydrophila invasion to monitor the dynamic changes in iron metabolism markers to evaluate the correlation between ferroptotic stress and A. hydrophila infection. A. hydrophila infection induces cytotoxicity and mitochondrial membrane damage via ferroptosis. This damage is attributed to the accumulation of lipid peroxides due to intracellular ferrous ion overload and glutathione depletion. Supplementation of Se-Met reduced mitochondrial damage, enhanced antioxidant enzyme activity and reduced ferroptosis by activating the Nrf2/HO1/GPX4 axis. These findings provide new insights into the regulatory mechanisms of A. hydrophila-induced ferroptosis in teleosts and suggest that targeted inhibition of ferroptosis may offer a novel therapeutic strategy for managing A. hydrophila infections.

Tissue factor pathway inhibitors disrupt structures of rhabdovirus/ranairidovirus and inhibit viral infection in Chinese perch, Siniperca chuatsi.

Viral diseases have caused great economic losses to the aquaculture industry. However, there are currently no specific drugs to treat these diseases. Herein, we utilized Siniperca chuatsi as an experimental model, and successfully extracted two tissue factor pathway inhibitors (TFPIs) that were highly distributed in different tissues. We then designed four novel peptides based on the TFPIs, named TS20, TS25, TS16, and TS30. Among them, TS25 and TS30 showed good biosafety and high antiviral activity. Further studies showed that TS25 and TS30 exerted their antiviral functions by preventing viruses from invading Chinese perch brain (CPB) cells and disrupting Siniperca chuatsi rhabdovirus (SCRV)/Siniperca chuatsi ranairidovirus (SCRIV) viral structures. Additionally, compared with the control group, TS25 and TS30 could significantly reduce the mortality of Siniperca chuatsi, the relative protection rates of TS25 against SCRV and SCRIV were 71.25 % and 53.85 % respectively, and the relative protection rate of TS30 against SCRIV was 69.23 %, indicating that they also had significant antiviral activity in vivo. This study provided an approach for designing peptides with biosafety and antiviral activity based on host proteins, which had potential applications in the prevention and treatment of viral diseases.

MicroRNA miR-722 Inhibits Cyprinid Herpesvirus 3 Replication via Targeting the Viral Immune Evasion Protein ORF89, Which Negatively Regulates IFN by Degrading IRF3.

Cyprinid herpesvirus 3 (CyHV-3) has caused severe economic losses to carp culture, but its pathogenicity is far from clear. Our previous study has revealed that microRNA (miR)-722 was upregulated during CyHV-3 infection, indicating that miR-722 might play an important role in CyHV-3 replication. In this study, we found that overexpression of miR-722 inhibited CyHV-3 replication and promoted IFN expression. The putative target gene of miR-722 was searched over the CyHV-3 genome, and ORF89 was identified and validated as a target gene of miR-722. Overexpression of ORF89 markedly reduced the expression of IFN and IFN-stimulated genes. Mechanistically, ORF89 interacted with and degraded IFN regulatory factor 3 (IRF3), and inhibited the entry of IRF3 into the nucleus by suppressing the dimerization of IRF3. Moreover, ORF89-mediated suppression of IFN expression could be restored by adding miR-722. To our knowledge, our findings confirm a novel virus-host combat, in which CyHV-3 evades host antiviral immunity by its ORF89 protein, whereas host miR-722, upregulated on CyHV-3 infection, targets ORF89 to impede CyHV-3 replication.

Insights into the Antibacterial Properties of Complement Peptides C3a, C4a, and C5a across Vertebrates.

Complement peptides C3a, C4a, and C5a are important components of innate immunity in vertebrates. Although they diverged from a common ancestor, only C3a and C4a can act as antibacterial peptides in Homo sapiens, suggesting that C5a has evolved into a purely chemotactic molecule; however, the antibacterial properties of C3a, C4a, and C5a across vertebrates still require elucidation. In this article, we show that, unlike those in H. sapiens, Mus musculus C3a, C4a, and C5a all possess antibacterial activities, implying that the antibacterial properties of C3a, C4a, and C5a have evolved divergently in vertebrates. The extremely different net charge, a key factor determining the antibacterial activities of cationic antimicrobial peptides, of vertebrate C3a, C4a, and C5a supports this speculation. Moreover, the antibacterial activity of overlapping peptides covering vertebrate C3a, C4a, and C5a further strongly supports the speculation, because their activity is positively correlated with the net charge of source molecules. Notably, the structures of C3a, C4a, and C5a are conserved in vertebrates, and the inactive overlapping peptides can become antibacterial peptides if mutated to possess enough net positive charges, indicating that net charge is the only factor determining the antibacterial properties of vertebrate C3a, C4a, and C5a. More importantly, many vertebrate C3a-, C4a-, and C5a-derived peptides possess high antibacterial activities yet exhibit no hemolytic activities, suggesting the application potential in anti-infective therapy. Taken together, our findings reveal that vertebrate C3a, C4a, and C5a are all sources of antibacterial peptides that will facilitate the design of excellent peptide antibiotics.

Insights into the Antibacterial Properties of Complement Peptides C3a, C4a, and C5a across Vertebrates.

Complement peptides C3a, C4a, and C5a are important components of innate immunity in vertebrates. Although they diverged from a common ancestor, only C3a and C4a can act as antibacterial peptides in Homo sapiens, suggesting that C5a has evolved into a purely chemotactic molecule; however, the antibacterial properties of C3a, C4a, and C5a across vertebrates still require elucidation. In this article, we show that, unlike those in H. sapiens, Mus musculus C3a, C4a, and C5a all possess antibacterial activities, implying that the antibacterial properties of C3a, C4a, and C5a have evolved divergently in vertebrates. The extremely different net charge, a key factor determining the antibacterial activities of cationic antimicrobial peptides, of vertebrate C3a, C4a, and C5a supports this speculation. Moreover, the antibacterial activity of overlapping peptides covering vertebrate C3a, C4a, and C5a further strongly supports the speculation, because their activity is positively correlated with the net charge of source molecules. Notably, the structures of C3a, C4a, and C5a are conserved in vertebrates, and the inactive overlapping peptides can become antibacterial peptides if mutated to possess enough net positive charges, indicating that net charge is the only factor determining the antibacterial properties of vertebrate C3a, C4a, and C5a. More importantly, many vertebrate C3a-, C4a-, and C5a-derived peptides possess high antibacterial activities yet exhibit no hemolytic activities, suggesting the application potential in anti-infective therapy. Taken together, our findings reveal that vertebrate C3a, C4a, and C5a are all sources of antibacterial peptides that will facilitate the design of excellent peptide antibiotics.

The oral antigen-adjuvant fusion vaccine P-MCP-FlaC provides effective protective effect against largemouth bass ranavirus infection.

Largemouth bass ranavirus (LMBV) is highly contagious and lethal to largemouth bass, causing significant economic losses to the aquaculture industry. Oral vaccination is generally considered the most ideal strategy for protecting fish from viral infection. In this study, the fusion protein MCP-FlaC, consisting of the main capsid protein (MCP) as the antigen and flagellin C (FlaC) as the adjuvant, was intracellularly expressed in Pichia pastoris. Subsequently, the recombinant P. pastoris was freeze-dried to prepare the oral vaccine P-MCP-FlaC. Transmission electron microscopy and scanning electron microscopy analysis showed that the morphology and structure of the freeze-dried recombinant P. pastoris vaccine remained intact. The experiment fish (n = 100) was divided into five groups (P-MCP-FlaC, P-MCP, P-FlaC, P-pPIC3.5K, control) to evaluate the protective efficacy of the recombinant vaccine. Oral P-MCP-FlaC vaccine effectively up-regulated the serum enzymes activity (total superoxide dismutase, lysozyme, total antioxidant capacity, and complement component 3). The survival rate of P-MCP-FlaC group was significantly higher than that of the other groups. The mRNA expression of crucial immune genes (IL-1ß, TNF-α, MHC-II, IFN-γ, Mx, IgM, IgT) was also signally elevated in P-MCP-FlaC group. Vaccine P-MCP-FlaC markedly inhibited the replication of LMBV in the spleen, head kidney, and intestine, while reducing the degree of lesion in the spleen. These results suggest that the oral P-MCP-FlaC vaccine could effectively control LMBV infection, proving an effective strategy for viral diseases prevention in aquaculture.

Vitamin D3 can effectively and rapidly clear largemouth bass ranavirus by immunoregulation.

Largemouth bass ranavirus (LMBV) is a highly destructive pathogen that causes significant mortality rates among largemouth bass populations. Unfortunately, there is a dearth of drug development efforts specifically aimed at treating LMBV. To address this, our study sought to investigate the potential effectiveness of incorporating varying doses of VD3 into the diet as a treatment for LMBV. Through qRT-PCR and semi-qPCR, we observed significant suppression and clearance of LMBV pathogens in largemouth bass fed with 15000 IU/Kg and 20000 IU/Kg of VD3 within 14 days. In addition, VD3 treatment significantly increased the expression levels of key immune-related genes such as IL-1ß, IFN-γ, Mx, and IgM. Encouragingly, we observed that VD3 significantly increased antioxidant and immune activities such as TSOD, TAOC and C3 in serum and maintained total protein levels. Additionally, tissue pathology sections highlighted a dose-dependent relationship between VD3 supplementation and tissue damage, with the 15000 IU and 20000 IU groups exhibiting minimal damage. In conclusion, a reasonable concentration of VD3 effectively reduced LMBV replication and tissue damages, while improved immune-related genes expression and serum biochemical indices. These findings declare the considerable therapeutic potential of VD3 supplementation for combating LMBV disease and provide an alternative treatment option for fish farming.

Teleost-Specific MxG, a Traitor in the Mx Family, Negatively Regulates Antiviral Responses by Targeting IPS-1 for Proteasomal Degradation and STING for Lysosomal Degradation.

IFN-ß promoter stimulator-1 (IPS-1)- and stimulator of IFN genes (STING)-mediated type I IFNs play a critical role in antiviral responses. Myxovirus resistance (Mx) proteins are pivotal components of the antiviral effectors induced by IFNs in many species. An unprecedented expansion of Mx genes has occurred in fish. However, the functions and mechanisms of Mx family members remain largely unknown in fish. In this study, we found that grass carp (Ctenopharyngodon idella) MxG, a teleost-specific Mx protein, is induced by IFNs and viruses, and it negatively regulates both IPS-1- and STING-mediated antiviral responses to facilitate grass carp reovirus, spring viremia of carp virus, and cyprinid herpesvirus-2 replication. MxG binds and degrades IPS-1 via the proteasomal pathway and STING through the lysosomal pathway, thereby negatively regulating IFN1 antiviral responses and NF-κB proinflammatory cytokines. MxG also suppresses the phosphorylation of STING IFN regulatory factor 3/7, and it subsequently downregulates IFN1 and NF-κB1 at the promoter, transcription, and protein levels. GTPase and GTPase effector domains of MxG contribute to the negative regulatory function. On the contrary, MxG knockdown weakens virus replication and cytopathic effect. Therefore, MxG can be an ISG molecule induced by IFNs and viruses, and degrade IPS-1 and STING proteins in a negative feedback manner to maintain homeostasis and avoid excessive immune responses after virus infection. To our knowledge, this is the first identification of a negative regulator in the Mx family, and our findings clarify a novel mechanism by which the IFN response is regulated.

CXCL20a, a Teleost-Specific Chemokine That Orchestrates Direct Bactericidal, Chemotactic, and Phagocytosis-Killing-Promoting Functions, Contributes to Clearance of Bacterial Infections.

The major role of chemokines is to act as a chemoattractant to guide the migration of immune cells to the infectious sites. In the current study, we found that CiCXCL20a, a teleost-specific chemokine from grass carp (Ctenopharyngodon idella), demonstrates broad-spectrum, potent, direct bactericidal activity and immunomodulatory functions to bacterial infections, apart from the chemotaxis. CiCXCL20a kills bacteria by binding, mainly targeting acid lipids, perforating bacterial membrane, resulting in bacterial cytoplasm leakage and death. CiCXCL20a aggregates and neutralizes LPS, agglutinates Gram-negative bacteria, and binds to peptidoglycan and Gram-positive bacteria, but not agglutinate them. All the complexes may be phagocytized and cleared away. CiCXCL20a chemoattracts leukocytes, facilitates phagocytosis of myeloid leukocytes, not lymphoid leukocytes, and enhances the bacteria-killing ability in leukocytes. We further identified its receptor CiCXCR3.1b1. Furthermore, we investigated the physiological roles of CiCXCL20a against Aeromonas hydrophila infection in vivo. The recombinant CiCXCL20a increases the survival rate and decreases the tissue bacterial loads, edema, and lesions. Then, we verified this function by purified CiCXCL20a Ab blockade, and the survival rate decreases, and the tissue bacterial burdens increase. In addition, zebrafish (Danio rerio) DrCXCL20, an ortholog of CiCXCL20a, was employed to verify the bactericidal function and mechanism. The results indicated that DrCXCL20 also possesses wide-spectrum, direct bactericidal activity through membrane rupture mechanism. The present study, to our knowledge, provides the first evidence that early vertebrate chemokine prevents from bacterial infections by direct bactericidal and phagocytosis-killing-promoting manners. The results also demonstrate the close functional relationship between chemokines and antimicrobial peptides.

Broad-Spectrum Robust Direct Bactericidal Activity of Fish IFNφ1 Reveals an Antimicrobial Peptide-like Function for Type I IFNs in Vertebrates.

Type I IFNs (IFN-Is) play pivotal roles in host defense against viral infections but remain enigmatic against bacterial pathogens. In this study, we recombinantly expressed and purified intact grass carp (Ctenopharyngodon idella) IFNφ1 (gcIFNφ1), a teleost IFN-I. gcIFNφ1 widely powerfully directly kills both Gram-negative and Gram-positive bacteria in a dose-dependent manner. gcIFNφ1 binds to LPS or peptidoglycan and provokes bacterial membrane depolarization and disruption, resulting in bacterial death. Furthermore, gcIFNφ1 can efficiently protect zebrafish against Aeromonas hydrophila infection and significantly reduce the bacterial loads in tissues by an infection model. In addition, we wonder whether antibacterial IFN-I members exist in other vertebrates. The amino acid compositions of representative IFN-Is with strong positive charges from Pisces, Amphibia, reptiles, Aves, and Mammalia demonstrate high similarities with those of 2237 reported cationic antimicrobial peptides in antimicrobial peptide database. Recombinant intact representative IFN-I members from the nonmammalian sect exhibit potent broad-spectrum robust bactericidal activity through bacterial membrane depolarization; in contrast, the bactericidal activity is very weak from mammalian IFN-Is. The findings display a broad-spectrum potent direct antimicrobial function for IFN-Is, to our knowledge previously unknown. The results highlight that IFN-Is are important and robust in host defense against bacterial pathogens, and unify direct antibacterial and indirect antiviral bifunction in nonmammalian jawed vertebrates.

Chitosan and anisodamine enhance the immersion immune efficacy of inactivated Elizabethkingia miricola vaccine in black spotted frogs.

Black spotted frogs have rich nutrition and delicious meat, and its market consumption has increased year by year. However, outbreaks of the diseases have caused huge losses to the breeding industry. The crooked head disease caused by Elizabethkingia miricola (E. miricola) is highly contagious and lethal, and there is no effective treatment method. Vaccination is the most promising strategy to prevent infectious diseases. Immersion vaccination has attracted many researchers because of its simplicity of operation in preventing infectious diseases. In addition, immersion vaccines can be more effective when used with adjuvants. In this study, we prepared inactivated E. miricola with 0.3% formaldehyde, and the black spotted frogs were vaccinated by soaking in inactivated E. miricola vaccine, anisodamine + vaccine mixture, ß-glucan + vaccine mixture, chitosan + vaccine mixture for 60 min. PBS was used as a control. After being challenged by E. miricola, the survival rate of anisodamine + vaccine (57%) and chitosan + vaccine group (63%) was significantly higher than that of the control group (17%). By analyzing pathological sections, we found that the chitosan + vaccine and anisodamine + vaccine groups protected the brain, eye, liver and kidney tissues of the black spotted frogs compared to the control group, which was consistent with the trend of survival rate. In addition, chitosan + vaccine and anisodamine + vaccine groups had better effects on LZM, TSOD and C3 in serum than control group. Meanwhile, the numbers of the percentage of leukocytes/haemocytes in the peripheral blood of immunized black spotted frogs increased. The anisodamine + vaccine group (5.3%) and chitosan + vaccine (5.38%) group were significantly higher than the blank control group (2.24%), which indicate that the two groups induced a more significant immune response and were more resistant to bacterial invasion. The tissue bacterial loads in liver, brain, kidney and eye were significantly lower in the anisodamine + vaccine and chitosan + vaccine groups than that of the control group. This study explored and demonstrated the good efficiency of chitosan and anisodamine as adjuvants for immunization by immersion and provided a reference for improving the efficiency of immunization by immersion.

Blunt snout bream (Megalobrama amblycephala) MaCSF-1 contributes to proliferation, phagocytosis and immunoregulation of macrophages via MaCSF-1R.

CSF-1 and CSF-1R have been well demonstrated in humans, regulating the differentiation, proliferation and survival of the mononuclear phagocyte system. However, the functional study on MaCSF-1 and MaCSF-1R from blunt snout bream (Megalobrama amblycephala) is still unknown. In the present study, we cloned and functionally characterized MaCSF-1 and MaCSF-1R. Multiple sequence alignment and phylogenetic tree analysis showed that both MaCSF-1 and MaCSF-1R were mostly close to the grass carp counterparts. Tissue distribution analysis showed that both MaCSF-1 and MaCSF-1R were widely distributed in all examined tissues, dominantly distributed in spleen, blood and head kidney tissues. Furthermore, confocal microscopy assay and flow cytometry assay showed that MaCSF-1R was the marker on the surface of macrophages. Recombinant MaCSF-1 promoted macrophage proliferation, phagocytosis and the production of IL-10. Through the pull-down experiments and indirect immunofluorescence experiments, the interaction between MaCSF-1 and MaCSF-1R was confirmed. To explore the relationship between MaCSF-1 and its receptor, MaCSF-1R and MaCSF-1R antibody was prepared. Then the MaCSF-1R blockage assay indicated that the role of MaCSF-1 on the macrophages proliferation and phagocytosis was weakened, leading the reduction of IL-10 expression level. In conclusion, MaCSF-1R is the marker on the surface of macrophage membrane; and MaCSF-1 promotes macrophage proliferation, phagocytosis, and significantly increased the expression levels of IL-10 depended on the interacting with MaCSF-1R. This study provides basal data for the biological function of MaCSF-1 and MaCSF-1R, and is valuable for the exploration of MaCSF-1 and MaCSF-1R molecular interactions.

Dietary supplementation with nanoparticle CMCS-20a enhances the resistance to GCRV infection in grass carp (Ctenopharyngodon idella).

Combination of antimicrobial proteins and nanomaterials provides a platform for the development of immunopotentiators. Oral administration of immunopotentiators can significantly enhance the immunity of organisms, which provides ideas for disease prevention. In this study, we confirmed that nanoparticles CMCS-20a can efficiently prevent grass carp reovirus (GCRV) infection. Firstly, we verified that CiCXCL20a is involved in the immune responses post GCRV challenge in vivo and alleviates the cell death post GCRV challenge in CIK cells. Then, we prepared nanoparticles CMCS-20a using carboxymethyl chitosan (CMCS) loaded with grass carp (Ctenopharyngodon idella) CXCL20a (CiCXCL20a). Meanwhile, we confirmed nanoparticles CMCS-20a can alleviate the degradation in intestine. Subsequently, we added it to the feed by low temperature vacuum drying method and high temperature spray drying method, respectively. Grass carp were oral administration for 28 days and challenged by GCRV. Low temperature vacuum drying group (LD-CMCS-20a) significantly improve grass carp survival rate, but not high temperature spray drying group (HD-CMCS-20a). To reveal the mechanisms, we investigated the serum biochemical indexes, intestinal mucus barrier, immune gene regulation and tissue damage. The complement component 3 content, lysozyme and total superoxide dismutase activities are highest in LD-CMCS-20a group. LD-CMCS-20a effectively attenuates the damage of GCRV to the number of intestinal villous goblet cells and mucin thickness. LD-CMCS-20a effectively regulates mRNA expressions of immune genes (IFN1, Mx2, Gig1 and IgM) in spleen and head kidney tissues. In addition, LD-CMCS-20a obviously alleviate tissue lesions and viral load in spleen. These results indicated that the nanoparticles CMCS-20a can enhance the disease resistance of fish by improving their immunity, which provides a new perspective for fish to prevent viral infections.