USP46 promotes the interferon antiviral signaling in black carp by deubiquitinating TBK1.

Ubiquitin-specific peptidase 46 (USP46) functions as a deubiquitinating enzyme, facilitating the removal of ubiquitin molecules attached to substrate proteins and playing a critical role in cancer and neurodegenerative diseases. However, its function in innate antiviral immunity is unknown. In this study we cloned and identified bcUSP46, a homolog of USP46 from black carp. We discovered that overexpression of bcUSP46 enhanced the transcription of interferon (IFN) promoters and increased the expression of IFN, PKR, and Mx1. In addition, bcUSP46 knockdown significantly inhibited the expression of ISG genes, as well as the antiviral activity of the host cells. Interestingly, when bcUSP46 was co-expressed with the RLR factors, it significantly enhanced the activity of the IFN promoter mediated by these factors, especially TANK-binding kinase 1 (TBK1). The subsequent co-immunoprecipitation (co-IP) and immunofluorescence (IF) assay confirmed the association between bcUSP46 and bcTBK1. Noteworthily, co-expression of bcUSP46 with bcTBK1 led to an elevation of bcTBK1 protein level. Further analysis revealed that bcUSP46 stabilized bcTBK1 by eliminating the K48-linked ubiquitination of bcTBK1. Overall, our findings highlight the unique role of USP46 in modulating TBK1/IFN signaling and enrich our knowledge of the function of deubiquitination in regulating innate immunity in vertebrates.

Mucosal immunity and microbiota change in the rainbow trout (Oncorhynchus mykiss) gills after being challenged with infectious hematopoietic necrosis virus.

Respiratory structures are crucial for vertebrate survival, as they serve not only to perform gas-exchange processes but also as entry points for opportunistic pathogens. Previous studies have demonstrated that fish contain gill mucosal-associated lymphoid tissue, and harbor a large number of commensal bacteria on their surface and contribute to maintaining fish health. However, by far, very limited information is known regarding the effects of viral infection on gill mucosal immunity and microbiota homeostasis. In this study, we conducted an infection model by bath with infectious hematopoietic necrosis virus (IHNV) and revealed a 27 % mortality rate among rainbow trout in the first two weeks after infection. Moreover, we found that diseased fish with the highest IHNV loads in gills exhibiting severe damage, as well as increased goblet cell counts in both primary lamellae (PL) and secondary lamellae (SL). Additionally, RT-qPCR and RNA-seq analyses revealed that IHNV infection induced a strong innate and adaptive antiviral immune responses. Interestingly, an antibacterial immune response was also observed, suggesting that a secondary bacterial infection occurred in trout gills after viral infection. Furthermore, 16S rRNA analysis of trout gills revealed a profound dysbiosis marked by a loss of beneficial taxa and expansion of pathobionts following IHNV infection. Overall, our finding demonstrates that IHNV infection induces significant changes of the microbial community in the fish respiratory surface, thus triggering local antiviral and bacterial mucosal immunity.

Teleost Eye Is the Portal of IHNV Entry and Contributes to a Robust Mucosal Immune Response.

The ocular mucosa (OM) is an important and unique part of the vertebrate mucosal immune system. The OM plays an important role in maintaining visual function and defending against foreign antigens or microorganisms, while maintaining a balance between the two through complex regulatory mechanisms. However, the function of ocular mucosal defense against foreign pathogens and mucosal immune response in bony fish are still less studied. To acquire deeper understanding into the mucosal immunity of the OM in teleost fish, we established a study of the immune response of rainbow trout (Oncorhynchus mykiss) infected with the infectious hematopoietic necrosis virus (IHNV). Our findings revealed that IHNV could successfully infiltrate the trout's OM, indicating that the OM could be an important portal for the IHNV. Furthermore, qPCR and RNA-Seq analysis results showed that a large number of immune-related genes were significantly upregulated in the OM of trout with IHNV infection. Critically, the results of our RNA-Seq analysis demonstrated that viral infection triggered a robust immune response, as evidenced by the substantial induction of antiviral, innate, and adaptive immune-related genes in the OM of infected fish, which underscored the essential role of the OM in viral infection. Overall, our findings revealed a previously unknown function of teleost OM in antiviral defense, and provided a theoretical basis for the study of the mucosal immunity of fish.

Gut mucosal immune responses and protective efficacy of oral yeast Cyprinid herpesvirus 2 (CyHV-2) vaccine in Carassius auratus gibelio.

Cyprinid herpesvirus 2 (CyHV-2) causes herpesviral hematopoietic necrosis (HVHN) disease outbreaks in farmed Cyprinid fish, which leads to serious economic losses worldwide. Although oral vaccination is considered the most suitable strategy for preventing infectious diseases in farmed fish, so far there is no commercial oral vaccine available for controlling HVNN in gibel carp (C. auratus gibelio). In the present study, we developed for the first time an oral vaccine against CyHV-2 by using yeast cell surface display technology and then investigated the effect of this vaccine in gibel carp. Furthermore, the protective efficacy was evaluated by comparing the immune response of a single vaccination with that of a booster vaccination (booster-vaccinated once 2 weeks after the initial vaccination). Critically, the activities of immune-related enzymes and genes expression in vaccine group, especially in the booster vaccine group, were higher than those in the control group. Moreover, strong innate and adaptive immune responses could be elicited in both mucosal and systemic tissues after receipt of the oral yeast vaccine. To further understand the protective efficacy of this vaccine in gibel carp, we successfully developed the challenge model with CyHV-2. Our results showed the relative percent survival was 66.7% in the booster vaccine group, indicating this oral yeast vaccine is a promising vaccine for controlling CyHV-2 disease in gibel carp aquaculture.

Molecular cloning and expression analysis of CD79a and CD79b in rainbow trout (Oncorhynchus mykiss) after bacterial, parasitic, and viral infection.

CD79a and CD79b heterodimers are important components that consist of B cell receptor compound, which play a crucial role in transduction activation signal of the antigen binding BCR, and B cell development and antibody production. In order to investigate the characters and potential functions of CD79a and CD79b in rainbow trout (Oncorhynchus mykiss), we firstly cloned and analyzed the expression of CD79a and CD79b and found that the cDNA sequences of CD79a and CD79b both contained open reading frame of 711 and 645 bp in length for encoding the protein of 237 and 215 amino acid residues, respectively. The predicted amino acid sequences from trout were highly conserved with those of other teleost fishes in structure. Phylogenetic tree was constructed to analyze the evolutionary relationship between the trout and other known species, the result indicated that CD79a and CD79b of trout clustered at high bootstrap values with Salmo salar. Moreover, three trout infection models with F. columnare G4, I. multifiliis and infectious hematopoietic necrosis virus (IHNV) were constructed, which resulted in morphological changes and serious lesions in skin and gills. Importantly, the high expression of CD79a and CD79b occurred in skin, gills, and followed by head kidney in response to bacterial, parasitic, and viral infection, as its expression was closely related to that of Igs. Our findings indicated that CD79a and CD79b play vital roles in both systemic and mucosal immune responses of rainbow trout during bacterial, parasitic, and viral infection, which will contribute to explore the roles of CD79 subunits in B cell signaling during ontogeny and disease.

Molecular Characterization and Expression Analysis of Intercellular Adhesion Molecule-1 (ICAM-1) Genes in Rainbow Trout (Oncorhynchus mykiss) in Response to Viral, Bacterial and Parasitic Challenge.

The intercellular adhesion molecule-1 (ICAM-1), known as CD54, is a transmembrane cell surface glycoprotein that interacts with two integrins (i.e., LFA-1 and Mac-l) important for trans-endothelial migration of leukocytes. The level of ICAM-1 expression is upregulated in response to some inflammatory stimulations, including pathogen infection and proinflammatory cytokines. Yet, to date, our knowledge regarding the functional role of ICAM-1 in teleost fish remains largely unknown. In this study, we cloned and characterized the sequence of ICAM-1 in rainbow trout (Oncorhynchus mykiss) for the first time, which exhibited that the molecular features of ICAM-1 in fishes were relatively conserved compared with human ICAM-1. The transcriptional level of ICAM-1 was detected in 12 different tissues, and we found high expression of this gene in the head kidney, spleen, gills, skin, nose, and pharynx. Moreover, upon stimulation with infectious hematopoietic necrosis virus (IHNV), Flavobacterium columnare G4 (F. columnare), and Ichthyophthirius multifiliis (Ich) in rainbow trout, the morphological changes were observed in the skin and gills, and enhanced expression of ICAM-1 mRNA was detected both in the systemic and mucosal tissues. These results indicate that ICAM-1 may be implicated in the mucosal immune responses to viral, bacterial, and parasitic infections in teleost fish, meaning that ICAM-1 emerges as a master regulator of mucosal immune responses against pathogen infections in teleost fish.

Dietary Supplementation of Probiotic Bacillus subtilis Affects Antioxidant Defenses and Immune Response in Grass Carp Under Aeromonas hydrophila Challenge.

This study investigated whether Bacillus subtilis can provide protection for grass carp against oxidative stress damage induced by Aeromonas hydrophila. A total of 240 healthy grass carp (Ctenopharyngodon idellus) (average weight of 71.42 ± 4.36g) were randomly divided into four groups with three replicates: control group, A. hydrophila group, B. subtilis + A. hydrophila group, and A. hydrophila + B. subtilis group. After challenge with A. hydrophila, the lipid oxidative damage, antioxidant defenses, and the gene expression of inflammatory cytokines of the grass carp were investigated. Our results showed that A. hydrophila caused lipid oxidative damage, led to significant decreases in antioxidant defenses, and induced inflammatory responses of grass carp. However, the grass carp group fed the probiotic B. subtilis diet for 42 days before the challenge and the group fed the probiotic B. subtilis diet immediately after the challenge both showed (i) a reduced level of oxidative stress with a decrease in the level of MDA; (ii) an increase in antioxidant defenses, including an increase in total antioxidant capacity (T-AOC), increased activities of SOD and CAT, increased levels of GSH, and upregulated gene expression of antioxidant enzymes (SOD, CAT, and Gpx); and (iii) an improved immune response with the level of antiinflammatory cytokines IL-10 messenger RNA (mRNA) upregulated and the levels of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-8 mRNA downregulated. Based on this study, B. subtilis can provide effective protection of fish against oxidative stress damage induced by A. hydrophila infection.

Effect of Bacillus subtilis on Aeromonas hydrophila-induced intestinal mucosal barrier function damage and inflammation in grass carp (Ctenopharyngodon idella).

Our study explored the effect of oral intubation of Bacillus subtilis on Aeromonas hydrophila-induced intestinal mucosal barrier function damage and inflammation in grass carp. The mid-intestine mucosal tissue was collected for ATPase activity measurement. Intestinal mucosa was also ultrastructurally examined with transmission electron microscope (TEM), and its permeability was determined using Evans blue (EB) and D-lactic acid. The mid-intestine pro-inflammation cytokine, MyD88 and tight junction (TJ) protein mRNA expression levels were measured using real-time quantitative PCR. The results revealed that B. subtilis was found to prevent the decrease in the activity of Na+, K+-ATPase and Ca2+, Mg2+-ATPase, as well as the increase in EB and D-lactic acid concentration and inflammation induced by A. hydrophila in grass carp. Compared with A. hydrophila groups, B. subtilis safeguarded the integrity of intestinal villi and tight junction structure and restrained A. hydrophila-induced down-regulation of TJ proteins zonula occludens-1 (ZO-1) and occludin. B. subtilis also restrained up-regulation of TJ protein claudin b, pro-inflammation cytokine tumour necrosis factor α (TNF-α), cytokine interleukin 8 (IL-8), IL-1ß, and adaptor protein myeloid differentiation factor 88 (MyD88) mRNA levels. Thus, oral intubation of B. subtilis could reduce A. hydrophila-induced intestinal mucosal barrier function damage and inflammation.

Antidesmone, a unique tetrahydroquinoline alkaloid, prevents acute lung injury via regulating MAPK and NF-κB activities.

Acute lung injury, characterized by inflammation, is a main cause of respiratory failure that affects patients worldwide. Antidesmone is one compound mainly isolated from Ajugade cumbens Thunb (Labiatae), an herb agent of Labiatae family. In this research, we investigated the anti-inflammation effect of antidesmone in vitro and in vivo. Antidesmone exerted none apparently cytotoxicity in vitro and toxic in vivo. In vitro results demonstrated that antidesmone suppressed the excess inflammatory cytokines production, including tumor necrosis factor-α, interleukin-6 and interleukin-1ß in lipopolysaccharide (LPS)-exposed RAW264.7 cells. In vivo results suggested that antidesmone inhibited inflammatory cytokines in the bronchoalveolar lavage fluid and lung tissue after LPS stimulation. Moreover, antidesmone attenuated the nuclear translocation of p65. Mechanism study revealed that mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways play important roles in antidesmone's action. Taken together, our data uncover a relative toxic anti-inflammatory drug, antidesmone, can inhibit inflammation on stimulated macrophages and thereby prevents acute lung injury by regulating MAPK and NF-κB signaling pathways.