Liver X Receptor LXRα Promotes Grass Carp Reovirus Infection by Attenuating IRF3-CBP Interaction and Inhibiting RLR Antiviral Signaling.

Liver X receptors (LXRs) are nuclear receptors involved in metabolism and the immune response. Different from mammalian LXRs, which include two isoforms, LXRα and LXRß, only a single LXRα gene exists in the piscine genomes. Although a study has suggested that piscine LXR inhibits intracellular bacterial survival, the functions of piscine LXRα in viral infection are unknown. In this study, we show that overexpression of LXRα from grass carp (Ctenopharyngodon idellus), which is named as gcLXRα, increases host susceptibility to grass carp reovirus (GCRV) infection, whereas gcLXRα knockdown in CIK (C. idellus kidney) cells inhibits GCRV infection. Consistent with these functional studies, gcLXRα knockdown promotes the transcription of antiviral genes involved in the RIG-I-like receptor (RLR) antiviral signaling pathway, including IFN regulatory factor (IRF3) and the type I IFN IFN1. Further results show that gcLXRα knockdown induces the expression of CREB-binding protein (CBP), a transcriptional coactivator. In the knockdown of CBP, the inhibitory effect of gcLXRα knockdown in limiting GCRV infection is completely abolished. gcLXRα also interacts with IRF3 and CBP, which impairs the formation of the IRF3/CBP transcription complex. Moreover, gcLXRα heterodimerizes with RXRg, which cooperatively impair the transcription of the RLR antiviral signaling pathway and promote GCRV infection. Taken together, to our knowledge, our findings provide new insight into the functional correlation between nuclear receptor LXRα and the RLR antiviral signaling pathway, and they demonstrate that gcLXRα can impair the RLR antiviral signaling pathway and the production of type I IFN via forming gcLXRα/RXRg complexes and attenuating IRF3/CBP complexes.

Interaction of Nmi and IFP35 Promotes Mutual Protein Stabilization and IRF3 and IRF7 Degradation to Suppress Type I IFN Production in Teleost Fish.

IFN-stimulated genes (ISGs) can act as effector molecules against viral infection and can also regulate pathogenic infection and host immune response. N-Myc and STAT interactor (Nmi) is reported as an ISG in mammals and in fish. In this study, the expression of Nmi was found to be induced significantly by the infection of Siniperca chuatsi rhabdovirus (SCRV), and the induced expression of type I IFNs after SCRV infection was reduced following Nmi overexpression. It is observed that Nmi can interact with IRF3 and IRF7 and promote the autophagy-mediated degradation of these two transcription factors. Furthermore, Nmi was found to be interactive with IFP35 through the CC region to inhibit IFP35 protein degradation, thereby enhancing the negative role in type I IFN expression after viral infection. In turn, IFP35 is also capable of protecting Nmi protein from degradation through its N-terminal domain. It is considered that Nmi and IFP35 in fish can also interact with each other in regulating negatively the expression of type I IFNs, but thus in enhancing the replication of SCRV.

Transcriptional Regulation and Signaling of Type IV IFN with Identification of the ISG Repertoire in an Amphibian Model, Xenopus laevis.

The type IV IFN (IFN-υ) is reported in vertebrates from fish to primary mammals with IFN-υR1 and IL-10R2 as receptor subunits. In this study, the proximal promoter of IFN-υ was identified in the amphibian model, Xenopus laevis, with functional IFN-sensitive responsive element and NF-κB sites, which can be transcriptionally activated by transcription factors, such as IFN regulatory factor (IRF)1, IRF3, IRF7, and p65. It was further found that IFN-υ signals through the classical IFN-stimulated gene (ISG) factor 3 (ISGF3) to induce the expression of ISGs. It seems likely that the promoter elements of the IFN-υ gene in amphibians is similar to type III IFN genes, and that the mechanism involved in IFN-υ induction is very much similar to type I and III IFNs. Using recombinant IFN-υ protein and the X. laevis A6 cell line, >400 ISGs were identified in the transcriptome, including ISGs homologous to humans. However, as many as 268 genes were unrelated to human or zebrafish ISGs, and some of these ISGs were expanded families such as the amphibian novel TRIM protein (AMNTR) family. AMNTR50, a member in the family, was found to be induced by type I, III, and IV IFNs through IFN-sensitive responsive element sites of the proximal promoter, and this molecule has a negative role in regulating the expression of type I, III, and IV IFNs. It is considered that the current study contributes to the understanding of transcription, signaling, and functional aspects of type IV IFN at least in amphibians.

T3SS protein EsrC binds to the lacI-like operator of type 1 fimbrial operon to suppress adhesion of Edwardsiella piscicida.

Type 1 fimbria, the short hair-like appendage assembled on the bacterial surface, plays a pivotal role in adhesion and invasion in Edwardsiella piscicida. The type III secretion system (T3SS), another bacterial surface appendage, facilitates E. piscicida's replication in vivo by delivering effectors into host cells. Our previous research demonstrated that E. piscicida T3SS protein EseJ inhibits adhesion and invasion of E. piscicida by suppressing type 1 fimbria. However, how EseJ suppresses type 1 fimbria remains elusive. In this study, a lacI-like operator (nt -245 to -1 of fimA) upstream of type 1 fimbrial operon in E. piscicida was identified, and EseJ inhibits type 1 fimbria through the lacI-like operator. Moreover, through DNA pull-down and electrophoretic mobility shift assay, an AraC-type T3SS regulator, EsrC, was screened and verified to bind to nt -145 to -126 and nt -50 to -1 of fimA, suppressing type 1 fimbria. EseJ is almost abolished upon the depletion of EsrC. EsrC and EseJ impede type 1 fimbria expression. Intriguingly, nutrition and microbiota-derived indole activate type 1 fimbria through downregulating T3SS, alleviating EsrC or EseJ's inhibitory effect on lacI-like operator of type 1 fimbrial operon. By this study, it is revealed that upon entering the gastrointestinal tract, rich nutrients and indole downregulate T3SS and thereof upregulate type 1 fimbria, stimulating efficient adhesion and invasion; upon being internalized into epithelium, the limit in indole and nutrition switches on T3SS and thereof switches off type 1 fimbria, facilitating effector delivery to guarantee E. piscicida's survival/replication in vivo.IMPORTANCEIn this work, we identified the lacI-like operator of type 1 fimbrial operon in E. piscicida, which was suppressed by the repressors-T3SS protein EseJ and EsrC. We unveiled that E. piscicida upregulates type 1 fimbria upon sensing rich nutrition and the microbiota-derived indole, thereof promoting the adhesion of E. piscicida. The increase of indole and nutrition promotes type 1 fimbria by downregulating T3SS. The decrease in EseJ and EsrC alleviates their suppression on type 1 fimbria, and vice versa.

Enabling AI-Generated Content for Gadolinium-Free Contrast-Enhanced Breast Magnetic Resonance Imaging.

BACKGROUND: There is increasing interest in utilizing AI-generated content for gadolinium-free contrast-enhanced breast MRI. PURPOSE: To develop a generative model for gadolinium-free contrast-enhanced breast MRI and evaluate the diagnostic utility of the generated scans. STUDY TYPE: Retrospective. POPULATION: Two hundred seventy-six women with 304 breast MRI examinations (49 ± 13 years, 243/61 for training/testing). FIELD STRENGTH/SEQUENCE: ZOOMit diffusion-weighted imaging (DWI), T1-weighted volumetric interpolated breath-hold examination (T1W VIBE), and axial T2 3D SPACE at 3.0 T. ASSESSMENT: A generative model was developed to generate contrast-enhanced scans using precontrast T1W VIBE and DWI images. The generated and real images were quantitatively compared using the structural similarity index (SSIM), mean absolute error (MAE), and Dice similarity coefficient. Three radiologists with 8, 5, and 5 years of experience independently rated the image quality and lesion visibility on AI-generated and real images within various subgroups using a five-point scale. Four breast radiologists, with 8, 8, 5, and 5 years of experience, independently and blindly interpreted four reading protocols: unenhanced MRI protocol alone and combined with AI-generated scans, abbreviated MRI protocol, and full-MRI protocol. STATISTICAL ANALYSIS: Results were assessed using t-tests and McNemar tests. Using pathology diagnosis as reference standard, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for each reading protocol. A P value <0.05 was considered significant. RESULTS: In the test set, the generated images showed similarity to the real images (SSIM: 0.935 ± 0.047 [SD], MAE: 0.015 ± 0.012 [SD], and Dice coefficient: 0.726 ± 0.177 [SD]). No significant difference in lesion visibility was observed between real and AI-generated scans of the mass, non-mass, and benign lesion subgroups. Adding AI-generated scans to the unenhanced MRI protocol slightly improved breast cancer detection (sensitivity: 92.86% vs. 85.71%, NPV: 76.92% vs. 70.00%); achieved non-inferior diagnostic utility compared to the AB-MRI protocol and full-protocol (sensitivity: 92.86%, 95.24%; NPV: 75.00%, 81.82%). DATA CONCLUSION: AI-generated gadolinium-free contrast-enhanced breast MRI has potential to improve the sensitivity of unenhanced MRI in detecting breast cancer. EVIDENCE LEVEL: 4 TECHNICAL EFFICACY: Stage 3.

IRF11 synergizes with STAT1 and STAT2 to promote type I IFN production.

Interferon regulatory factor 11 (IRF11), a fish specific member of IRF family, is a transcription factor known for its positive role in teleost antiviral defense by regulating IFN expression. Despite its recognized function, the precise mechanism of IRF11 in type I IFNs production remains largely unknown. In this study, we identified IRF11 in Japanese eel, Anguilla japonica, (AjIRF11) and determined its involvement in the later phase of fish IFN production. Our results demonstrate that IRF11-induced IFN production operates through ISRE binding. Mutations in each ISRE site within the promoter of AjIFN2 or AjIFN4 abolished IRF11-mediated activation of IFN promoters. In addition, the overexpression of AjIRF11 does not significantly impact the activation of AjIFN promoters induced by RLR-related signaling pathway proteins. Furthermore, IRF11-knockdown in ZFLs (zebrafish liver cells) has no effect on the RLRs-induced expression of zebrafish IFN-φ1 and IFN-φ3, indicating that IRF11 is not involved in the RLR-mediated IFN production. However, AjIRF11 can form transcription complexes with AjSTAT1 or AjSTAT2, or form homo- or heterodimers with AjIRF1 to stimulate the transcription of type I IFNs. Overall, it is shown in this study that IRF11 can act synergistically with STAT1 and/or STAT2 for the induction of IFN.

SIRT6 positively regulates antiviral response in a bony fish, the Chinese perch Siniperca chuatsi.

SIRT6, a key member of the sirtuin family, plays a pivotal role in regulating a number of vital biological processes, including energy metabolism, oxidative stress, and immune system modulation. Nevertheless, the function of SIRT6 in bony fish, particularly in the context of antiviral immune response, remains largely unexplored. In this study, a sirt6 was cloned and characterized in a commercial fish, the Chinese perch (Siniperca chuatsi). The SIRT6 possesses conserved SIR2 domain with catalytic core region when compared with other vertebrates. Tissue distribution analysis indicated that sirt6 was expressed in all detected tissues, and the sirt6 was significantly induced following infection of infectious haemorrhagic syndrome virus (IHSV). The overexpression of SIRT6 resulted in significant upregulation of interferon-stimulated genes (ISGs), such as viperin, mx, isg15, irf3 and ifp35, and inhibited viral replication. It was further found that SIRT6 was located in nucleus and could enhance the expression of ISGs induced by type I and II IFNs. These findings may provide new information in relation with the function of SIRT6 in vertebrates, and with viral prevention strategy development in aquaculture.

Molecular cloning and functional analyses of C-C motif chemokine ligand 3 (CCL3) in mandarin fish Siniperca chuatsi.

Chemokines are critical molecules involved in immune reaction and immune system homeostasis, and some chemokines play a role in antiviral immunity. It is not known if the C-C motif chemokine ligand 3 (CCL3), a member of the CC chemokine family, possesses antiviral properties in fish. In this study, a ccl3 was cloned from the mandarin fish (Siniperca chuatsi), and it has an open reading frame (ORF) of 276 base pairs, which are predicted to encode a 91-amino acid peptide. Mandarin fish CCL3 revealed conserved sequence features with four cysteine residues and closely relationships with the CCL3s from other vertebrates based on the sequence alignment and phylogenetic analysis. The transcripts of ccl3 were notably enriched in immune-related organs, such as spleen and gills in healthy mandarin fish, and the ccl3 was induced in the isolated mandarin fish brain (MFB) cells following infection with infectious spleen and kidney necrosis virus (ISKNV). Moreover, in MFB cells, overexpression of CCL3 induced immune factors, such as IL1ß, TNFα, MX, IRF1 and IFNh, and exhibited antiviral activity against ISKNV. This study sheds light on the immune role of CCL3 in immune response of mandarin fish, and its antiviral defense mechanism is of interest for further investigation.

Grass Carp Reovirus Nonstructural Proteins Avoid Host Antiviral Immune Response by Targeting the RLR Signaling Pathway.

Grass carp reovirus (GCRV) is a highly virulent RNA virus that mainly infects grass carp and causes hemorrhagic disease. The roles of nonstructural proteins NS38 and NS80 of GCRV-873 in the viral replication cycle and viral inclusion bodies have been established. However, the strategies that NS38 and NS80 used to avoid host antiviral immune response are still unknown. In this study, we report the negative regulations of NS38 and NS80 on the RIG-I-like receptors (RLRs) antiviral signaling pathway and the production of IFNs and IFN-stimulated genes. First, both in the case of overexpression and GCRV infection, NS38 and NS80 inhibited the IFN promoter activation induced by RIG-I, MDA5, MAVS, TBK1, IRF3, and IRF7 and mRNA abundance of key antiviral genes involved in the RLR-mediated signaling. Second, both in the case of overexpression and GCRV infection, NS38 interacted with piscine TBK1 and IRF3, but not with piscine RIG-I, MDA5, MAVS, and TNF receptor-associated factor (TRAF) 3. Whereas NS80 interacted with piscine MAVS, TRAF3, and TBK1, but not with piscine RIG-I, MDA5, and IRF3. Finally, both in the case of overexpression and GCRV infection, NS38 inhibited the formation of the TBK1-IRF3 complex, but NS80 inhibited the formation of the TBK1-TRAF3 complex. Most importantly, NS38 and NS80 could hijack piscine TBK1 and IRF3 into the cytoplasmic viral inclusion bodies and inhibit the translocation of IRF3 into the nucleus. Collectively, all of these data demonstrate that GCRV nonstructural proteins can avoid host antiviral immune response by targeting the RLR signaling pathway, which prevents IFN-stimulated gene production and facilitates GCRV replication.

Isolation and identification of vapA-absent Aeromonas salmonicida in diseased snakehead Channa argus in China.

Aeromonas salmonicida is the typical pathogen causing furunculosis, reported widely in salmonids. Because of multiple serotypes, the control of A. salmonicida-caused disease has increasingly received much attention. Recently, A. salmonicida infection was reported in non-salmonid fish species. Here, a pathogenic A. salmonicida, named as As-s, was isolated from cultured snakehead (Channa argus) in a local fish farm in Shandong, China. As-s displayed clear hemolysis, amylase, and positive catalase activities, and grew at a wide range of temperatures (10-37 °C) and pH values (5.5-8.5). As-s was highly sensitive to cefuroxime sodium, ceftriaxone, ceftazidime, piperacillin, and cefoperazone and also apparently sensitive to chloramphenicol, erythromycin, and 25% cinnamaldehyde. The Virulence array protein gene cloning' results suggested that As-s has this gene compared with the other two vapA-containing strains, despite a close relationship of these strains via phylogenetic analysis. Severe ulcers on skin, muscle, and abnormal liver, and hemorrhage in pectoral/ventral fins and anal region were observed, and exophthalmos were also noticed in infected juvenile snakehead, as well as necrosis and infiltration of blood cells emerged in the internal organs using pathological section. In addition, As-s caused high mortality in snakehead, consistently with its immune gene response. This study reports the first isolation of vapA-absent A. salmonicida in snakehead.

Molecular cloning and functional analysis of polymeric immunoglobulin receptor, pIgR, gene in mandarin fish Siniperca chuatsi.

Polymeric immunoglobulin receptor (pIgR) can bind and transport immunoglobulins (Igs), thus playing a role in mucosal immunity. In this study, pIgR gene was cloned in mandarin fish, Siniperca chuatsi, with the open reading frame (ORF) of 1011 bp, encoding 336 amino acids. The pIgR protein consists of a signal peptide, an extracellular domain, a transmembrane domain and an intracellular region, with the presence of two Ig-like domains (ILDs) in the extracellular domain, as reported in other species of fish. The pIgR gene was expressed in all organs/tissues of healthy mandarin fish, with higher level observed in liver and spleen. Following the immersion infection of Flavobacterium columnare, pIgR transcripts were detected in immune related, especially mucosal tissues, with significantly increased transcription during the first two days of infection. Through transfection of plasmids expressing pIgR, IgT and IgM, pIgR was found to be interacted with IgT and IgM as revealed by co-immunoprecipitation and immunofluorescence.

Retroposition of the Long Transcript from Multiexon IFN-ß Homologs in Ancestry Vertebrate Gave Rise to the Proximal Transcription Elements of Intronless IFN-ß Promoter in Humans.

IFN-ß is a unique member of type I IFN in humans and contains four positive regulatory domains (PRDs), I-II-III-IV, in its promoter, which are docking sites for transcription factors IFN regulatory factor (IRF) 3/7, NF-κB, IRF3/7, and activating transcription factor 2/Jun proto-oncogene, respectively. In chicken IFN-ß and zebrafish IFNφ1 promoters, a conserved PRD or PRD-like sequences have been reported. In this study, a type I IFN gene, named as xl-IFN1 in the amphibian model Xenopus laevis, was found to contain similar PRD-like sites, IV-III/I-II, in its promoter, and these PRD-like sites were proved to be functionally responsive to activating transcription factor 2/Jun proto-oncogene, IRF3/IRF7, and p65, respectively. The xl-IFN1, as IFNφ1 in zebrafish, was transcribed into a long and a short transcript, with the long transcript containing all of the transcriptional elements, including PRD-like sites and TATA box in its proximal promoter. A retroposition model was then proposed to explain the transcriptional conservation of IFNφ1, xl-IFN1, and IFN-ß in chicken and humans.

Development of a hyper-adhesive and attenuated Edwardsiella ictaluri strain as a novel immersion vaccine candidate in yellow catfish (Pelteobagrus fulvidraco).

Edwardsiella ictaluri, a Gram-negative intracellular pathogen, is the causative agent of enteric septicemia in channel catfish, and catfish aquaculture in China suffers heavy economic losses due to E. ictaluri infection. Vaccination is an effective control measure for this disease. In this study, an attenuated E. ictaluri strain was acquired through deletion mutation of the T3SS protein eseJei, and the ΔeseJei strain fails to replicate in the epithelioma papillosum of carp cells. The type 1 fimbria plays a pivotal role in the adhesion of E. ictaluri, and it was found in this study that deletion of -245 to -50 nt upstream of fimA increases its adhesion to around five times that of the WT strain. A hyper-adhesive and highly attenuated double mutant (ΔeseJeiΔfimA-245--50 strain) was constructed, and it was used as a vaccine candidate in yellow catfish via bath immersion at a dosage of 1 × 105 CFU/mL. It was found that this vaccine candidate can stimulate protection when challenged with E. ictaluri HSN-1 at 5 × 107 CFU/mL (∼20 × LD50). The survival rate was 83.61% for the vaccinated group and 33.33% for the sham-vaccinated group. The RPS (relative percent of survival) of the vaccination trial reached 75.41%. In conclusion, the ΔeseJeiΔfimA-245--50 strain developed in this study can be used as a vaccine candidate. It excels in terms of ease of delivery (via bath immersion) and is highly efficient in stimulating protection against E. ictaluri infection.

Orf1B controls secretion of T3SS proteins and contributes to Edwardsiella piscicida adhesion to epithelial cells.

Edwardsiella piscicida is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish. The type III secretion system (T3SS) is one of its two most important virulence islands. T3SS protein EseJ inhibits E. piscicida adhesion to epithelioma papillosum cyprini (EPC) cells by negatively regulating type 1 fimbria. Type 1 fimbria helps E. piscicida to adhere to fish epithelial cells. In this study, we characterized a functional unknown protein (Orf1B) encoded within the T3SS gene cluster of E. piscicida. This protein consists of 122 amino acids, sharing structural similarity with YscO in Vibrio parahaemolyticus. Orf1B controls secretion of T3SS translocon and effectors in E. piscicida. By immunoprecipitation, Orf1B was shown to interact with T3SS ATPase EsaN. This interaction may contribute to the assembly of the ATPase complex, which energizes the secretion of T3SS proteins. Moreover, disruption of Orf1B dramatically decreased E. piscicida adhesion to EPC cells due to the increased steady-state protein level of EseJ within E. piscicida. Taken together, this study partially unraveled the mechanisms through which Orf1B promotes secretion of T3SS proteins and contributes to E. piscicida adhesion. This study helps to improve our understanding on molecular mechanism of E. piscicida pathogenesis.

Molecular and functional characterization of zinc finger aspartate-histidine-histidine-cysteine (DHHC)-type containing 1, ZDHHC1 in Chinese perch Siniperca chuatsi.

In the present study, the zinc finger aspartate-histidine-histidine-cysteine (DHHC)-type containing 1 (ZDHHC1) gene was identified in a commercial fish, the Chinese perch Siniperca chuatsi. The ZDHHC1 has five putative transmembrane motifs and conserved DHHC domain, showing high amino-acid identity with other teleost fish, and vertebrate ZDHHC1 loci are conserved from fish to human. In vivo expression analysis indicated that ZDHHC1 gene was constitutively transcribed in all the examined organs/tissues, and was induced following infectious spleen and kidney necrosis virus (ISKNV) infection. It is further observed that ZDHHC1 interacts with MITA and the overexpression of ZDHHC1 in cells resulted in the upregulated expression of ISGs, such as Mx, RSAD2, IRF3 and type I IFNs such as IFNh and IFNc, exhibiting its antiviral function in fish as reported in mammals.

Identification of PGRP2 and its three splice variants in grass carp Ctenopharyngodon idella.

In this study, peptidoglycan recognition protein 2 (PGRP2) gene was cloned in grass carp Ctenopharyngodon idella, with the open reading frame (ORF) of PGRP2 being 1452 bp, encoding a protein of 483 amino acids. Three splice variants, PGRP2a, PGRP2b, and PGRP2c, were found also in grass carp with the absence of entire exon two and partial exon two of the PGRP2, and were predicted to have 124, 371 and 311 amino acids. But, they all have PGRP domain and signal peptide, except PGRP2a. The PGRP2 and its variants were expressed in all organs/tissues examined, and stimulated following PGN injection. It is further detected that the expression of gcPGRP2 and its variants was up-regulated after the single transfection of each of gcPGRP2 and its variant expression plasmids in CO cells. It is considered that the cloning of PGRP2 in grass carp provides a compositional completeness of PGRP members in this fish with the inclusion of previously reported PGRP5 and PGRP6.

NOD1 Promotes Antiviral Signaling by Binding Viral RNA and Regulating the Interaction of MDA5 and MAVS.

Nucleotide oligomerization domain-like receptors (NLRs) and RIG-I-like receptors (RLRs) detect diverse pathogen-associated molecular patterns to activate the innate immune response. The role of mammalian NLR NOD1 in sensing bacteria is well established. Although several studies suggest NOD1 also plays a role in sensing viruses, the mechanisms behind this are still largely unknown. In this study, we report on the synergism and antagonism between NOD1 and MDA5 isoforms in teleost. In zebrafish, the overexpression of NOD1 enhances the antiviral response and mRNA abundances of key antiviral genes involved in RLR-mediated signaling, whereas the loss of NOD1 has the opposite effect. Notably, spring viremia of carp virus-infected NOD1-/- zebrafish exhibit reduced survival compared with wild-type counterparts. Mechanistically, NOD1 targets MDA5 isoforms and TRAF3 to modulate the formation of MDA5-MAVS and TRAF3-MAVS complexes. The cumulative effects of NOD1 and MDA5a (MDA5 normal form) were observed for the binding with poly(I:C) and the formation of the MDA5a-MAVS complex, which led to increased transcription of type I IFNs and ISGs. However, the antagonism between NOD1 and MDA5b (MDA5 truncated form) was clearly observed during proteasomal degradation of NOD1 by MDA5b. In humans, the interactions between NOD1-MDA5 and NOD1-TRAF3 were confirmed. Furthermore, the roles that NOD1 plays in enhancing the binding of MDA5 to MAVS and poly(I:C) are also evolutionarily conserved across species. Taken together, our findings suggest that mutual regulation between NOD1 and MDA5 isoforms may play a crucial role in the innate immune response and that NOD1 acts as a positive regulator of MDA5/MAVS normal form-mediated immune signaling in vertebrates.

In Primitive Zebrafish, MHC Class II Expression Is Regulated by IFN-γ, IRF1, and Two Forms of CIITA.

Mammalian CIITA isoforms are tightly regulated by independent promoters. These promotors are induced by IFN-γ through JAK-STAT signaling pathway. The induction of CIITA controls the expression of MHC class II (MHCII) and Ag presentation to the adaptive immune system. In the current study, to our knowledge, we first identified two independent promoters, p1 and p2, in the zebrafish (Danio rerio) that control the expression of the two variants of CIITA, CIITA variant 1 (CIITAv1), and CIITA variant 2 (CIITAv2), respectively. Moreover, although IRF1 in an IFN-γ signaling pathway induced CIITAv2, which has two ISRE motifs in its promoter, CIITAv1 expression was not induced by this signal. Further, the transcription of MHCII DAB was controlled by IRF1 via two distinct mechanisms: 1) the transcription of MHCII DAB was controlled by IRF1 indirectly through the two ISREs in p2; and 2) directly via the ISRE in MHCII DAB promoter. We also found that IRF1 associated with CIITAv1 and CIITAv2 via protein-protein interactions to synergistically drive the transcription of MHCII DAB. The IFN-γ-IRF1-CIITA-MHCII signaling cascade was functional in early life stages of CIITA-/- and IRF1-/- zebrafish. Our findings imply that the immune system develops early in fishes and that the IFN-γ signaling cascade-induced CIITA and MHCII DAB is conserved in teleost fishes and mammals.

Edwardsiella piscicida type III protein EseJ suppresses apoptosis through down regulating type 1 fimbriae, which stimulate the cleavage of caspase-8.

The type III secretion system effector EseJ plays a regulatory role inside bacteria. It suppresses the adherence of Edwardsiella piscicida (E. piscicida) to host epithelial cells by down regulating type 1 fimbriae. In this study, we observed that more macrophages infected with ΔeseJ strain of E. piscicida detached as compared with those infected with the wild-type (WT) strain. Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining and cleaved caspase-3 examination revealed that the detachment is due to increased apoptosis, suggesting that EseJ suppresses macrophage apoptosis. However, apoptosis inhibition by EseJ is not relative to a type III secretion system (T3SS) and is not related to EseJ's translocation. Since EseJ negatively regulates type 1 fimbriae, murine J774A.1 cells were infected with ΔeseJΔfimA or ΔeseJΔfimH strains. It was demonstrated that ΔeseJ stimulates macrophage apoptosis through type 1 fimbriae. Moreover, we found that infecting J774A.1 cells with the ΔeseJ strain increased levels of cleaved caspase-8, caspase-9, and caspase-3, demonstrating that EseJ inhibits apoptosis through either an extrinsic or a combination of extrinsic and intrinsic pathways. Pre-treatment of macrophages with caspase-8 inhibitor prior to infection with the ΔeseJ strain decreased the levels of cleaved caspase-8, caspase-9, and caspase-3, indicating that the ΔeseJ strain stimulates apoptosis, mainly through an extrinsic pathway by up regulating type 1 fimbriae. Zebrafish larvae or blue gourami fish infected with the ΔeseJ strain consistently exhibited higher apoptosis than those infected with the E. piscicida WT strain or ΔeseJΔfimA strain. Taken together, we revealed that the T3SS protein EseJ of E. piscicida inhibits host apoptosis, mainly through an extrinsic pathway by down regulating type 1 fimbriae.

Tilapia dsRNA-activated protein kinase R (PKR): An interferon-induced antiviral effector with translation inhibition activity.

The dsRNA-activated protein kinase R (PKR) is one of key antiviral effectors induced by interferons (IFNs), and its functions are largely unknown in tilapia, an important commercial fish species suffering from several viral infectious diseases. In the present study, a PKR gene named On-PKR was identified and cloned from Nile tilapia, Oreochromis niloticus. On-PKR gene was constitutively expressed in all tissues examined, with the highest expression level observed in head kidney and liver, and was rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). Importantly, the expression of On-PKR is induced by group I and group II IFNs with distinct induction kinetics in vivo: group I IFN elicits a relative delayed but sustained induction of On-PKR, whereas group II IFN triggers a rapid and transient expression of On-PKR. Moreover, the overexpression of On-PKR has been proven to inhibit the protein translation and virus replication in fish cells. The present study thus contributes to a better understanding of the functions of antiviral effectors in tilapia, and may provide clues for the prevention and therapy of viral diseases in fish.