Tripartite motif 2b (trim2b) restricts spring viremia of carp virus by degrading viral proteins and negative regulators NLRP12-like receptors.

Tripartite motif (TRIM) proteins are involved in different cellular functions, including regulating virus infection. In teleosts, two orthologous genes of mammalian TRIM2 are identified. However, the functions and molecular mechanisms of piscine TRIM2 remain unclear. Here, we show that trim2b-knockout zebrafish are more susceptible to spring viremia of carp virus (SVCV) infection than wild-type zebrafish. Transcriptomic analysis demonstrates that NOD-like receptor (NLR), but not RIG-I-like receptor (RLR), signaling pathway is significantly enriched in the trim2b-knockout zebrafish. In vitro, overexpression of Trim2b fails to degrade RLRs and those key proteins involved in the RLR signaling pathway but does for negative regulators NLRP12-like proteins. Zebrafish Trim2b degrades NLRP12-like proteins through its NHL_TRIM2_like and IG_FLMN domains in a ubiquitin-proteasome degradation pathway. SVCV-N and SVCV-G proteins are also degraded by NHL_TRIM2_like domains, and the degradation pathway is an autophagy lysosomal pathway. Moreover, zebrafish Trim2b can interfere with the binding between NLRP12-like protein and SVCV viral RNA and can completely block the negative regulation of NLRP12-like protein on SVCV infection. Taken together, our data demonstrate that the mechanism of action of zebrafish trim2b against SVCV infection is through targeting the degradation of host-negative regulators NLRP12-like receptors and viral SVCV-N/SVCV-G genes.IMPORTANCESpring viremia of carp virus (SVCV) is a lethal freshwater pathogen that causes high mortality in cyprinid fish. In the present study, we identified zebrafish trim2b, NLRP12-L1, and NLRP12-L2 as potential pattern recognition receptors (PRRs) for sensing and binding viral RNA. Zebrafish trim2b functions as a positive regulator; however, NLRP12-L1 and NLRP12-L2 function as negative regulators during SVCV infection. Furthermore, we find that zebrafish trim2b decreases host lethality in two manners. First, zebrafish Trim2b promotes protein degradations of negative regulators NLRP12-L1 and NLRP12-L2 by enhancing K48-linked ubiquitination and decreasing K63-linked ubiquitination. Second, zebrafish trim2b targets viral RNAs for degradation. Therefore, this study reveals a special antiviral mechanism in lower vertebrates.

Zebrafish nos2a benefits bacterial proliferation via suppressing ROS and inducing NO production to impair the expressions of inflammatory cytokines and antibacterial genes.

The enzyme nitric oxide synthase 2 or inducible NOS (NOS2), reactive oxygen species (ROS) and nitric oxide (NO) are important participants in various inflammatory and immune responses. However, the functional significances of the correlations among piscine NOS2, ROS and NO during pathogen infection remain unclear. In teleost, there are two nos2 genes (nos2a and nos2b). It has been previously reported that zebrafish nos2a behaves as a classical inducible NOS, and nos2b exerts some functions similar to mammalian NOS3. In the present study, we reported the functional characterization of zebrafish nos2a during bacterial infection. We found that zebrafish nos2a promoted bacterial proliferation, accompanied by an increased susceptibility to Edwardsiella piscicida infection. The nagative regulation of zebrafish nos2a during E. piscicida infection was characterized by the impaired ROS levels, the induced NO production and the decreased expressions of proinflammatory cytokines, antibacterial genes and oxidant factors. Furthermore, although both inducing ROS and inhibiting NO production significantly inhibited bacterial proliferation, only inhibiting NO production but not inducing ROS significantly increased resistance to E. piscicida infection. More importantly, ROS supplementation and inhibition of NO completely abolished this detrimental consequence mediated by zebrafish nos2a during E. piscicida infection. All together, these results firstly demonstrate that the innate response mediated by zebrafish nos2a in promoting bacterial proliferation is dependent on the lower ROS level and higher NO production. The present study also reveals that inhibition of NO can be effective in the protection against E. piscicida infection.

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.

Association between IL12B polymorphisms and inflammatory bowel disease in Caucasian population: A meta-analysis.

BACKGROUND: Published studies on association between IL12B (G/A) rs10045431, (T/C)rs6887695 polymorphisms and inflammatory bowel disease (IBD) risk in Caucasian population have yielded conflicting results. The aim of this study was to potentially provide more reliable conclusions by conducting a meta-analysis. METHODS: Published studies concerned association between IL12B rs10045431, rs6887695 polymorphisms and IBD were searched from the Wiley Online Library, PubMed, Web of Science and the CNKI database. The odds ratio (OR) with 95% confidence interval (CI) was calculated to evaluate the strength of the relationship. The false positive report probabilities (FPRPs) test and trial sequential analysis (TSA) was performed to investigated the reliability of results. RESULTS: A total of 20 studies comprising 10761 Crohn's disease (CD), 10921 ulcerative colitis (UC) and 18381 controls were included in this meta-analysis. Overall, the pooled results showed that IL12B rs6887695 polymorphism significantly increased both CD and UC risk under all model, while IL12B rs10045431 polymorphism dramatically decreased both CD and UC risk under all model. FPRP and TSA demonstrated that above associations was confirmed in the present study. CONCLUSION: The results of meta-analysis indicate IL12B rs10045431 and rs6887695 polymorphisms significantly associate with IBD in Caucasian population.

An ultra-sensitive "turn-off" fluorescent sensor for the trace detection of rifampicin based on glutathione-stabilized copper nanoclusters.

Rifampicin is a common antibiotic used in human and veterinary medicine to treat tuberculosis and other diseases caused by numerous pathogenic bacteria. However, the excessive or improper use of rifampicin usually leads to a series of problems, including bacterial resistance, excessive drug-resistance and water pollution. Thus, it is of great importance to develop selective and sensitive assays for monitoring rifampicin in biological systems. In this study, we designed a fluorescence "turn-off" strategy for the trace detection of rifampicin based on a glutathione-stabilized copper nanoclusters (GSH-Cu NC) sensor. In an aqueous solution, the fluorescence of the GSH-Cu NCs at 632 nm can be quenched effectively and selectively by rifampicin due to the inner-filter effect (IFE) of fluorescence mechanism. Distinctively, this GSH-Cu NC sensor exhibited excellent fluorescence sensing capability for the trace detection of rifampicin with a very low limit of detection (LOD) of 16 pM in a wide linear range from 50 to 10 000 pM. It is not only more sensitive than the other methods previously reported for the detection of rifampicin, but also has an outstanding selectivity and strong anti-interference in complex samples. Furthermore, the as-developed GSH-Cu NCs were also successfully applied to determine rifampicin in different real samples with quantitative spike recoveries ranging from 97% to 105%.

Transcriptomic characterization of adult zebrafish infected with Streptococcus agalactiae.

Streptococcus agalactiae is a major aquaculture pathogen infecting various saltwater and freshwater fish. To better understand the mechanism of the immune responses to S. agalactiae in wildtype zebrafish, the transcriptomic profiles of two organs containing mucosal-associated lymphoid tissues from S. agalactiae-infected and non-infected groups were obtained using RNA-seq techniques. In the intestines, 6735 and 12908 differently expressed genes (DEGs) were identified at 24 hpi and 48 hpi, respectively. Among 66 and 116 significantly enriched pathways, 15 and 21 pathways were involved in immune system or signal transduction at 24 hpi and 48 hpi, respectively. A number of genes involved in Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, T cell receptor signaling pathway, B cell receptor signaling pathway, Antigen processing and presentation, NF-kappa B signaling pathway and PI3K-Akt signaling pathway were significantly downregulated. In the skins, 3113 and 4467 DEGs were identified at 24 hpi and 48 hpi, respectively. Among 24 and 56 significantly enriched pathways, 4 and 13 pathways were involved in immune system or signal transduction at 24 hpi and 48 hpi, respectively. More immune-related signaling pathways including Leukocyte transendothelial migration, Cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, IL-17 signaling pathway, MAPK signaling pathway, TNF signaling pathway, Complement and coagulation cascades, Hematopoietic cell lineage and Jak-STAT signaling pathway were differently enriched for upregulated DEGs at 48 hpi, which were completely different from that in the intestines. Furthmore, comparative transcriptome analysis revealed that the downregulated 1618 genes and upregulated 1622 genes existed both at 24 hpi and 48 hpi for the intestine samples. In the skins, the downregulated 672 genes and upregulated 428 genes existed both at 24 hpi and 48 hpi. Three pathways related to immune processes were significantly enriched for downregulated DEGs both in the intestines and skins collected at 24 hpi and 48 hpi, which included Antigen processing and presentation, Intestinal immune network for IgA production and Hematopoietic cell lineage. Interaction network analysis of DEGs identified the main DEGs in the sub-network of complement and coagulation cascades both in the intestines and skins. Twenty of DEGs involved in complement and coagulation cascades were further validated by Real-time quantitative PCR. Altogether, the results obtained in this study will provide insight into the immune response of zebrafish against S. agalactiae XQ-1 infection in fatal conditions, and reveal the discrepant expression pattern of complement and coagulation cascades in the intestines and skins.

Involvement of polyphosphate kinase in virulence and stress tolerance of uropathogenic Proteus mirabilis.

Proteus mirabilis (P. mirabilis), a gram-negative enteric bacterium, frequently causes urinary tract infections. Many virulence factors of uropathogenic P. mirabilis have been identified, including urease, flagella, hemolysin and fimbriae. However, the functions of polyphosphate kinase (PPK), which are related to the pathogenicity of many bacteria, remain entirely unknown in P. mirabilis. In this study, a ppk gene encoding the PPK insertional mutant in P. mirabilis strain HI4320 was constructed, and its biological functions were examined. The results of survival studies demonstrated that the ppk mutant was deficient in resistance to oxidative, hyperosmotic and heat stress. The swarming and biofilm formation abilities of P. mirabilis were also attenuated after the ppk interruption. In vitro and in vivo experiments suggested that ppk was required for P. mirabilis to invade the bladder. The negative phenotypes of the ppk mutant could be restored by ppk gene complementation. Furthermore, two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry were used to analyze the proteomes of the wild-type strain and the ppk mutant. Compared with the wild-type strain, seven proteins including TonB-dependent receptor, universal stress protein G, major mannose-resistant/Proteus-like fimbrial protein (MR/P fimbriae), heat shock protein, flagellar capping protein, putative membrane protein and multidrug efflux protein were down-regulated, and four proteins including exported peptidase, repressor protein for FtsI, FKBP-type peptidyl-prolyl cis-trans isomerase and phosphotransferase were up-regulated in the ppk mutant. As a whole, these results indicate that PPK is an important regulator and plays a crucial role in stress tolerance and virulence in uropathogenic P. mirabilis.

TBK1-like transcript negatively regulates the production of IFN and IFN-stimulated genes through RLRs-MAVS-TBK1 pathway.

TANK-binding kinase 1 (TBK1) is an essential serine/threonine-protein kinase required for Toll-like receptor (TLR)- and retinoic acid-inducible gene I (RIG-I) -mediated induction of type I IFN and host antiviral defense. In the present study, TBK1-like transcript, namely TBK1L, was cloned from zebrafish. Compared with TBK1, TBK1L contains an incomplete S_TKc domain, and lacks UBL_TBK1_like domain. Realtime PCR showed that TBK1L was constitutively produced in embryos, early larvae and ZF4 cells, and unchanged in ZF4 cells following SVCV infection. Overexpression of TBK1 but not TBK1L resulted in significant activation of zebrafish IFN1 and IFN3 promoters. Similarly, TBK1L had little impact on the antiviral state of the cells. However, the overexpression of TBK1L negatively regulated the induction of zebrafish IFN1 and/or IFN3 promoters mediated by the retinoic acid-inducible gene I-like receptors (RLRs), MAVS and TBK1. In addition, the overexpression of TBK1L in zebrafish embryos led to the decreased production of many IFN-stimulated genes induced by TBK1. Collectively, these data support that zebrafish TBK1L negatively regulates RLRs-MAVS-TBK1 pathway.

Immunoprotective Effects of Two Histone H2A Variants in the Grass Carp Against Flavobacterium columnare Infection.

In teleost fish, the nucleotide polymorphisms of histone H2A significantly affect the resistance or susceptibility of zebrafish to Edwardsiella piscicida infection. Whether histone H2A variants can enhance the resistance of grass carp to Flavobacterium columnare infection remains unclear. Here, the effects of 7 previously obtained variants (gcH2A-1~gcH2A-7) and 5 novel histone H2A variants (gcH2A-11, gcH2A-13~gcH2A-16) in response to F. columnare infection were investigated. It was found that these histone H2A variants could be divided into type I and II. Among them, 5 histone H2A variants had no any effects on the F. columnare infection, however 7 histone H2A variants had antibacterial activity against F. columnare infection. The gcH2A-4 and gcH2A-11, whose antibacterial activity was the strongest in type I and II histone H2A variants respectively, were picked out for yeast expression. Transcriptome data for the samples from the intestines of grass carp immunized with the engineered Saccharomyces cerevisiae expressing PYD1, gcH2A-4 or gcH2A-11 revealed that 5 and 12 immune-related signaling pathways were significantly enriched by gcH2A-4 or gcH2A-11, respectively. For the engineered S. cerevisiae expressing gcH2A-4, NOD-like receptor and Toll-like receptor signaling pathways were enriched for up-regulated DEGs. Besides NOD-like receptor and Toll-like receptor signaling pathways, the engineered S. cerevisiae expressing gcH2A-11 also activated Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway and C-type lectin receptor signaling pathway. Furthermore, grass carp were immunized with the engineered S. cerevisiae expressing PYD1, gcH2A-4 or gcH2A-11 for 1 month and challenged with F. columnare. These grass carp immunized with gcH2A-4 or gcH2A-11 showed lower mortality and fewer numbers of F. columnare than did the control group. All these results suggest that gcH2A-4 and gcH2A-11 play important roles in evoking the innate immune responses and enhancing disease resistance of grass carp against F. columnare infection.

CD44a functions as a regulator of p53 signaling, apoptosis and autophagy in the antibacterial immune response.

The cell adhesion molecule CD44 has been implicated in diverse biological functions including the pathological responses to infections and inflammatory diseases. The variable forms of CD44 contribute to functional variations, which are not yet defined in teleost. Here, we show that zebrafish CD44a plays a protective role in the host defense against Edwardsiella piscicida infection. Zebrafish CD44a deficiency inhibits cell growth and proliferation, impairs cell growth and death pathways, and regulates the expression levels of many genes involved in p53 signaling, apoptosis and autophagy. In addition, CD44a gene disruption in zebrafish leads to inhibition of apoptosis and induction of autophagy, with the increased susceptibility to E. piscicida infection. Furthermore, we show that zebrafish CD44a variants including CD44a_tv1 and CD44a_tv2 promote the translocation of p53 from the nucleus to the cytoplasm and interact with p53 in the cytoplasm. Mechanistically, zebrafish CD44a_tv1 mediates the beneficial effect for larvae survival infected with E. piscicida is depending on the CASP8-mediated apoptosis. However, the antibacterial effect of zebrafish CD44a_tv2 depends on the cytoplasmic p53-mediated inhibition of autophagy. Collectively, our results identify that different mechanisms regulate CD44a variants-mediated antibacterial responses.

Effects and Molecular Regulation Mechanisms of Salinity Stress on the Health and Disease Resistance of Grass Carp.

Though some freshwater fish have been successfully cultivated in saline-alkali water, the survival rates of freshwater fish are greatly affected by different saline-alkali conditions. The mechanisms of immune adaptation or immunosuppression of freshwater fish under different saline-alkali stress remain unclear. Here, grass carp were exposed to 3‰ and 6‰ salinity for 30 days. It was observed that salinity treatments had no obvious effects on survival rates, but significantly increased the percent of unhealthy fish. Salinity treatments also increased the susceptibility of grass carp against Flavobacterium columnare infection. The fatality rate (16.67%) of grass carp treated with 6‰ salinity was much lower than that treated with 3‰ salinity (40%). In the absence of infection, higher numbers of immune-related DEGs and signaling pathways were enriched in 6‰ salinity-treated asymptomatic fish than in 3‰ salinity-treated asymptomatic fish. Furthermore different from salinity-treated symptomatic fish, more DEGs involved in the upstream sensors of NOD-like receptor signaling pathway, such as NLRs, were induced in the gills of 6‰ salinity-treated asymptomatic fish. However in the case of F. columnare infection, more immune-related signaling pathways were impaired by salinity treatments. Among them, only NOD-like receptor signaling pathway was significantly enriched at early (1 and/or 2 dpi) and late (7 dpi) time points of infection both for 3‰ salinity-treated and 6‰ salinity-treated fish. Besides the innate immune responses, the adaptive immune responses such as the production of Ig levels were impaired by salinity treatments in the grass carp infected with F. columnare. The present study also characterized two novel NLRs regulated by salinity stress could inhibit bacterial proliferation and improve the survival rate of infected cells. Collectively, the present study provides the insights into the possible mechanisms why the percent of unhealthy fish in the absence of infection and mortality of grass carp in the case of F. columnare infection were much lower in the 6‰ salinity-treated grass carp than in 3‰ salinity-treated grass carp, and also offers a number of potential markers for sensing both environmental salinity stress and pathogen.

The expanding and function of NLRC3 or NLRC3-like in teleost fish: Recent advances and novel insights.

The nucleotide-binding domain and leucine-rich repeat-containing family (NLR) proteins are innate immune sensors which recognize highly conserved pathogen-associated molecular patterns (PAMPs). Mammals have small numbers of NLR proteins, whereas in some species such as in invertebrates and jawless vertebrates, NLRs have expanded into very large families. Nearly 400 NLR proteins are identified in the zebrafish genome. Members of the NLR family can be divided into two functional sub-groups based on their ability to either positively or negatively regulate host immune response or inflammatory signaling cascades. Mammalian NLRC3 has been identified as an inhibitory NLR, and serves as a negative regulator in the NF-κB-mediated inflammatory response, STING-mediated DNA sensing and PI3K-mTOR pathways. Different from mammalian NLRC3, the analysis from genomes or transcriptomes revealed that the expansions of NLRC3 existed in different species of fish. Furthermore, piscine NLRC3-like genes were confirmed to have a negative or positive regulatory function in response to different kinds of pathogen infections and in the production of proinflammatory cytokines. In this review, we summarize recent advances in our understanding of the expanding and function of NLRC3 or NLRC3-like genes in teleost fish, and give our view of important directions for future studies. The knowledge of piscine NLRC3 or expansive NLRC3-like genes-mediated biological functions in homeostasis and diseases will shed new light on the prevention and control of inflammatory and/or infectious diseases.

The function of zebrafish gpbar1 in antiviral response and lipid metabolism.

G protein-coupled bile acids receptor 1 (GPBAR1 or TGR5) has been widely studied as a metabolic regulator involved in bile acids synthesis, glucose metabolism and energy homeostasis. Several recent studies have shown that mammalian GPBAR1 is also involved in antiviral innate immune responses. However, the functions of piscine GPBAR1 in antibacterial or antiviral immune responses and lipid metabolism remain unclear. In the present study, we report the functional characterization of zebrafish gpbar1. Similar to mammalian GPBAR1, zebrafish gpbar1 contains similar domain composition, shows a dose-dependent activation by bile acids including INT777, LCA, DCA, CDCA and CA, and can be induced by viral infection. Compared with corresponding control groups, a significant antiviral activity against spring viremia of carp virus (SVCV) infection was observed in ZF4 cells overexpressing zebrafish gpbar1 with INT777 treatment, but not in ZF4 cells overexpressing zebrafish gpbar1 without INT777 treatment. The activation of zebrafish gpbar1 had no significant antibacterial effect against Edwardsiella piscicida infection in ZF4 cells in vitro. Transcriptome analysis revealed that zebrafish gpbar1 activation played a crucial role in activating RLR signaling pathway and inducing the production of ISGs, but not for bile acid biosynthesis and transportation. The co-occurrence analysis for antiviral-related and bile acids metabolism-related DEGs suggested a strong interaction among 2 bile acid receptors (gpbar1 and nr1h4), slco2b1 and the antiviral DEGs. The lipidomic analysis showed that zebrafish gpbar1 activation in ZF4 cells resulted a change of glycerophospholipids, but none of bile acids nor their derivatives, which were different from mammalian GPBAR1. All together, these results firstly demonstrate the conserved antiviral role of gpbar1 and its function in regulating glycerophospholipids metabolism in teleost.

Histone H2A Nuclear/Cytoplasmic Trafficking Is Essential for Negative Regulation of Antiviral Immune Response and Lysosomal Degradation of TBK1 and IRF3.

Histone H2A is a nuclear molecule tightly associated in the form of the nucleosome. Our previous studies have demonstrated the antibacterial property of piscine H2A variants against gram-negative bacteria Edwardsiella piscicida and Gram-positive bacteria Streptococcus agalactiae. In this study, we show the function and mechanism of piscine H2A in the negative regulation of RLR signaling pathway and host innate immune response against spring viremia of carp virus (SVCV) infection. SVCV infection significantly inhibits the expression of histone H2A during an early stage of infection, but induces the expression of histone H2A during the late stage of infection such as at 48 and 72 hpi. Under normal physiological conditions, histone H2A is nuclear-localized. However, SVCV infection promotes the migration of histone H2A from the nucleus to the cytoplasm. The in vivo studies revealed that histone H2A overexpression led to the increased expression of SVCV gene and decreased survival rate. The overexpression of histone H2A also significantly impaired the expression levels of those genes involved in RLR antiviral signaling pathway. Furthermore, histone H2A targeted TBK1 and IRF3 to promote their protein degradation via the lysosomal pathway and impair the formation of TBK1-IRF3 functional complex. Importantly, histone H2A completely abolished TBK1-mediated antiviral activity and enormously impaired the protein expression of IRF3, especially nuclear IRF3. Further analysis demonstrated that the inhibition of histone H2A nuclear/cytoplasmic trafficking could relieve the protein degradation of TBK1 and IRF3, and blocked the negative regulation of histone H2A on the SVCV infection. Collectively, our results suggest that histone H2A nuclear/cytoplasmic trafficking is essential for negative regulation of RLR signaling pathway and antiviral immune response in response to SVCV infection.

A Novel Transcript Isoform of TBK1 Negatively Regulates Type I IFN Production by Promoting Proteasomal Degradation of TBK1 and Lysosomal Degradation of IRF3.

TANK-binding kinase 1 (TBK1), an IKK-related serine/threonine kinase, is pivotal for the induction of antiviral type I interferon (IFN) by TLR and RLR signaling pathways. In a previous study, we demonstrated that TBK1 spliced isoforms (TBK1_tv1 and TBK1_tv2) from zebrafish were dominant negative regulators in the RLR antiviral pathway by targeting the functional TBK1-IRF3 complex formation. In this study, we show that the third TBK1 isoform (namely TBK1_tv3) inhibits zebrafish type I IFN production by promoting TBK1 and IRF3 degradation. First, ectopic expression of TBK1_tv3 suppresses poly(I:C)- and Spring viremia of carp virus-induced type I IFN response, and also inhibits the up-regulation of IFN promoter activities stimulated by RIG-I, MDA5, MAVS, TBK1, and IRF3. Second, TBK1_tv3 targets TBK1 and IRF3 to impair the formation of TBK1 dimer, TBK1-IRF3 complex, and IRF3 dimer. Notably, TBK1_tv3 promotes the degradation of TBK1 through the ubiquitin-proteasome pathway and the degradation of IRF3 through the lysosomal pathway. Further analysis demonstrates that TBK1_tv3 promotes the degradation of TBK1 for K48-linked ubiquitination by targeting the K251, K256, and K271 sites of TBK1. Collectively, our results suggest a novel TBK1 isoform-mediated negative regulation mechanism, which serves to balance the production of type I IFN and ISGs.

NLRC3-like 1 inhibits NOD1-RIPK2 pathway via targeting RIPK2.

Both NLRC3 and NOD1 belong to regulatory NLR subfamily based on their best-characterized function. In mammals, NLRC3 was reported to function by attenuating signaling cascades initiated by other families of PRRs. In teleosts, multiple NLRC3-like genes were identified through transcriptome sequencing. However, the functions of many NLRC3-like genes, especially the fish-specific NLRC3-like genes, remain unclear. In the present study, we report the functional characterization of a novel category of NLRC3-like proteins (named as NLRC3-like 1) from the zebrafish, which consists of a fish-specific FISNA, a conserved NACHT and five C-terminal LRRs domains. The expression of zebrafish NLRC3-like 1 was inducible in response to Edwardsiella piscicida infection. During bacterial infection, the in vitro and in vivo studies revealed that zebrafish NLRC3-like 1 overexpression facilitated bacterial growth and dissemination, together with the decreased survival rate of zebrafish larvae infected with E. piscicida. The attenuated response by zebrafish NLRC3-like 1 in response to bacterial infection were characterized by the impaired expression of antibacterial genes, proinflammatory cytokines and Nox genes. Furthermore, zebrafish NLRC3-like 1 interacted with the adaptor protein RIPK2 of NODs signaling via the FISNA (Fish-specific NACHT associated domain) and NACHT domains. However, the interaction between zebrafish NLRC3-like 1 and RIPK2 inhibited the assembly of the NOD1-RIPK2 complex. Importantly, zebrafish NLRC3-like 1 inhibited NOD1-mediated antibacterial activity, NF-κB and MAPK pathways and proinflammatory cytokine production. All together, these results firstly demonstrate that zebrafish NLRC3-like 1 inhibits NOD1-RIPK2 antibacterial pathway via targeting the adaptor protein RIPK2.

Cytokine activin C ameliorates chronic neuropathic pain in peripheral nerve injury rodents by modulating the TRPV1 channel.

BACKGROUND AND PURPOSE: The cytokine activin C is mainly expressed in small-diameter dorsal root ganglion (DRG) neurons and suppresses inflammatory pain. However, the effects of activin C in neuropathic pain remain elusive. EXPERIMENTAL APPROACH: Male rats and wild-type and TRPV1 knockout mice with peripheral nerve injury - sciatic nerve axotomy and spinal nerve ligation in rats; chronic constriction injury (CCI) in mice - provided models of chronic neuropathic pain. Ipsilateral lumbar (L)4-5 DRGs were assayed for activin C expression. Chronic neuropathic pain animals were treated with intrathecal or locally pre-administered activin C or the vehicle. Nociceptive behaviours and pain-related markers in L4-5 DRGs and spinal cord were evaluated. TRPV1 channel modulation by activin C was measured. KEY RESULTS: Following peripheral nerve injury, expression of activin ßC subunit mRNA and activin C protein was markedly up-regulated in L4-5 DRGs of animals with axotomy, SNL or CCI. [Correction added on 26 November 2020, after first online publication: The preceding sentence has been corrected in this current version.] Intrathecal activin C dose-dependently inhibited neuropathic pain in spinal nerve ligated rats. Local pre-administration of activin C decreased neuropathic pain, macrophage infiltration into ipsilateral L4-5 DRGs and microglial reaction in L4-5 spinal cords of mice with CCI. In rat DRG neurons, activin C enhanced capsaicin-induced TRPV1 currents. Pre-treatment with activin C reduced capsaicin-evoked acute hyperalgesia and normalized capsaicin-evoked persistent hypothermia in mice. Finally, the analgesic effect of activin C was abolished in TRPV1 knockout mice with CCI. CONCLUSION AND IMPLICATIONS: Activin C inhibits neuropathic pain by modulating TRPV1 channels, revealing potential analgesic applications in chronic neuropathic pain therapy.

The negative regulation of piscine CD44c in viral and bacterial infection.

CD44 gene is a cell surface receptor which undergoes complex alternative splicing and extensive post-translational modifications. Although many studies have showed that CD44 is involved in the process of host defense, the function of piscine CD44 in antibacterial or antiviral defense response remains unclear. In the present study, we report the functional characterization of zebrafish CD44c, which is more similar to CD44b antigen isoforms rather than CD44a based on amino acid composition and phylogenetic analysis. The expression of zebrafish CD44c was inducible in response to bacterial and viral infections. During SVCV infection, the in vivo studies revealed that CD44c overexpression led to the increased virus loads and decreased survival rate. The attenuated response by zebrafish CD44c in response to SVCV infection were characterized by the impaired production of inflammatory cytokines and the impaired expressions of IFNs, IFN-stimulated genes, MHC class I and II genes. During Edwardsiella piscicida infection, the overexpression of zebrafish CD44c facilitated bacterial growth and dissemination, but did not impact on larvae survival. The detrimental role of CD44c in host defense against E. piscicida infection was supported by a decreased production of several antibacterial molecules including defbl2, defbl3, NK-lysin and RNase3. All together, these results firstly demonstrate the negative regulation of piscine CD44c in viral and bacterial infection.

Histone H2A cooperates with RIP2 to induce the expression of antibacterial genes and MHC related genes.

An octamer consisting of two copies of histones H2A, H2B, H3 and H4 is the nucleosome core. It is well established that histone derived antimicrobial peptides (AMPs) have anti-microbial properties in various invertebrate and vertebrate species. Different from well-known histone H2A-derived AMPs, the antimicrobial properties of the complete histone H2A are rather limited. In the present study, we report the functional characterization of the complete histone H2A from zebrafish. The expression of zebrafish histone H2A was higher in embryos than in larvae, and inducible in response to bacterial infection. Furthermore, the expression of zebrafish histone H2A was decreased by RIP2 deficiency with and/or without bacterial infection. During Edwardsiella piscicida infection, the overexpression of zebrafish histone H2A inhibited bacterial proliferation and increased the survival rate of zebrafish larvae. The overexpression of zebrafish histone H2A demonstrated an increased transcription of many antibacterial genes and MHC related genes, which was dependent on RIP2, an adaptor protein for signal propagation of the NLRs-mediated antibacterial immune response. In line with this, zebrafish histone H2A cooperated with RIP2 to induce the transcription of many antibacterial genes and MHC related genes. All together, these results firstly demonstrate the antibacterial property of the complete histone H2A against gram-negative bacteria E. piscicida in vivo and the correlation between zebrafish histone H2A and RIP2 adaptor protein on the transcriptional regulation of antibacterial genes and MHC related genes.

Facile synthesis of near-infrared emitting dBSA-templated Cu nanoclusters for sensitive detection of heparin.

Heparin is the most widely studied glycosaminoglycan. It plays an important role in regulating several normal physiological and pathological processes, including inhibition of thrombocytopenia, lipid regulation, metabolism and electrostatic attraction with various proteins. Hence, it is crucial to develop selective and sensitive assays for monitoring heparin levels in biological systems. In the present study, we designed a facile synthesis method to prepare near-infrared emitting denatured bovine serum albumin-templated copper nanoclusters (dBSA-Cu NCs) for the trace detection of heparin. In aqueous solution, the bovine serum albumin (BSA) was denatured by heating at a temperature of 70 °C to prepare dBSA, which was employed as the template for fabricating the dBSA-Cu NCs. Then, Cu2+ ions were directly reduced to dBSA-Cu NCs by hydrazine hydrate at room temperature. The synthetic process was very simple to control due to the lack of any complicated procedure, such as heating or adjusting pH. In addition, the fluorescence intensity of dBSA-Cu NCs at 642 nm was quenched by heparin samples. Hence, the dBSA-Cu NCs can be used as a probe for the trace detection of heparin with a very low limit of detection (LOD) of 0.26 ng mL-1 in a linear range from 1.25 ng mL-1 to 250 ng mL-1. Furthermore, the as-developed dBSA-Cu NCs were also successfully applied to determine heparin in human plasma samples with quantitative spike recoveries from 95% to 104%.