RESUMO
In mammals, NLRX1 is a unique member of the nucleotide-binding domain and leucine-rich repeat (NLR) family showing an ability to negatively regulate IFN antiviral immunity. Intron-containing genes, including NLRX1, have more than one transcript due to alternative splicing; however, little is known about the function of its splicing variants. Here, we identified a transcript variant of NLRX1 in zebrafish (Danio rerio), termed NLRX1-tv4, as a negative regulator of fish IFN response. Zebrafish NLRX1-tv4 was slightly induced by viral infection, with an expression pattern similar to the full-length NLRX1. Despite the lack of an N-terminal domain that exists in the full-length NLRX1, overexpression of NLRX1-tv4 still impaired fish IFN antiviral response and promoted viral replication in fish cells, similar to the full-length NLRX1. Mechanistically, NLRX1-tv4 targeted STING for proteasome-dependent protein degradation by recruiting an E3 ubiquitin ligase RNF5 to drive the K48-linked ubiquitination, eventually downregulating the IFN antiviral response. Mapping of NLRX1-tv4 domains showed that its N-terminal and C-terminal regions exhibited a similar potential to inhibit STING-mediated IFN antiviral response. Our findings reveal that like the full-length NLRX1, zebrafish NLRX-tv4 functions as an inhibitor to shape fish IFN antiviral response.IMPORTANCEIn this study, we demonstrate that a transcript variant of zebrafish NLRX1, termed NLRX1-tv4, downregulates fish IFN response and promotes virus replication by targeting STING for protein degradation and impairing the interaction of STING and TBK1 and that its N- and C-terminus exhibit a similar inhibitory potential. Our results are helpful in clarifying the current contradictory understanding of structure and function of vertebrate NLRX1s.
Assuntos
Proteínas de Membrana , Proteínas Mitocondriais , Proteínas de Peixe-Zebra , Animais , Imunidade Inata , Domínios Proteicos , Isoformas de Proteínas/genética , Ubiquitina-Proteína Ligases , Ubiquitinação , Peixe-Zebra/imunologia , Peixe-Zebra/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Membrana/metabolismo , Interferons/metabolismoRESUMO
Tripartite motif (TRIM) family proteins have come forth as important modulators of innate signaling dependent on of E3 ligase activity. Recently, several human TRIM proteins have been identified as unorthodox RNA-binding proteins by RNA interactome analyses; however, their targets and functions remain largely unknown. FTRCA1 is a crucian carp (Carassius auratus)-specific finTRIM (fish novel TRIM) member and negatively regulates the IFN antiviral response by targeting two retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) pathway molecules, that is, TANK-binding kinase 1 (TBK1) and IFN regulatory factor 7 (IRF7). In this study, we identify FTRCA1 as an RNA-binding E3 ligase and characterize the contribution of its RNA-binding activity and E3 ligase activity to fish IFN response. Besides targeting TBK1 and IRF7, FTRCA1 downregulates fish IFN response also by targeting stimulator of IFN response cGAMP interactor 1 (STING1). E3 ligase activity is required for full inhibition on the TBK1- and IRF7-mediated IFN response, but partial inhibition on the STING1-mediated IFN response. However, FTRCA1 has a general binding potential to mRNAs in vitro, it selectively binds STING1 and IRF7 mRNAs in vivo to attenuate mRNA levels, and it directly interacts with TBK1 protein to target protein degradation for downregulating the IFN response. Our results present an interesting example of a fish species-specific finTRIM protein that has acquired RNA-binding activity and E3 ligase activity to fine-tune fish IFN response.
Assuntos
Fator VII , RNA , Animais , Antivirais , Proteínas de Peixes/genética , Humanos , Imunidade Inata , RNA Mensageiro , Tretinoína , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
The small HERC family currently comprises four members (HERC3-6) involved in the regulation of various physiological activities. Little is known about the role of HERCs in IFN response. In this study, we identify a novel fish HERC member, named crucian carp HERC7, as a negative regulator of fish IFN response. Genome-wide search of homologs and comprehensive phylogenetic analyses reveal that the small HERC family, apart from HERC3-6 that have been well-characterized in mammals, contains a novel HERC7 subfamily exclusively in nonmammalian vertebrates. Lineage-specific and even species-specific expansion of HERC7 subfamily in fish indicates that crucian carp HERC7 might be species-specific. In virally infected fish cells, HERC7 is induced by IFN and selectively targets three retinoic acid-inducible gene-I-like receptor signaling factors for degradation to attenuate IFN response by two distinct strategies. Mechanistically, HERC7 delivers mediator of IFN regulatory factor 3 activator and mitochondrial antiviral signaling protein for proteasome-dependent degradation at the protein level and facilitates IFN regulatory factor 7 transcript decay at the mRNA level, thus abrogating cellular IFN induction to promote virus replication. Whereas HERC7 is a putative E3 ligase, the E3 ligase activity is not required for its negative regulatory function. These results demonstrate that the ongoing expansion of the small HERC family generates a novel HERC7 to fine-tune fish IFN antiviral response.
Assuntos
Fator Regulador 7 de Interferon/metabolismo , Interferons/imunologia , Reoviridae/imunologia , Rhabdoviridae/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Carpas , Linhagem Celular , Proteínas de Peixes/genética , Células HEK293 , Humanos , Fator Regulador 7 de Interferon/genética , Proteínas de Membrana/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , Transdução de Sinais/imunologia , Transativadores/genéticaRESUMO
In humans, four small HERCs (HERC3-6) exhibit differential degrees of antiviral activity toward HIV-1. Recently we revealed a novel member HERC7 of small HERCs exclusively in non-mammalian vertebrates and varied copies of herc7 genes in distinct fish species, raising a question of what is the exact role for a certain fish herc7 gene. Here, a total of four herc7 genes (named HERC7a-d sequentially) are identified in the zebrafish genome. They are transcriptionally induced by a viral infection, and detailed promoter analyses indicate that zebrafish herc7c is a typical interferon (IFN)-stimulated gene. Overexpression of zebrafish HERC7c promotes SVCV (spring viremia of carp virus) replication in fish cells and concomitantly downregulates cellular IFN response. Mechanistically, zebrafish HERC7c targets STING, MAVS, and IRF7 for protein degradation, thus impairing cellular IFN response. Whereas the recently-identified crucian carp HERC7 has an E3 ligase activity for the conjugation of both ubiquitin and ISG15, zebrafish HERC7c only displays the potential to transfer ubiquitin. Considering the necessity for timely regulation of IFN expression during viral infection, these results together suggest that zebrafish HERC7c is a negative regulator of fish IFN antiviral response.
Assuntos
Doenças dos Peixes , Infecções por Rhabdoviridae , Animais , Humanos , Peixe-Zebra/genética , Interferons/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Antivirais , UbiquitinasRESUMO
In mammals, transcription factors of IFN-regulatory factors (IRFs) family translate viral recognition into IFN antiviral responses through translocating to nucleus and subsequently binding to the promoters of IFN and IFN-stimulated genes (ISGs). In addition to IRF1-9 conserved across vertebrates and IRF10 in teleost fish and bird, teleost fish has another novel member, IRF11; however, little is known about its role in IFN response. In this study, we provide evidence that IRF11 is present only in Osteichthyes (bony fish) but lost in tetrapods and subsequently characterize the stimulatory potential of zebrafish IRF11 to IFN antiviral response relevant to its subcellular localization and promoter binding. Overexpression of zebrafish IRF11 restricts virus replication through induction of IFN and ISGs. Zebrafish IRF11 is constitutively localized to nucleus, which is driven by a tripartite NLS motif, consisting of three interdependent basic clusters, two in DNA binding domain (DBD) and one in the region immediately C-terminal to DBD. Nuclear IRF11 binds to the IRF-binding element/IFN-stimulated response element motifs of zebrafish IFN promoters depending on the two conserved amino acids (K78, R82) within DBD helix α3. K78 and R82 also benefit zebrafish IRF11 nuclear import as two key residues positioned at the first basic cluster of the tripartite NLS motif. Such features enable zebrafish IRF11 to function as a positive transcription factor for fish IFN antiviral response. Our results identify a unique tripartite NLS motif that integrates DNA-binding activity and nuclear import ability, allowing zebrafish IRF11 to initiate IFN and ISG expression.
Assuntos
Fator Regulador 1 de Interferon/metabolismo , Interferons/genética , Infecções por Rhabdoviridae/veterinária , Fatores de Transcrição TFII/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/imunologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Sequência Conservada/genética , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Fator Regulador 1 de Interferon/genética , Interferons/metabolismo , Regiões Promotoras Genéticas/genética , Domínios Proteicos/genética , Elementos de Resposta , Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/virologia , Transdução de Sinais/genética , Fatores de Transcrição TFII/genética , Replicação Viral/imunologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/virologiaRESUMO
Fish mucus acts as a physiological and immunological barrier for maintaining normal fish physiology and conferring defense against pathogens infection. Here we report proteomic profiling of skin mucus of yellow catfish before and after E. ictaluri infection by Label-free LC-MS/MS approach. A total of 918 non-redundant proteins were identified from 54443 spectra referring to yellow catfish genome database. Further annotation via GO and KEGG database revealed complex protein composition of yellow catfish mucus. Besides structural proteins in mucus, a lot of immune-related proteins were retrieved, such as lectins, complement components, antibacterial peptides and immunoglobins. 133 differentially-expressed proteins (DEPs), including 76 up-regulated and 57 down-regulated proteins, were identified, most of which were enriched into 17 pathways centering on "immune system" category with 33 proteins involved. Consistently, significant proliferation of mucus-secreting goblet cells and CYPA-expressing cells were observed along outside of yellow catfish skin after E. ictaluri infection, indicating an enhanced immune response to E. ictaluri infection in yellow catfish skin mucus. The proteomic data provide systematic protein information to comprehensively understand the biological function of yellow catfish skin mucus in response to bacterial infection.
Assuntos
Peixes-Gato/genética , Infecções por Enterobacteriaceae/veterinária , Proteínas de Peixes/genética , Muco/imunologia , Pele/imunologia , Animais , Peixes-Gato/imunologia , Cromatografia Líquida , Edwardsiella ictaluri , Infecções por Enterobacteriaceae/imunologia , Doenças dos Peixes/imunologia , Perfilação da Expressão Gênica , Masculino , Muco/microbiologia , Proteoma , Pele/microbiologia , Espectrometria de Massas em TandemRESUMO
In mammals and fish, emerging evidence highlights that TRIM family members play important roles in the interferon (IFN) antiviral immune response. Fish TRIM family has undergone an unprecedented expansion leading to generation of finTRIM subfamily, which is exclusively specific to fish. Our recent results have shown that FTRCA1 (finTRIM C. auratus 1) is likely a fish species-specific finTRIM member in crucian carp C. auratus and acts as a negative modulator to downregulate fish IFN response by autophage-lysosomal degradation of protein kinase TBK1. In the present study, we found that FTRCA1 also impedes the activation of crucian carp IFN promoter by IRF7 but not by IRF3. Mechanistically, FTRCA1 attenuates IRF7 transcription levels likely due to enhanced decay of IRF7 mRNA, leading to reduced IRF7 protein levels and subsequently reduced fish IFN expression. E3 ligase activity is required for FTRCA1 to negatively regulate IRF7-mediated IFN response, because ligase-inactive mutants and the RING-deleted mutant of FTRCA1 lose the ability to block the activation of crucian carp IFN promoter by IRF7. These results together indicate that FTRCA1 is a multifaceted modulator to target different signaling factors for shaping fish IFN response in crucian carp.
Assuntos
Carpas/genética , Carpas/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Fator Regulador 7 de Interferon/metabolismo , Interferons/metabolismo , Transcrição Gênica , Animais , Carpas/metabolismo , Proteínas de Peixes/metabolismoRESUMO
The development of CRISPR-Cas9 technology introduces an efficient tool for precise engineering of fish genomes. With a short reproduction cycle, zebrafish infection mode can be referenced as antiviral breeding researches in aquaculture fish. Previously we identified a crucian carp-specific gene ftrca1 as an inhibitor of interferon response in vitro. Here, we demonstrate that genome editing of zebrafish ftr42, a homolog of ftrca1, generates a zebrafish mutant (ftr42lof/lof) with an improved resistance to SVCV infection. Zebrafish ftr42 acts as a virus-induced E3 ligase and downregulates IFN antiviral response by facilitating TBK1 protein degradation and also IRF7 mRNA decay. Genome editing results in loss of function of zebrafish ftr42, which enables zebrafish to have enhanced interferon response, thus improving zebrafish survival against virus infection. Our results suggest that fine-tuning fish IFN innate immunity through genome editing of negative regulators can genetically improve viral resistance in fish.
RESUMO
Running cold and hot water in buildings is a widely established commodity. However, interests regarding hygiene and microbiological aspects had so far been focussed on cold water. Little attention has been given to the microbiology of domestic hot-water installations (DHWIs), except for aspects of pathogenic Legionella. World-wide, regulations consider hot (or warm) water as 'heated drinking water' that must comply (cold) drinking water (DW) standards. However, the few reports that exist indicate presence and growth of microbial flora in DHWIs, even when supplied with water with disinfectant residual. Using flow cytometric (FCM) total cell counting (TCC), FCM-fingerprinting, and 16S rRNA-gene-based metagenomic analysis, the characteristics and composition of bacterial communities in cold drinking water (DW) and hot water from associated boilers (operating at 50 - 60 °C) was studied in 14 selected inhouse DW installations located in Switzerland and Austria. A sampling strategy was applied that ensured access to the bulk water phase of both, supplied cold DW and produced hot boiler water. Generally, 1.3- to 8-fold enhanced TCCs were recorded in hot water compared to those in the supplied cold DW. FCM-fingerprints of cold and corresponding hot water from individual buildings indicated different composition of cold- and hot-water microbial floras. Also, hot waters from each of the boilers sampled had its own individual FCM-fingerprint. 16S rRNA-gene-based metagenomic analysis confirmed the marked differences in composition of microbiomes. E.g., in three neighbouring houses supplied from the same public network pipe each hot-water boiler contained its own thermophilic bacterial flora. Generally, bacterial diversity in cold DW was broad, that in hot water was restricted, with mostly thermophilic strains from the families Hydrogenophilaceae, Nitrosomonadaceae and Thermaceae dominating. Batch growth assays, consisting of cold DW heated up to 50 - 60 °C and inoculated with hot water, resulted in immediate cell growth with doubling times between 5 and 10 h. When cold DW was used as an inoculum no significant growth was observed. Even boilers supplied with UVC-treated cold DW contained an actively growing microbial flora, suggesting such hot-water systems as autonomously operating, thermophilic bioreactors. The generation of assimilable organic carbon from dissolved organic carbon due to heating appears to be the driver for growth of thermophilic microbial communities. Our report suggests that a man-made microbial ecosystem, very close to us all and of potential hygienic importance, may have been overlooked so far. Despite consumers having been exposed to microbial hot-water flora for a long time, with no major pathogens so far been associated specifically with hot-water usage (except for Legionella), the role of harmless thermophiles and their interaction with potential human pathogens able to grow at elevated temperatures in DHWIs remains to be investigated.
Assuntos
Água Potável , Legionella , Humanos , Água Potável/microbiologia , RNA Ribossômico 16S , Ecossistema , Abastecimento de Água , Bactérias/genética , Microbiologia da ÁguaRESUMO
Assembly of a complete Y chromosome is a significant challenge in animals with an XX/XY sex-determination system. Recently, we created YY-supermale yellow catfish by crossing XY males with sex-reversed XY females, providing a valuable model for Y-chromosome assembly and evolution. Here, we assembled highly homomorphic Y and X chromosomes by sequencing genomes of the YY supermale and XX female in yellow catfish, revealing their nucleotide divergences with only less than 1% and with the same gene compositions. The sex-determining region (SDR) was identified to locate within a physical distance of 0.3 Mb by FST scanning. Strikingly, the incipient sex chromosomes were revealed to originate via autosome-autosome fusion and were characterized by a highly rearranged region with an SDR downstream of the fusion site. We found that the Y chromosome was at a very early stage of differentiation, as no clear evidence of evolutionary strata and classical structure features of recombination suppression for a rather late stage of Y-chromosome evolution were observed. Significantly, a number of sex-antagonistic mutations and the accumulation of repetitive elements were discovered in the SDR, which might be the main driver of the initial establishment of recombination suppression between young X and Y chromosomes. Moreover, distinct three-dimensional chromatin organizations of the Y and X chromosomes were identified in the YY supermales and XX females, as the X chromosome exhibited denser chromatin structure than the Y chromosome, while they respectively have significantly spatial interactions with female- and male-related genes compared with other autosomes. The chromatin configuration of the sex chromosomes as well as the nucleus spatial organization of the XX neomale were remodeled after sex reversal and similar to those in YY supermales, and a male-specific loop containing the SDR was found in the open chromatin region. Our results elucidate the origin of young sex chromosomes and the chromatin remodeling configuration in the catfish sexual plasticity.
RESUMO
In mammals, right open reading frame kinases (RIOKs) are initially reported to participate in cancer cell proliferation, apoptosis, migration and invasion, and recently they have been related to host immune response. Little is known about the homologs of RIOKs in fish. In the current study, we cloned three homologous genes of RIOK family in yellow catfish (Pelteobagrus fulvidraco), termed Pfriok1, Pfriok2 and Pfriok3. Pfriok1, Pfriok2 and Pfriok3 were constitutively expressed at relatively high levels in yellow catfish tissues, and their mRNA levels were not changed under viral infection. Individual overexpression of PfRIOK1, PfRIOK2 and PfRIOK3 attenuated fish interferon (IFN) response, thereby promoting viral replication in fish cells. Mechanistically, yellow catfish RIOK proteins downregulated fish IFN response through attenuating TBK1 protein levels in cytoplasm. Our findings suggest that yellow catfish RIOK1, RIOK2 and RIOK3 are involved in downregulating fish IFN antiviral response.
Assuntos
Peixes-Gato , Animais , Peixes-Gato/genética , Interferons , Antivirais , Proteínas de Peixes/genética , MamíferosRESUMO
Retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) are viral RNA sensors that regulate host interferon (IFN)-mediated antiviral signaling. LGP2 (laboratory genetics and physiology 2) lacks the N-terminal caspase activation and recruitment domains (CARDs) responsible for signaling transduction in the other two RLR proteins, RIG-I and melanoma differentiation associated gene-5 (MDA5). How LGP2 regulates IFN signaling is controversial, and inconsistent results have often been obtained in overexpression assays when performed in fish cells and mammalian cells. Here we report that the differential sensitivity of fish cells and mammalian cells to poly(I:C) transfection conceals the function conservation of zebrafish and human LGP2. In fish cells, overexpression of zebrafish or human LGP2 initially activates IFN signaling in a dose-dependent manner, followed by inhibition at a critical threshold of LGP2 expression. A similar trend exists for LGP2-dependent IFN induction in response to stimulation by low and high concentrations of poly(I:C). In contrast, overexpression of zebrafish or human LGP2 alone in mammalian cells does not activate IFN signaling, but co-stimulation with very low or very high concentrations of poly(I:C) shows LGP2-dependent enhancement or inhibition of IFN signaling, respectively. Titration assays show that LGP2 promotes MDA5 signaling in mammalian cells mainly under low concentration of poly(I:C) and inhibits RIG-I/MDA5 signaling mainly under high concentration of poly(I:C). Our results suggest that fish and human LGP2s switch regulatory roles from a positive one to a negative one in increasing concentrations of poly(I:C)-triggered IFN response.
Assuntos
Poli I-C , RNA Helicases , Peixe-Zebra , Animais , Antivirais/metabolismo , Humanos , Helicase IFIH1 Induzida por Interferon/genética , Interferons , Mamíferos/metabolismo , Poli I-C/farmacologia , RNA Helicases/genética , RNA Helicases/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Exosomes participate in many physiological and pathological processes by regulating cell-to-cell communication. This affects the etiology and development of diseases, such as osteoarthritis (OA). Although exosomes in the OA tissue microenvironment are involved in the progression of OA, exosomes derived from therapeutic cells represent a new therapeutic strategy for OA treatment. Recent studies have shown that exosomes participate in OA treatment by regulating the proliferation, apoptosis, inflammation, and extracellular matrix synthesis of chondrocytes. However, studies in this field are scant. This review summarizes the therapeutic properties of exosomes on chondrocytes in OA and their underlying molecular mechanisms. We also discuss the challenges and prospects of exosome-based OA treatment.
RESUMO
Interferon regulatory factors (IRFs) constitute a family of transcription factors that synchronize interferon (IFN) antiviral response through translocating to nucleus and binding to the promoters of IFN and IFN-stimulated genes (ISGs). Fish contain 11 IRF members; however, whether or how fish IRF family genes function in IFN response remains limited. Herein, we determine the regulatory roles of 11 zebrafish IRF family members in IFN response relevant to their subcellular localization and promoter binding. Zebrafish IRF family members display three patterns of constitutive localization, only in nucleus (IRF1/2/9/11), only in cytoplasm (IRF3/5/7), and largely in nucleus with small amounts in cytoplasm (IRF4b/6/8/10). DNA pull-down assays confirm that all zebrafish IRF proteins are capable to bind fish IFN promoters, albeit to various degrees, thus regulating IFN gene transcription as activators (IRF1/3/5/6/7/8/9/11) or repressors (IRF2/4b/10). Further characterization of distinct IFN gene activation reveals that IRF1/3/5/6/7/8/9/11 efficiently stimulate zebrafish IFNφ1 expression, and IRF1/7/11 are responsible for zebrafish IFNφ3 expression. Two conserved basic residues within the helix α3 of DNA binding domains (DBDs) contribute to constitutive or inducible nuclear import for all zebrafish IRF family members and DNA binding for most members, thereby enabling them to function as transcription factors. Our results reveal a conserved and general mechanism that specifies zebrafish IRF family proteins to nuclear import and DNA binding, thereby regulating fish IFN response.
Assuntos
Interferons , Peixe-Zebra , Animais , Núcleo Celular/metabolismo , Fatores Reguladores de Interferon/metabolismo , Interferons/genética , Interferons/metabolismo , Regiões Promotoras Genéticas , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
In mammals, LGP2 is the enigmatic RLR family member, being initially believed as an inhibitor of RLR-triggered IFN response but subsequently as an activator of MDA5 signaling and an inhibitor of RIG-I signaling. The contradiction happens to fish LGP2. Here, we generate a lgp2 loss-of-function (lgp2 lof/lof ) zebrafish mutant, which is highly susceptible to SVCV infection, displaying an initially decreased activation of IFN response and a following increased one. Mechanistically, zebrafish LGP2 functions as the essential activator of IFN response dependent on MDA5 at the early stage of viral infection but as a negative regulator by impairing mRNA levels of tbk1 and ikki at the late stage of viral infection. The function switch of LGP2 is related to cellular IFN production during viral infection. Our data demonstrate that zebrafish LGP2 is a key homeostatic regulator of IFN response and thus essential for zebrafish survival against SVCV infection.
RESUMO
Objective: Osteoarthritis (OA) is a degenerative joint disease. Excessive nitric oxide (NO) mediates the chondrocyte inflammatory response, apoptosis, and extracellular matrix (ECM) degradation during the occurrence and development of OA. NO in chondrocytes is mainly produced by inducible nitric oxide synthase (iNOS). The aim of this study was to design and synthesize an iNOS dimerization inhibitor and evaluate its effects on chondrocyte inflammation and articular cartilage injury in OA via in vitro and in vivo experiments. Design: The title compound 22o was designed, synthesized, and screened based on a previous study. The effects of different concentrations (5, 10, and 20 µM) of compound 22o on chondrocyte inflammatory response and ECM anabolism or catabolism were evaluated by Western blot and real-time quantitative reverse transcription-polymerase chain reaction using the rat chondrocyte model of IL-1ß-induced OA. Furthermore, different doses (40 and 80 mg/kg) of compound 22o were administered by gavage to a rat OA model induced by anterior cruciate ligament transection (ACLT), and their protective effects on the articular cartilage were evaluated by histopathology and immunohistochemistry. Results: Compound 22o showed effective iNOS inhibitory activity by inhibiting the dimerization of iNOS. It inhibited the IL-1ß-induced expression of cyclooxygenase-2 (COX-2) and matrix metalloproteinase 3 (MMP3) in the chondrocytes, decreased NO production, and significantly increased the expression levels of the ECM anabolic markers, aggrecan (ACAN), and collagen type II (COL2A1). Gavage with compound 22o was found to be effective in the rat OA model induced by ACLT, wherein it regulated the anabolism and catabolism and exerted a protective effect on the articular cartilage. Conclusions: Compound 22o inhibited the inflammatory response and catabolism of the chondrocytes and reduced articular cartilage injury in the rat OA model, indicating its potential as a disease-modifying OA drug.
RESUMO
Recent studies have related the membrane-associated RING-CH-type finger (MARCH) family proteins to host innate immune response. Zebrafish (Danio rerio) MARCH8 is reported to target SVCV glycoprotein for degradation; however, little is known about whether fish MARCH8 is involved in innate interferon (IFN) response. In this study, zebrafish march8 was significantly induced by SVCV infection. Overexpression of MARCH8 diminished fish IFN-mediated antiviral response, thus promoting the replication of SVCV and GCRV in fish cells. Mechanistically, MARCH8 interacts with and degrades MITA and TBK1 proteins to inhibit IFN response. Moreover, MARCH8 has an E3 ligase activity and enhances MITA and TBK1 polyubiquitination. Our findings reveal a mechanism whereby zebrafish MARCH8 downregulates fish IFN response and facilitates viral replication by targeting MITA and TBK1 for protein degradation.
Assuntos
Interferons , Peixe-Zebra , Animais , Antivirais , Imunidade Inata , Interferons/metabolismo , Proteólise , Replicação ViralRESUMO
It is well known that RA (Rheumatoid arthritis) is an autoimmune disease characterized by multiple and symmetric arthropathy. The main pathological features of RA are synovial hyperplasia, angiogenesis, pannus formation, inflammatory cell infiltration, articular cartilage, bone destruction, and ultimately joint dysfunction, even deformity. IL-35 (Interleukin-35) is a new member of the IL-12 (Interleukin-12) family, which is an immunosuppressive and anti-inflammatory cytokine secreted mainly by Treg (T regulatory cells). There is evidence suggested that IL-35 can attenuate the progression of RA through influencing the immune and pathological process. It suggests that IL-35 played an important role in the pathogenesis of RA, and can be used as a potential target for the future treatment of RA. This review summarizes the recent advances of IL-35 in the pathological roles and the therapeutic potential roles in RA.
RESUMO
Primordial germ cells (PGCs) are singled out from somatic cells very early during embryogenesis, then they migrate towards the genital ridge and differentiate into gametes through oogenesis or spermatogenesis. Labeling PGCs with Localized RNAexpression (LRE) technique by fluorescent proteins has been widely applied among teleost species to study the germ cell development and gonad differentiation. In this study, we first cloned and characterized the 3' untranslated regions (3'UTRs) of nanos homolog 1-like (nos1l), dead end (dnd), and vasa in yellow catfish (Pelteobagrus fulvidraco), and then synthesized the GFP-nos1l/dnd/vasa 3'UTR mRNAs. Each of these three 3'UTRs could label PGCs in yellow catfish embryos, of which, vasa 3'UTR exhibited the highest labeling efficiency. To identify the differences in PGCs at embryonic stage, XX all-female and XY all-male yellow catfish embryos were produced and injected with GFP-vasa 3'UTR mRNA. We observed the PGC migration route in these two monosex embryos from 24 hpf to 7 dpf, and found there was no difference between them. Besides, the PGC number was counted at 48 hpf, and the result showed that the average PGC number in XX females (11.3) was significantly larger than that in XY males (8.1).These findings provide an insight into the development of PGCs in yellow catfish embryos and the relationship between embryonicPGCnumberand thelatergonaddifferentiation.
Assuntos
Peixes-Gato/genética , Gametogênese/genética , Células Germinativas/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Movimento Celular/genética , RNA Helicases DEAD-box/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Gônadas/metabolismo , Masculino , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Edwardsiella ictaluri (E. ictaluri) is one of the main bacterial pathogens in catfish which has caused serious economic loss to yellow catfish (Pelteobagrus fulvidraco) in China. In our previous work, we demonstrated that CypA was up-regulated at the early stage of E. ictaluri infection in yellow catfish and displayed strong chemotactic activity for leukocytes in vitro. However, the effect of CypA on E. ictaluri is unknown in vivo. Therefore, two homozygous transgenic zebrafish lines expressing yellow catfish CypA (TG-CypA-1 and TG-CypA-2) were generated. After challenged with E. ictaluri at a dose of 1.0 × 104 CFU per adult fish, both two transgenic lines exhibited a higher resistance to bacterial infection than the wildtype zebrafish. Herein, CypA gene in E. ictaluri-challenged yellow catfish was screened for presence of polymorphisms by sequencing and six single nucleotide polymorphisms (SNPs) were identified. SNP association analysis revealed that 528T/C SNP in the first intron was significantly different in disease-susceptible and -resistant groups, which was confirmed in two independent populations of yellow catfish. Moreover, the relative expression of CypA in the resistant group (CC genotype in 528T/C SNP) was significantly higher than that in the susceptible group (TT genotype in 528T/C SNP) in different immune organs of yellow catfish including spleen, head kidney, body kidney and liver. Our results reveal the potential function of CypA in host defense to bacterial infection and suggest the SNP marker in CypA gene associated with the resistance to E. ictaluri may facilitate the selective breeding of disease-resistant yellow catfish in the future.