RESUMEN

The innate immune system relies on a variety of pathogen recognition receptors (PRRs) as the first line of defense against pathogenic invasions. Viruses have evolved multiple strategies to evade the host immune system through coevolution with hosts. The CRISPR-Cas system is an adaptive immune system in bacteria or archaea that defends against viral reinvasion by targeting nucleic acids for cleavage. Based on the characteristics of Cas proteins and their variants, the CRISPR-Cas system has been developed into a versatile gene-editing tool capable of gene knockout or knock-in operations to achieve genetic variations in organisms. It is now widely used in the study of viral immune evasion mechanisms. This chapter will introduce the use of the CRISPR-Cas9 system for editing herpes simplex virus 1 (HSV-1) genes to explore the mechanisms by which HSV-1 evades host innate immunity and the experimental procedures involved.

Asunto(s)

Sistemas CRISPR-Cas , Técnicas de Inactivación de Genes , Herpesvirus Humano 1 , Evasión Inmune , Inmunidad Innata , Sistemas CRISPR-Cas/genética , Inmunidad Innata/genética , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/genética , Evasión Inmune/genética , Humanos , Edición Génica/métodos , Animales , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genética , Herpes Simple/inmunología , Herpes Simple/virología , Herpes Simple/genética

RESUMEN

The application of CRISPR-mediated library screening has fundamentally transformed functional genomics by revealing the complexity of virus-host interactions. This protocol describes the use of CRISPR-mediated library screening to identify key functional genes regulating the innate immune response to PEDV infection. We detail a step-by-step process, starting from the design and construction of a customized CRISPR knockout library targeting genes involved in innate immunity to the effective delivery of these constructs into cells using lentiviral vectors. Subsequently, we outline the process of identifying functional genes postviral attack, including the use of next-generation sequencing (NGS), to analyze and identify knockout cells that exhibit altered responses to infection. This integrated approach provides researchers in immunology and virology with a resource and a robust framework for uncovering the genetic basis of host-pathogen interactions and the arsenal of the innate immune system against viral invasions.

Asunto(s)

Sistemas CRISPR-Cas , Técnicas de Inactivación de Genes , Biblioteca de Genes , Inmunidad Innata , Inmunidad Innata/genética , Sistemas CRISPR-Cas/genética , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genética , Línea Celular , Lentivirus/genética

RESUMEN

Transcriptomics is an extremely important area of molecular biology and is a powerful tool for studying all RNA molecules in an organism. Conventional transcriptomic technologies include microarrays and RNA sequencing, and the rapid development of single-cell sequencing and spatial transcriptomics in recent years has provided an enormous scope for research in this field. This chapter describes the application, significance, and experimental procedures of a variety of transcriptomic technologies in antiviral natural immunity.

Asunto(s)

Perfilación de la Expresión Génica , Inmunidad Innata , Transcriptoma , Inmunidad Innata/genética , Humanos , Perfilación de la Expresión Génica/métodos , Animales , Virosis/inmunología , Virosis/genética , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos

RESUMEN

With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.

Asunto(s)

Sistemas CRISPR-Cas , Edición Génica , Inmunidad Innata , Ratones Noqueados , ARN Guía de Sistemas CRISPR-Cas , Animales , Inmunidad Innata/genética , Ratones , ARN Guía de Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Virosis/inmunología , Virosis/genética

RESUMEN

Zebrafish is a widely used model organism in genetics, developmental biology, pathology, and immunology research. Due to their fast reproduction, large numbers, transparent early embryos, and high genetic conservation with the human genome, zebrafish have been used as a model for studying human and fish viral diseases. In particular, the ability to easily perform forward and reverse genetics and lacking a functional adaptive immune response during the early period of development establish the zebrafish as a favored option to assess the functional implication of specific genes in the antiviral innate immune response and the pathogenesis of viral diseases. In this chapter, we detail protocols for the antiviral innate immunity analysis using the zebrafish model, including the generation of gene-overexpression zebrafish, generation of gene-knockout zebrafish by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, methods of viral infection in zebrafish larvae, analyzing the expression of antiviral genes in zebrafish larvae using qRT-PCR, Western blotting and transcriptome sequencing, and in vivo antiviral assays. These experimental protocols provide effective references for studying the antiviral immune response in the zebrafish model.

Asunto(s)

Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Inmunidad Innata , Pez Cebra , Animales , Pez Cebra/inmunología , Pez Cebra/genética , Pez Cebra/virología , Inmunidad Innata/genética , Virosis/inmunología , Virosis/genética , Técnicas de Inactivación de Genes , Animales Modificados Genéticamente

RESUMEN

BACKGROUND: Nod2 is involved in innate immune responses to bacteria, regulation of metabolism, and sensitivity to cancer. A Nod2 polymorphism is associated with breast cancer, but the role of Nod2 in the development and progression of breast cancer is unknown. METHODS: Here, we tested the hypothesis that Nod2 protects mice from breast cancer using the 4T1 orthotopic model of mammary tumorigenesis. WT and Nod2-/- mice were injected with 4T1 mammary carcinoma cells and the development of tumors was monitored. A detailed analysis of the tumor transcriptome was performed and genes that were differentially expressed and pathways that were predicted to be altered between WT and Nod2-/- mice were identified. The activation of key signaling molecules involved in metabolism and development of cancer was studied. RESULTS: Our data demonstrate that Nod2-/- mice had a higher incidence and larger tumors than WT mice. Nod2-/- mice had increased expression of genes that promote DNA replication and cell division, and decreased expression of genes required for lipolysis, lipogenesis, and steroid biosynthesis compared with WT mice. Nod2-/- mice also had lower expression of genes required for adipogenesis and reduced levels of lipids compared with WT mice. The tumors in Nod2-/- mice had decreased expression of genes associated with PPARα/γ signaling, increased activation of STAT3, decreased activation of STAT5, and no change in the activation of ERK compared with WT mice. CONCLUSIONS: We conclude that Nod2 protects mice from the 4T1 orthotopic breast tumor, and that tumors in Nod2-/- mice are predicted to have increased DNA replication and cell proliferation and decreased lipid metabolism compared with WT mice.

Asunto(s)

Neoplasias de la Mama , Inmunidad Innata , Proteína Adaptadora de Señalización NOD2 , Animales , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/metabolismo , Ratones , Femenino , Inmunidad Innata/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Ratones Noqueados , Línea Celular Tumoral , Transducción de Señal/genética , Regulación Neoplásica de la Expresión Génica , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Ratones Endogámicos BALB C , Transcriptoma/genética , Perfilación de la Expresión Génica/métodos

RESUMEN

The innate immune response is triggered rapidly after injury and its spatiotemporal dynamics are critical for regeneration; however, many questions remain about its exact role. Here we show that MyD88, a key component of the innate immune response, controls not only the inflammatory but also the fibrotic response during zebrafish cardiac regeneration. We find in cryoinjured myd88-/- ventricles a significant reduction in neutrophil and macrophage numbers and the expansion of a collagen-rich endocardial population. Further analyses reveal compromised PI3K/AKT pathway activation in the myd88-/- endocardium and increased myofibroblasts and scarring. Notably, endothelial-specific overexpression of myd88 reverses these neutrophil, fibrotic and scarring phenotypes. Mechanistically, we identify the endocardial-derived chemokine gene cxcl18b as a target of the MyD88 signaling pathway, and using loss-of-function and gain-of-function tools, we show that it controls neutrophil recruitment. Altogether, these findings shed light on the pivotal role of MyD88 in modulating inflammation and fibrosis during tissue regeneration.

Asunto(s)

Fibrosis , Inmunidad Innata , Factor 88 de Diferenciación Mieloide , Regeneración , Transducción de Señal , Proteínas de Pez Cebra , Pez Cebra , Animales , Animales Modificados Genéticamente , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Endocardio/metabolismo , Endocardio/patología , Endocardio/inmunología , Corazón/fisiopatología , Inmunidad Innata/genética , Macrófagos/metabolismo , Macrófagos/inmunología , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patología , Infiltración Neutrófila , Neutrófilos/metabolismo , Neutrófilos/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Regeneración/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo

RESUMEN

Male infertility can be caused by chromosomal abnormalities, mutations and epigenetic defects. Epigenetic modifiers pre-program hundreds of spermatogenic genes in spermatogonial stem cells (SSCs) for expression later in spermatids, but it remains mostly unclear whether and how those genes are involved in fertility. Here, we report that Wfdc15a, a WFDC family protease inhibitor pre-programmed by KMT2B, is essential for spermatogenesis. We found that Wfdc15a is a non-canonical bivalent gene carrying both H3K4me3 and facultative H3K9me3 in SSCs, but is later activated along with the loss of H3K9me3 and acquisition of H3K27ac during meiosis. We show that WFDC15A deficiency causes defective spermiogenesis at the beginning of spermatid elongation. Notably, depletion of WFDC15A causes substantial disturbance of the testicular protease-antiprotease network and leads to an orchitis-like inflammatory response associated with TNFα expression in round spermatids. Together, our results reveal a unique epigenetic program regulating innate immunity crucial for fertility.

Asunto(s)

Homeostasis , Espermátides , Espermatogénesis , Masculino , Animales , Espermatogénesis/genética , Ratones , Espermátides/metabolismo , Testículo/metabolismo , Histonas/metabolismo , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/genética , Epigénesis Genética , Infertilidad Masculina/genética , Ratones Endogámicos C57BL , Meiosis/genética , Células Madre Germinales Adultas/metabolismo , Ratones Noqueados , Inmunidad Innata/genética , Espermatogonias/metabolismo

RESUMEN

MicroRNAs (miR) are a group of small, non-coding RNAs of 17-25 nucleotides that regulate gene expression at the post-transcriptional level. Dysregulation of miRNA expression or function may contribute to abnormal gene expression and signaling pathways, leading to disease pathology. Lagovirus europaeus (L. europaeus) causes severe disease in rabbits called rabbit hemorrhagic disease (RHD). The symptoms of liver, lung, kidney, and spleen degeneration observed during RHD are similar to those of acute liver failure (ALF) and multi-organ failure (MOF) in humans. In this study, we assessed the expression of miRs and their target genes involved in the innate immune and inflammatory response. Also, we assessed their potential impact on pathways in L. europaeus infection-two genotypes (GI.1 and GI.2)-in the liver, lungs, kidneys, and spleen. The expression of miRs and target genes was determined using quantitative real-time PCR (qPCR). We assessed the expression of miR-155 (MyD88, TAB2, p65, NLRP3), miR-146a (IRAK1, TRAF6), miR-223 (TLR4, IKKα, NLRP3), and miR-125b (MyD88). We also examined biomarkers of inflammation: IL-1ß, IL-6, TNF-α, and IL-18 in four tissues at the mRNA level. Our study shows that the main regulators of the innate immune and inflammatory response in L. europaeus/GI.1 and GI.2 infection, as well as RHD, are miR-155, miR-223, and miR-146a. During infection with L. europaeus/RHD, miR-155 has both pro- and anti-inflammatory effects in the liver and anti-inflammatory effects in the kidneys and spleen; miR-146a has anti-inflammatory effects in the liver, lungs and kidneys; miR-223 has anti-inflammatory effects in all tissues; however, miR-125b has anti-inflammatory effects only in the liver. In each case, such an effect may be a determinant of the pathogenesis of RHD. Our research shows that miRs may regulate three innate immune and inflammatory response pathways in L. europaeus infection. However, the result of this regulation may be influenced by the tissue microenvironment. Our research shows that infection of rabbits with L. europaeus/GI.1 and GI.2 genotypes causes an overexpression of two critical acute phase cytokines: IL-6 in all examined tissues and TNF-α (in the liver, lungs, and spleen). IL-1ß was highly expressed only in the lungs after L. europaeus infection. These facts indicate a strong and rapid involvement of the local innate immune and inflammatory response in L. europaeus infection-two genotypes (GI.1 and GI.2)-and in the pathogenesis of RHD. Profile of biomarkers of inflammation in rabbits infected with L. europaeus/GI.1 and GI.2 genotypes are similar regarding the nature of changes but are different for individual tissues. Therefore, we propose three inflammation profiles for L. europaeus infection for both GI.1 and GI.2 genotypes (pulmonary, renal, liver, and spleen).

Asunto(s)

Infecciones por Caliciviridae , Genotipo , Virus de la Enfermedad Hemorrágica del Conejo , Inmunidad Innata , MicroARNs , Animales , MicroARNs/genética , Inmunidad Innata/genética , Conejos , Infecciones por Caliciviridae/genética , Infecciones por Caliciviridae/inmunología , Infecciones por Caliciviridae/virología , Virus de la Enfermedad Hemorrágica del Conejo/genética , Virus de la Enfermedad Hemorrágica del Conejo/inmunología , Inflamación/genética , Inflamación/inmunología , Regulación de la Expresión Génica , Hígado/metabolismo , Hígado/patología , Hígado/virología

RESUMEN

BACKGROUND: Fibrinogen-related protein 1 (frep1) is a member of the pattern-recognizing receptor family (PRR) which generates an innate immune response after recognizing the pattern associated molecular pattern (PAMP) that occurs on the surface of microorganisms. The main objective of this study is to characterize frep1 and its in-silico analysis in Anopheles stephensi. METHODS AND RESULT: The DNA was extracted from female Anopheles stephensi. PCR was performed for complete analysis of frep1 using specific primers. The gene sequence of frep1 was identified by Sanger sequencing. The bioinformatics structure analysis approach revealed the presence of 3 exons and 4 introns in the frep1. The sequence of frep1 was submitted to NCBI GeneBank with accession number ON817187.1. Quantitative real-time PCR was performed to analyze frep1 expression. At the developmental stage, frep1 is highly expressed in the L1 stage, egg, and adult female mosquito. In addition, frep1 is highly expressed in the tissue fat body, midgut, and salivary gland. After blood-fed, an upregulation of frep1 at 48 h in the midgut, and downregulation in fat body were observed at different time intervals. CONCLUSION: The genomic data of frep1 is encoded by 12,443 bp. The frep1 has a significant role in the early metamorphosis. Its expression in fat body and midgut suggests it could be important for fat metabolism and post-blood digestion. The conserved domain could be targeted for vector control. Further study is required to elucidate its function against malaria parasites to confirm its agonist role in malaria transmission.

Asunto(s)

Anopheles , Proteínas de Insectos , Malaria , Mosquitos Vectores , Anopheles/genética , Anopheles/metabolismo , Animales , Mosquitos Vectores/genética , Femenino , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Malaria/parasitología , Simulación por Computador , Fibrinógeno/metabolismo , Fibrinógeno/genética , Filogenia , Inmunidad Innata/genética , Secuencia de Aminoácidos

RESUMEN

Innate immune parameters, a first line of defense against invading pathogens like bacteria, parasites, fungi, etc, play a significant role in the prevention and elimination of aetiological agents primarily by recognition of invading pathogen-specific molecules by different pattern recognition receptors. Toll-like receptors (TLRs), a type-I transmembrane glycoprotein, cause innate immune responses mainly by produing inflammatory cytokines, chemokines and interferons. The objective of present study was to determine the role of TLRs in parasite resistance in Malpura sheep. In the current study, transcript variation of TLRs and its downstream signalling molecules namely MyD88, TRIF, IRF-3, TRAF, TGF-ß, NFκB, and CD14 were ascertained by real-time PCR in Haemonchus contortus resistant (R) and susceptible (S) Malpura sheep. Results have shown significantly (P<0.05) up-regulated expression of TLR-2, TLR-4, TLR-5, TLR-8 and TLR-10 in July however down-regulated patterns were observed in August and September in R-line sheep compared to S-line sheep. This indicates that at more or less equal parasite load, the TLR genes in R sheep produce more transcripts, but after parasite loads have increased hugely in the S line, they easily surpass the levels seen in the S line. Result suggests that transcriptional activity of the TLR genes was related to parasite load and there were differences between the lines at different infection intensities. Three-point transcript expression observation of the signalling molecules namely TRIF, IRF-3, TRAF, a similar pattern was observed in R sheep compared with S sheep.

Asunto(s)

Hemoncosis , Haemonchus , Inmunidad Innata , Enfermedades de las Ovejas , Receptores Toll-Like , Animales , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Haemonchus/inmunología , Ovinos/inmunología , Hemoncosis/veterinaria , Hemoncosis/inmunología , Hemoncosis/parasitología , Inmunidad Innata/genética , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/parasitología , Enfermedades de las Ovejas/genética , Resistencia a la Enfermedad/inmunología , Resistencia a la Enfermedad/genética

RESUMEN

BACKGROUND: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell-type composition. RESULTS: Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019-2020 and 2020-2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. CONCLUSIONS: Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.

Asunto(s)

Metilación de ADN , Inmunidad Innata , Vacunas contra la Influenza , Gripe Humana , Humanos , Metilación de ADN/genética , Metilación de ADN/efectos de los fármacos , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/administración & dosificación , Inmunidad Innata/genética , Femenino , Masculino , Gripe Humana/prevención & control , Gripe Humana/inmunología , Gripe Humana/genética , Persona de Mediana Edad , Adulto , Transducción de Señal , Linfocitos T/inmunología , Estudios Longitudinales , Epigénesis Genética , Vacunación , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo

RESUMEN

Bronchiolitis is the leading cause of infant hospitalization. However, the molecular networks driving bronchiolitis pathobiology remain unknown. Integrative molecular networks, including the transcriptome and metabolome, can identify functional and regulatory pathways contributing to disease severity. Here, we integrated nasopharyngeal transcriptome and metabolome data of 397 infants hospitalized with bronchiolitis in a 17-center prospective cohort study. Using an explainable deep network model, we identified an omics-cluster comprising 401 transcripts and 38 metabolites that distinguishes bronchiolitis severity (test-set AUC, 0.828). This omics-cluster derived a molecular network, where innate immunity-related metabolites (e.g., ceramides) centralized and were characterized by toll-like receptor (TLR) and NF-κB signaling pathways (both FDR < 0.001). The network analyses identified eight modules and 50 existing drug candidates for repurposing, including prostaglandin I2 analogs (e.g., iloprost), which promote anti-inflammatory effects through TLR signaling. Our approach facilitates not only the identification of molecular networks underlying infant bronchiolitis but the development of pioneering treatment strategies.

Asunto(s)

Bronquiolitis , Humanos , Bronquiolitis/genética , Bronquiolitis/metabolismo , Lactante , Estudios Prospectivos , Transcriptoma/genética , Masculino , Femenino , Transducción de Señal/genética , Metaboloma/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Recién Nacido , Inmunidad Innata/genética , Metabolómica/métodos

RESUMEN

Tuberculous meningitis, a severe form of tuberculosis caused by Mycobacterium tuberculosis (BK), remains a major public health challenge worldwide. In addition to the complex mechanisms of the innate and adaptive immune response against Mycobacterium tuberculosis, there is a crucial genetic dimension to consider. Individuals with specific genetic variations may have altered immune responses that make them more susceptible to this form of tuberculosis. Genetic mutations in genes encoding surface receptors, adaptor proteins, kinases, transcription factors, nucleic receptors and other molecules involved in cellular interactions and molecular mechanisms have been associated with susceptibility to TB. Understanding the molecular mechanisms of immune interactions in host response to Mycobacterium tuberculosis is crucial to understanding the genetic dimension in susceptibility to tuberculosis, particularly its dreaded form of tuberculous meningitis. The aim of this update is to explore in details the key interactions between the main players in innate and adaptive immunity during infection with Mycobacterium tuberculosis, with particular emphasis on the genetic factors associated with susceptibility to tuberculosis, especially its dreaded form of tuberculous meningitis.

Asunto(s)

Predisposición Genética a la Enfermedad , Mycobacterium tuberculosis , Tuberculosis Meníngea , Humanos , Tuberculosis Meníngea/genética , Tuberculosis Meníngea/inmunología , Mycobacterium tuberculosis/inmunología , Inmunidad Innata/genética , Inmunidad Adaptativa/genética

RESUMEN

The production of type I interferon is tightly regulated to prevent excessive immune activation. However, the role of selective autophagy receptor SQSTM1 in this regulation in teleost remains unknown. In this study, we cloned the triploid fish SQSTM1 (3nSQSTM1), which comprises 1371 nucleotides, encoding 457 amino acids. qRT-PCR data revealed that the transcript levels of SQSTM1 in triploid fish were increased both in vivo and in vitro following spring viraemia of carp virus (SVCV) infection. Immunofluorescence analysis confirmed that 3nSQSTM1 was mainly distributed in the cytoplasm. Luciferase reporter assay results showed that 3nSQSTM1 significantly blocked the activation of interferon promoters induced by 3nMDA5, 3nMAVS, 3nTBK1, and 3nIRF7. Co-immunoprecipitation assays further confirmed that 3nSQSTM1 could interact with both 3nTBK1 and 3nIRF7. Moreover, upon co-transfection, 3nSQSTM1 significantly inhibited the antiviral activity mediated by TBK1 and IRF7. Mechanistically, 3nSQSTM1 decreased the TBK1 phosphorylation and its interaction with 3nIRF7, thereby suppressing the subsequent antiviral response. Notably, we discovered that 3nSQSTM1 also interacted with SVCV N and P proteins, and these viral proteins may exploit 3nSQSTM1 to further limit the host's antiviral innate immune responses. In conclusion, our study demonstrates that 3nSQSTM1 plays a pivotal role in negatively regulating the interferon signaling pathway by targeting 3nTBK1 and 3nIRF7.

Asunto(s)

Carpas , Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Factor 7 Regulador del Interferón , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Inmunidad Innata/genética , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Rhabdoviridae/fisiología , Infecciones por Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/veterinaria , Carpas/inmunología , Carpas/genética , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Regulación de la Expresión Génica/inmunología , Transducción de Señal/inmunología , Triploidía , Filogenia , Secuencia de Aminoácidos , Alineación de Secuencia/veterinaria , Perfilación de la Expresión Génica/veterinaria

RESUMEN

The c-Jun N-terminal kinase (JNK) constitutes an evolutionarily conserved family of serine/threonine protein kinases, pivotal in regulating various physiological processes in vertebrates, encompassing apoptosis and antibacterial immunity. Nevertheless, the involvement of JNK in the innate immune response remains largely unexplored in pathogen-induced echinoderms. We isolated and characterized the JNK gene from Apostichopus japonicus (AjJNK) in our investigation. The full-length cDNA sequences of AjJNK spanned 1806 bp, comprising a 1299 bp open reading frame (ORF) encoding 432 amino acids, a 274 bp 5'-untranslated region (UTR), and a 233 bp 3'-UTR. Structural analysis revealed the presence of a classical S_TKc domain (37-335 amino acids) within AjJNK and contains several putative immune-related transcription factor-binding sites, including Elk-1, NF-κB, AP-1, and STAT5. Spatial expression analysis indicated ubiquitous expression of AjJNK across all examined tissues, with the highest expression noted in coelomocytes. The mRNA, protein, and phosphorylation levels of AjJNK were obviously induced in coelomocytes upon V. splendidus challenge and lipopolysaccharide stimulation. Immunofluorescence analysis demonstrated predominant cytoplasmic localization of AjJNK in coelomocytes with subsequent nuclear translocation following the V. splendidus challenge in vivo. Moreover, siRNA-mediated knockdown of AjJNK led to a significant increase in intracellular bacterial load, as well as elevated levels of Ajcaspase 3 and coelomocyte apoptosis post V. splendidus infection. Furthermore, the phosphorylation levels of AjJNK inhibited by its specific inhibitor SP600125 and also significantly suppressed the expression of Ajcaspase 3 and coelomocyte apoptosis during pathogen infection. Collectively, these data underscored the pivotal role of AjJNK in immune defense, specifically in the regulation of coelomocyte apoptosis in V. splendidus-challenged A. japonicus.

Asunto(s)

Secuencia de Aminoácidos , Inmunidad Innata , Proteínas Quinasas JNK Activadas por Mitógenos , Filogenia , Stichopus , Vibrio , Animales , Stichopus/inmunología , Stichopus/genética , Stichopus/microbiología , Vibrio/fisiología , Inmunidad Innata/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/inmunología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Alineación de Secuencia/veterinaria , Perfilación de la Expresión Génica/veterinaria , Secuencia de Bases , Regulación de la Expresión Génica/inmunología , Vibriosis/inmunología , Vibriosis/veterinaria

RESUMEN

Blood transcriptomics has emerged as a vital tool for tracking the immune system and supporting disease diagnosis, prognosis, treatment, and research. The present study was conducted to analyze the gene expression profile and potential biomarker candidates using the whole blood of mandarin fish (Siniperca chuatsi) infected with LPS or poly (I:C) at 0 h, 3 h, 6 h, and 12 h. Our data suggest that 310 shared differentially expressed genes (DEGs) were identified among each comparison group after LPS infection, and 137 shared DEGs were identified after poly (I:C) infection. A total of 62 shared DEGs were differentially expressed in all compared groups after LPS or poly (I:C) infection. Pathways analysis for DEGs in all different compared groups showed that cytokine-cytokine receptor interaction was the most enrichment pathway. The expression levels of genes C-X-C chemokine receptor type 2-like (cxcr2), chemokine (C-C motif) receptor 9a (ccr9a), chemokine (C-C motif) receptor 9b (ccr9b), chemokine (C-X-C motif) receptor 4b (cxcr4b), and interleukin 10 receptor alpha (il10ra) were significantly different in all compared groups and most enriched in cytokine-cytokine receptor interaction pathway. The protein-protein interactions analysis among all shared DEGs showed that cxcr4 was the hub gene with the highest degree. The biomarker candidates discovered in this study may, following validation, prove effective as diagnostic tools in monitoring mandarin fish diseases.

Asunto(s)

Biomarcadores , Enfermedades de los Peces , Proteínas de Peces , Lipopolisacáridos , Perciformes , Poli I-C , Transcriptoma , Animales , Enfermedades de los Peces/inmunología , Poli I-C/farmacología , Biomarcadores/sangre , Lipopolisacáridos/farmacología , Perciformes/genética , Perciformes/inmunología , Perciformes/sangre , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/sangre , Perfilación de la Expresión Génica/veterinaria , Inmunidad Innata/genética , Regulación de la Expresión Génica/inmunología , Regulación de la Expresión Génica/efectos de los fármacos

RESUMEN

The ivory shell (Babylonia areolata) is one of the most promising high quality marine products. However, ivory shell is susceptible to Vibrio harveyi infection during the culture period. In this study, we investigated the biochemical indicators, histological changes and transcriptomic response in the hepatopancreas of ivory shells from the PBS control group (PC) and infection group (A3) with 1 × 109 CFU/mL V. harveyi after 24 h. Results showed that compared to the PC group, biochemical indicators, including malondialdehyde (MDA), reactive oxygen species (ROS), acid phosphatase (ACP), and Caspase 3 (Casp-3) were significantly increased (p < 0.05) in A3 group after V. harveyi infection for 24 h. Compared with the PC group, the hepatopancreas of A3 group were seriously damaged, the columnar epithelial cells of the tissue were enlarged, the space of digestive cells was increased, and vacuolar cavities appeared. A total of 95,581 unigenes were obtained and 2949 (1787 up-regulated and 1162 down-regulated) differential expressed genes (DEGs) were identified in the A3 group. GO and KEGG enrichment analysis showed that DEGs were mainly enriched in immune system process (GO:0002376), antioxidant activity (GO:0016209), lysosome (ko04142), toll and IMD signaling pathway (ko04624), and etc. These biological functions and pathways are associated with immune and inflammatory responses and apoptosis. 12 DEGs were randomly selected for real-time quantitative PCR (RT-qPCR) validation, and the expression profiles of these DEGs were consistent with the transcriptome data, confirming the accuracy and reliability of the transcriptome results. In summary, V. harveyi infection of ivory shells inducing oxidative stress, leading to severe hepatopancreatic damage, stimulating glutathione production to neutralize excessive ROS, and stimulating antimicrobial peptides production to counteract the deleterious effects of bacterial infection, which in turn modifying the immune and inflammatory response, ultimately resulting in apoptosis. This study provided valuable information to explore the immune regulation mechanism after V. harveyi infection and established molecular basis to support the prevention of V. harveyi infection.

Asunto(s)

Hepatopáncreas , Transcriptoma , Vibrio , Animales , Vibrio/fisiología , Hepatopáncreas/inmunología , Hepatopáncreas/patología , Inmunidad Innata/genética

RESUMEN

Takifugu obscurus is a farmed fish of great economic importance in China. The rapid development of T. obscurus aquaculture industry has been accompanied by disease and low-temperature stress, resulting in huge economic losses. Cell lines are used extensively in teleost physiology and pathology as the most cost-effective platform for in vitro research. A novel gill cell line of T. obscurus (named TOG) was first successfully established, and passed through 52 generations. The optimal conditions for TOG growth were 20 % FBS concentration and 24 °C, TOG could be grown in both hypotonic (150 mOsmol-kg-1) and hypertonic (600 mOsmol-kg-1) environments. TOG was determined to be derived from T. obscurus by sequencing the mitochondrial COI gene. Karyotype analysis revealed that the chromosome number of TOG was 44 (2n = 44). Transfection experiment showed that TOG was able to express foreign genes. Furthermore, several immune-related genes were significantly up-regulated in TOG after LPS and poly (I:C) stimulation, including tlr3, isg15, il1ß and il10. Additionally, transcriptome analysis of TOG under low-temperature stress (24 °C, 18 °C, 12 °C, 10 °C and 8 °C) found that differentially expressed genes (DEGs) were significantly clustered in several immunological and energy metabolic pathways, and cold stress could disrupt the immune barrier and reduce immunity by downregulating the immune-related pathways. Additionally, weighted gene co-expression network analysis (WGCNA) revealed that bule module and turquoise module, which were closely correlated with low temperature and the degree of fish damage, were both predominantly found in PPAR, NOD-like receptor and Toll-like receptor signaling pathway. Hub genes were identified in these two modules, including mre11, clpb, dhx15, ddx18 and utp15. TOG cell line will become an effective experimental platform for genetic and immunological research, and our results would help us gain a deeper insight into the molecular mechanism of cold tolerance in teleost.

Asunto(s)

Frío , Perfilación de la Expresión Génica , Branquias , Takifugu , Transcriptoma , Animales , Takifugu/genética , Branquias/metabolismo , Línea Celular , Perfilación de la Expresión Génica/veterinaria , Frío/efectos adversos , Inmunidad Innata/genética , Proteínas de Peces/genética

RESUMEN

Interferon-related developmental regulator 1 (IFRD1) is a viral responsive gene associated with interferon-gamma. Herein, we identified the IFRD1 gene (EaIFRD1) from red-spotted grouper (Epinephelus akaara), evaluated its transcriptional responses, and investigated its functional features using various biological assays. EaIFRD1 encodes a protein comprising 428 amino acids with a molecular mass of 48.22 kDa. It features a substantial domain belonging to the interferon-related developmental regulator superfamily. Spatial mRNA expression of EaIFRD1 demonstrated the highest expression levels in the brain and the lowest in the skin. Furthermore, EaIFRD1 mRNA expression in grouper tissues exhibited significant modulation in response to immune stimulants, including poly (I:C), LPS, and nervous necrosis virus (NNV) infection. We analyzed downstream gene regulation by examining type Ⅰ interferon pathway genes following EaIFRD1 overexpression. The results demonstrated a significant upregulation in cells overexpressing EaIFRD1 compared to the control after infection with viral hemorrhagic septicemia virus (VHSV). A subcellular localization assay confirmed the nuclear location of the EaIFRD1 protein, consistent with its role as a transcriptional coactivator. Cells overexpressing EaIFRD1 exhibited increased migratory activity, enhancing wound-healing capabilities compared to the control. Additionally, under H2O2 exposure, EaIFRD1 overexpression protected cells against oxidative stress. Overexpression of EaIFRD1 also reduced poly (I:C)-mediated NO production in RAW267.4 macrophage cells. In FHM cells, EaIFRD1 overexpression significantly reduced VHSV virion replication. Collectively, these findings suggest that EaIFRD1 plays a crucial role in the antiviral immune response and immunological regulation in E. akaara.

Asunto(s)

Secuencia de Aminoácidos , Lubina , Enfermedades de los Peces , Proteínas de Peces , Regulación de la Expresión Génica , Inmunidad Innata , Filogenia , Poli I-C , Animales , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Lubina/inmunología , Lubina/genética , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Poli I-C/farmacología , Nodaviridae/fisiología , Alineación de Secuencia/veterinaria , Perfilación de la Expresión Génica/veterinaria , Novirhabdovirus/fisiología , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/veterinaria , Lipopolisacáridos/farmacología