Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 6.218
Filter
1.
Transl Psychiatry ; 14(1): 289, 2024 Jul 16.
Article in English | MEDLINE | ID: mdl-39009558

ABSTRACT

Prenatal exposure to infections is a risk factor for neurodevelopmental disorders in offspring, and alterations in mitochondrial function are discussed as a potential underlying factor. Here, using a mouse model of viral-like maternal immune activation (MIA) based on poly(I:C) (POL) treatment at gestational day (GD) 12, we show that adult offspring exhibit behavioral deficits, such as reduced levels of social interaction. In addition, we found increased nicotinamidadenindinucleotid (NADH)- and succinate-linked mitochondrial respiration and maximal electron transfer capacity in the prefrontal cortex (PFC) and in the amygdala (AMY) of males and females. The increase in respiratory capacity resulted from an increase in mitochondrial mass in neurons (as measured by complex IV activity and transcript expression), presumably to compensate for a reduction in mitochondrion-specific respiration. Moreover, in the PFC of control (CON) male offspring a higher excess capacity compared to females was observed, which was significantly reduced in the POL-exposed male offspring, and, along with a higher leak respiration, resulted in a lower mitochondrial coupling efficiency. Transcript expression of the uncoupling proteins (UCP4 and UCP5) showed a reduction in the PFC of POL male mice, suggesting mitochondrial dysfunction. In addition, in the PFC of CON females, a higher expression of the antioxidant enzyme superoxide dismutase (SOD1) was observed, suggesting a higher antioxidant capacity as compared to males. Finally, transcripts analysis of genes involved in mitochondrial biogenesis and dynamics showed reduced expression of fission/fusion transcripts in PFC of POL offspring of both sexes. In conclusion, we show that MIA causes alterations in neuronal mitochondrial function and mass in the PFC and AMY of adult offspring with some effects differing between males and females.


Subject(s)
Mitochondria , Prefrontal Cortex , Prenatal Exposure Delayed Effects , Animals , Female , Prenatal Exposure Delayed Effects/immunology , Pregnancy , Mitochondria/metabolism , Mice , Male , Prefrontal Cortex/metabolism , Prefrontal Cortex/immunology , Poly I-C/pharmacology , Disease Models, Animal , Brain/immunology , Brain/metabolism , Amygdala/metabolism , Amygdala/immunology , Behavior, Animal , Mice, Inbred C57BL , Neurons/metabolism , Neurons/immunology
2.
PLoS Pathog ; 20(7): e1012320, 2024 Jul.
Article in English | MEDLINE | ID: mdl-39012849

ABSTRACT

Apoptosis, a major form of programmed cell death, is an essential component of host defense against invading intracellular pathogens. Viruses encode inhibitors of apoptosis to evade host responses during infection, and to support their own replication and survival. Therefore, hosts and their viruses are entangled in a constant evolutionary arms race to control apoptosis. Until now, apoptosis in the context of the antiviral immune system has been almost exclusively studied in vertebrates. This limited phyletic sampling makes it impossible to determine whether a similar mechanism existed in the last common ancestor of animals. Here, we established assays to probe apoptosis in the sea anemone Nematostella vectensis, a model species of Cnidaria, a phylum that diverged approximately 600 million years ago from the rest of animals. We show that polyinosinic:polycytidylic acid (poly I:C), a synthetic long double-stranded RNA mimicking viral RNA and a primary ligand for the vertebrate RLR melanoma differentiation-associated protein 5 (MDA5), is sufficient to induce apoptosis in N. vectensis. Furthermore, at the transcriptomic level, apoptosis related genes are significantly enriched upon poly(I:C) exposure in N. vectensis as well as bilaterian invertebrates. Our phylogenetic analysis of caspase family genes in N. vectensis reveals conservation of all four caspase genes involved in apoptosis in mammals and revealed a cnidarian-specific caspase gene which was strongly upregulated. Altogether, our findings suggest that apoptosis in response to a viral challenge is a functionally conserved mechanism that can be traced back to the last common ancestor of Bilateria and Cnidaria.


Subject(s)
Apoptosis , RNA, Double-Stranded , Sea Anemones , Animals , Sea Anemones/genetics , RNA, Double-Stranded/metabolism , RNA, Double-Stranded/genetics , Phylogeny , Poly I-C/pharmacology , Cnidaria/genetics , Biological Evolution
3.
Mol Med Rep ; 30(3)2024 Sep.
Article in English | MEDLINE | ID: mdl-38963029

ABSTRACT

Viral infections in the respiratory tract are common, and, in recent years, severe acute respiratory syndrome coronavirus 2 outbreaks have highlighted the effect of viral infections on antiviral innate immune and inflammatory reactions. Specific treatments for numerous viral respiratory infections have not yet been established and they are mainly treated symptomatically. Therefore, understanding the details of the innate immune system underlying the airway epithelium is crucial for the development of new therapies. The present study aimed to investigate the function and expression of interferon (IFN)­stimulated gene (ISG)60 in non­cancerous bronchial epithelial BEAS­2B cells exposed to a Toll­like receptor 3 agonist. BEAS­2B cells were treated with a synthetic TLR3 ligand, polyinosinic­polycytidylic acid (poly IC). The mRNA and protein expression levels of ISG60 were analyzed using reverse transcription­quantitative PCR and western blotting, respectively. The levels of C­X­C motif chemokine ligand 10 (CXCL10) were examined using an enzyme­linked immunosorbent assay, and the effects of knockdown of IFN­ß, ISG60 and ISG56 were examined using specific small interfering RNAs. Notably, ISG60 expression was increased in proportion to poly IC concentration, and recombinant human IFN­ß also induced ISG60 expression. By contrast, knockdown of IFN­ß and ISG56 decreased ISG60 expression, and ISG60 knockdown reduced CXCL10 and ISG56 expression. These findings suggested that ISG60 is partly implicated in CXCL10 expression and that ISG60 may serve a role in the innate immune response of bronchial epithelial cells. The present study highlights ISG60 as a potential target for new therapeutic strategies against viral infections in the airway.


Subject(s)
Bronchi , Chemokine CXCL10 , Epithelial Cells , Poly I-C , Signal Transduction , Toll-Like Receptor 3 , Humans , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 3/genetics , Chemokine CXCL10/metabolism , Chemokine CXCL10/genetics , Epithelial Cells/metabolism , Epithelial Cells/drug effects , Bronchi/cytology , Bronchi/metabolism , Poly I-C/pharmacology , Signal Transduction/drug effects , Cell Line , Immunity, Innate , Interferon-beta/metabolism , Interferon-beta/genetics , Gene Expression Regulation/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , RNA-Binding Proteins , Adaptor Proteins, Signal Transducing , Apoptosis Regulatory Proteins
4.
Front Immunol ; 15: 1398955, 2024.
Article in English | MEDLINE | ID: mdl-38994355

ABSTRACT

Introduction: STAT1a is an essential signal transduction protein involved in the interferon pathway, playing a vital role in IFN-alpha/beta and gamma signaling. Limited information is available about the STAT protein in fish, particularly in Indian major carps (IMC). This study aimed to identify and characterize the STAT1a protein in Labeo rohita (LrSTAT1a). Methods: The full-length CDS of LrSTAT1a transcript was identified and sequenced. Phylogenetic analyses were performed based on the nucleotide sequences. The in-vivo immune stimulant poly I: C was used to treat various tissues, and the expression of LrSTAT1a was determined using quantitative real-time polymerase chain reaction (qRT-PCR). A 3D model of the STAT1a protein was generated using close structure homologs available in the database and checked using molecular dynamics (MD) simulations. Results: The full-length CDS of Labeo rohita STAT1a (LrSTAT1a) transcript consisted of 3238 bp that encoded a polypeptide of 721 amino acids sequence was identified. Phylogenetic analyses were performed based on the nucleotide sequences. Based on our findings, other vertebrates share a high degree of conservation with STAT1a. Additionally, we report that the in vivo immune stimulant poly I: C treatment of various tissues resulted in the expression of LrSTAT1a as determined by quantitative real-time polymerase chain reaction (qRT-PCR). In the current investigation, treatment with poly I: C dramatically increased the expression of LrSTAT1a in nearly every organ and tissue, with the brain, muscle, kidney, and intestine showing the highest levels of expression compared to the control. We made a 3D model of the STAT1a protein by using close structure homologs that were already available in the database. The model was then checked using molecular dynamics (MD) simulations. Consistent with previous research, the MD study highlighted the significance of the STAT1a protein, which is responsible for Src homology 2 (SH2) recognition. An important H-bonding that successfully retains SH2 inside the STAT1a binding cavity was determined to be formed by the conserved residues SER107, GLN530, SER583, LYS584, MET103, and ALA106. Discussion: This study provides molecular insights into the STAT1a protein in Rohu (Labeo rohita) and highlights the potential role of STAT1a in the innate immune response in fish. The high degree of conservation of STAT1a among other vertebrates suggests its crucial role in the immune response. The in-vivo immune stimulation results indicate that STAT1a is involved in the immune response in various tissues, with the brain, muscle, kidney, and intestine being the most responsive. The 3D model and MD study provide further evidence of the significance of STAT1a in the immune response, specifically in SH2 recognition. Further research is necessary to understand the specific mechanisms involved in the IFN pathway and the role of STAT1a in the immune response of IMC.


Subject(s)
Fish Proteins , Phylogeny , Poly I-C , STAT1 Transcription Factor , Animals , Poly I-C/immunology , STAT1 Transcription Factor/metabolism , STAT1 Transcription Factor/genetics , Fish Proteins/genetics , Fish Proteins/immunology , Fish Proteins/metabolism , src Homology Domains , Protein Binding , Amino Acid Sequence , Molecular Dynamics Simulation , Carps/immunology , Carps/genetics , Carps/metabolism , Gene Expression Profiling , Cyprinidae/immunology , Cyprinidae/genetics , Cyprinidae/metabolism
5.
Int J Mol Sci ; 25(13)2024 Jul 08.
Article in English | MEDLINE | ID: mdl-39000607

ABSTRACT

Natural killer (NK) cells play a crucial role in innate immunity, particularly in combating infections and tumors. However, in hematological cancers, NK cells often exhibit impaired functions. Therefore, it is very important to activate its endosomal Toll-like receptors (TLRs) as a potential strategy to restore its antitumor activity. We stimulated NK cells from the peripheral blood mononuclear cells from children with acute lymphoblastic leukemia and NK cells isolated, and the NK cells were stimulated with specific TLR ligands (Poly I:C, Imiquimod, R848, and ODN2006) and we evaluated changes in IFN-γ, CD107a, NKG2D, NKp44 expression, Granzyme B secretion, cytokine/chemokine release, and cytotoxic activity. Results revealed that Poly I:C and Imiquimod enhanced the activation of both immunoregulatory and cytotoxic NK cells, increasing IFN-γ, CD107a, NKG2D, and NKp44 expression. R848 activated immunoregulatory NK cells, while ODN2006 boosted CD107a, NKp44, NKG2D, and IFN-γ secretion in cytotoxic NK cells. R848 also increased the secretion of seven cytokines/chemokines. Importantly, R848 and ODN 2006 significantly improved cytotoxicity against leukemic cells. Overall, TLR stimulation enhances NK cell activation, suggesting TLR8 (R848) and TLR9 (ODN 2006) ligands as promising candidates for antitumor immunotherapy.


Subject(s)
Imiquimod , Killer Cells, Natural , Lymphocyte Activation , Poly I-C , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Toll-Like Receptors , Humans , Killer Cells, Natural/immunology , Killer Cells, Natural/drug effects , Killer Cells, Natural/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Poly I-C/pharmacology , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Imiquimod/pharmacology , Toll-Like Receptors/metabolism , Toll-Like Receptors/agonists , Child , Oligodeoxyribonucleotides/pharmacology , Cytokines/metabolism , Female , Interferon-gamma/metabolism , Male , Imidazoles/pharmacology , Cytotoxicity, Immunologic/drug effects , Child, Preschool , Toll-Like Receptor Agonists
6.
EMBO Mol Med ; 16(7): 1579-1602, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38890537

ABSTRACT

Mucopolysaccharidosis type IIIA (MPS IIIA) is a rare paediatric lysosomal storage disorder, caused by the progressive accumulation of heparan sulphate, resulting in neurocognitive decline and behavioural abnormalities. Anecdotal reports from paediatricians indicate a more severe neurodegeneration in MPS IIIA patients, following infection, suggesting inflammation as a potential driver of neuropathology. To test this hypothesis, we performed acute studies in which WT and MPS IIIA mice were challenged with the TLR3-dependent viral mimetic poly(I:C). The challenge with an acute high poly(I:C) dose exacerbated systemic and brain cytokine expression, especially IL-1ß in the hippocampus. This was accompanied by an increase in caspase-1 activity within the brain of MPS IIIA mice with concomitant loss of hippocampal GFAP and NeuN expression. Similar levels of cell damage, together with exacerbation of gliosis, were also observed in MPS IIIA mice following low chronic poly(I:C) dosing. While further investigation is warranted to fully understand the extent of IL-1ß involvement in MPS IIIA exacerbated neurodegeneration, our data robustly reinforces our previous findings, indicating IL-1ß as a pivotal catalyst for neuropathological processes in MPS IIIA.


Subject(s)
Disease Models, Animal , Mucopolysaccharidosis III , Poly I-C , Animals , Mucopolysaccharidosis III/pathology , Mucopolysaccharidosis III/immunology , Mucopolysaccharidosis III/metabolism , Mice , Interleukin-1beta/metabolism , Neurodegenerative Diseases/pathology , Neurodegenerative Diseases/immunology , Brain/pathology , Brain/metabolism , Cytokines/metabolism , Mice, Inbred C57BL , Hippocampus/pathology , Hippocampus/metabolism
7.
Biol Pharm Bull ; 47(6): 1144-1147, 2024.
Article in English | MEDLINE | ID: mdl-38866523

ABSTRACT

Long non-coding RNAs (lncRNAs) are sequences longer than 200 nucleotides, but they do not encode proteins. Nevertheless, they have significant roles in diverse biological functions. It remains unclear how viral infections trigger the expression of lncRNAs. In our previous research, we revealed a distinct type of lncRNAs with a lifespan under 4 h in human HeLa cells. These short-lived lncRNAs might be associated with numerous regulatory roles. Given their potential impact on human physiology, these short-lived lncRNAs could be key indicators to measure polyinosinic:polycytidylic acid (poly I:C) stimulation. In our recent work, we discovered three lncRNAs: IDI2-AS1, OIP5-AS1, and LITATS1. After exposure to poly I:C, imitating viral assault in human A549 cells, IDI2-AS1 levels dropped significantly while OIP5-AS1 and LITATS1 levels rose markedly. Our results indicate that short-lived lncRNAs respond to poly I:C stimulation, exhibiting substantial changes in expression. This indicates that the understanding the role of lncRNAs in the host response to viral infection and the potential for these molecules to serve as novel therapeutic targets.


Subject(s)
Poly I-C , RNA, Long Noncoding , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Humans , Poly I-C/pharmacology , A549 Cells , HeLa Cells
8.
Int J Mol Sci ; 25(11)2024 May 24.
Article in English | MEDLINE | ID: mdl-38891899

ABSTRACT

In aquaculture, viral diseases pose a significant threat and can lead to substantial economic losses. The primary defense against viral invasion is the innate immune system, with interferons (IFNs) playing a crucial role in mediating the immune response. With advancements in molecular biology, the role of non-coding RNA (ncRNA), particularly microRNAs (miRNAs), in gene expression has gained increasing attention. While the function of miRNAs in regulating the host immune response has been extensively studied, research on their immunomodulatory effects in teleost fish, including silver carp (Hyphthalmichthys molitrix), is limited. Therefore, this research aimed to investigate the immunomodulatory role of microRNA-30b-5p (miR-30b-5p) in the antiviral immune response of silver carp (Hypophthalmichthys molitrix) by targeting cytokine receptor family B5 (CRFB5) via the JAK/STAT signaling pathway. In this study, silver carp were stimulated with polyinosinic-polycytidylic acid (poly (I:C)), resulting in the identification of an up-regulated miRNA (miR-30b-5p). Through a dual luciferase assay, it was demonstrated that CRFB5, a receptor shared by fish type I interferon, is a novel target of miR-30b-5p. Furthermore, it was found that miR-30b-5p can suppress post-transcriptional CRFB5 expression. Importantly, this study revealed for the first time that miR-30b-5p negatively regulates the JAK/STAT signaling pathway, thereby mediating the antiviral immune response in silver carp by targeting CRFB5 and maintaining immune system stability. These findings not only contribute to the understanding of how miRNAs act as negative feedback regulators in teleost fish antiviral immunity but also suggest their potential therapeutic measures to prevent an excessive immune response.


Subject(s)
Carps , Fish Proteins , MicroRNAs , Poly I-C , Signal Transduction , Animals , Carps/genetics , Carps/immunology , Carps/virology , Carps/metabolism , Fish Diseases/immunology , Fish Diseases/virology , Fish Diseases/genetics , Fish Proteins/genetics , Fish Proteins/metabolism , Gene Expression Regulation/drug effects , Immunity, Innate/genetics , Janus Kinases/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Poly I-C/pharmacology , STAT Transcription Factors/metabolism , STAT Transcription Factors/genetics
9.
Biomolecules ; 14(6)2024 Jun 14.
Article in English | MEDLINE | ID: mdl-38927097

ABSTRACT

MicroRNAs (miRNAs) are highly conserved endogenous single-stranded non-coding RNA molecules that play a crucial role in regulating gene expression to maintain normal physiological functions in fish. Nevertheless, the specific physiological role of miRNAs in lower vertebrates, particularly in comparison to mammals, remains elusive. Additionally, the mechanisms underlying the control of antiviral responses triggered by viral stimulation in fish are still not fully understood. In this study, we investigated the regulatory impact of miR-1388 on the signaling pathway mediated by IFN regulatory factor 3 (IRF3). Our findings revealed that following stimulation with the viral analog poly(I:C), the expression of miR-1388 was significantly upregulated in primary immune tissues and macrophages. Through a dual luciferase reporter assay, we corroborated a direct targeting relationship between miR-1388 and tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3). Furthermore, our study demonstrated a distinct negative post-transcriptional correlation between miR-1388 and TRAF3. We observed a significant negative post-transcriptional regulatory association between miR-1388 and the levels of antiviral genes following poly(I:C) stimulation. Utilizing reporter plasmids, we elucidated the role of miR-1388 in the antiviral signaling pathway activated by TRAF3. By intervening with siRNA-TRAF3, we validated that miR-1388 regulates the expression of antiviral genes and the production of type I interferons (IFN-Is) through its interaction with TRAF3. Collectively, our experiments highlight the regulatory influence of miR-1388 on the IRF3-mediated signaling pathway by targeting TRAF3 post poly(I:C) stimulation. These findings provide compelling evidence for enhancing our understanding of the mechanisms through which fish miRNAs participate in immune responses.


Subject(s)
Carps , MicroRNAs , Poly I-C , TNF Receptor-Associated Factor 3 , Animals , MicroRNAs/genetics , MicroRNAs/metabolism , Poly I-C/pharmacology , Carps/genetics , Carps/metabolism , Carps/virology , TNF Receptor-Associated Factor 3/genetics , TNF Receptor-Associated Factor 3/metabolism , Down-Regulation/drug effects , Down-Regulation/genetics , Interferon Regulatory Factor-3/metabolism , Interferon Regulatory Factor-3/genetics , Gene Expression Regulation/drug effects , Fish Proteins/genetics , Fish Proteins/metabolism , Signal Transduction
11.
Cells ; 13(11)2024 May 26.
Article in English | MEDLINE | ID: mdl-38891051

ABSTRACT

Respiratory viruses cause airway inflammation, resulting in epithelial injury and repair. miRNAs, including miR-149-5p, regulate different pathological conditions. We aimed to determine how miR-149-5p functions in regulating pro-inflammatory IL-6 and p63, key regulators of airway epithelial wound repair, in response to viral proteins in bronchial (BEAS-2B) and alveolar (A549) epithelial cells. BEAS-2B or A549 cells were incubated with poly (I:C, 0.5 µg/mL) for 48 h or SARS-CoV-2 spike protein-1 or 2 subunit (S1 or S2, 1 µg/mL) for 24 h. miR-149-5p was suppressed in BEAS-2B challenged with poly (I:C), correlating with IL-6 and p63 upregulation. miR-149-5p was down-regulated in A549 stimulated with poly (I:C); IL-6 expression increased, but p63 protein levels were undetectable. miR-149-5p remained unchanged in cells exposed to S1 or S2, while S1 transfection increased IL-6 expression in BEAS-2B cells. Ectopic over-expression of miR-149-5p in BEAS-2B cells suppressed IL-6 and p63 mRNA levels and inhibited poly (I:C)-induced IL-6 and p63 mRNA expressions. miR-149-5p directly suppressed IL-6 mRNA in BEAS-2B cells. Hence, BEAS-2B cells respond differently to poly (I:C), S1 or S2 compared to A549 cells. Thus, miR-149-5p dysregulation may be involved in poly (I:C)-stimulated but not S1- or S2-stimulated increased IL-6 production and p63 expression in BEAS-2B cells.


Subject(s)
Epithelial Cells , Interleukin-6 , MicroRNAs , Poly I-C , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Interleukin-6/metabolism , A549 Cells , Epithelial Cells/metabolism , Epithelial Cells/drug effects , Epithelial Cells/virology , Poly I-C/pharmacology , SARS-CoV-2 , COVID-19/metabolism , COVID-19/virology , Tumor Suppressor Proteins/metabolism , Tumor Suppressor Proteins/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Gene Expression Regulation/drug effects
12.
Front Immunol ; 15: 983686, 2024.
Article in English | MEDLINE | ID: mdl-38827742

ABSTRACT

Recently, OTULIN haploinsufficiency was linked to enhanced susceptibility to Staphylococcus aureus infections accompanied by local necrosis and systemic inflammation. The pathogenesis observed in haploinsufficient patients differs from the hyperinflammation seen in classical OTULIN-related autoinflammatory syndrome (ORAS) patients and is characterized by increased susceptibility of dermal fibroblasts to S. aureus alpha toxin-inflicted cytotoxic damage. Immunological abnormalities were not observed in OTULIN haploinsufficient patients, suggesting a non-hematopoietic basis. In this research report, we investigated an Otulin+/- mouse model after in vivo provocation with lipopolysaccharide (LPS) to explore the potential role of hematopoietic-driven inflammation in OTULIN haploinsufficiency. We observed a hyperinflammatory signature in LPS-provoked Otulin+/- mice, which was driven by CD64+ monocytes and macrophages. Bone marrow-derived macrophages (BMDMs) of Otulin+/- mice demonstrated higher proinflammatory cytokine secretion after in vitro stimulation with LPS or polyinosinic:polycytidylic acid (Poly(I:C)). Our experiments in full and mixed bone marrow chimeric mice suggest that, in contrast to humans, the observed inflammation was mainly driven by the hematopoietic compartment with cell-extrinsic effects likely contributing to inflammatory outcomes. Using an OTULIN haploinsufficient mouse model, we validated the role of OTULIN in the regulation of environmentally directed inflammation.


Subject(s)
Haploinsufficiency , Inflammation , Lipopolysaccharides , Macrophages , Animals , Mice , Inflammation/genetics , Macrophages/immunology , Macrophages/metabolism , Disease Models, Animal , Cytokines/metabolism , Poly I-C , Mice, Inbred C57BL , Mice, Knockout , Humans
13.
Methods Mol Biol ; 2813: 235-244, 2024.
Article in English | MEDLINE | ID: mdl-38888782

ABSTRACT

Double-stranded RNA is produced by viruses during their replicative cycle. It is a potent immune modulator and indicator of viral infection within the body. Extracellular vesicles (EVs) are lipid-bound particles released from cells homeostatically. Recent studies have shown that a commercially available dsRNA, poly inosinic: poly cytidylic acid (poly IC), can be detected within EVs. This finding opens the door for studying EVs as (1) carriers for dsRNA and (2) indicators of viral infection. To study dsRNA-containing EVs, we must have reliable methods for producing, isolating, and detecting them. This chapter uses U937, a pro-monocytic, human myeloid leukemia cell line, as the EV producer following poly IC treatment, and an immunoblot using an anti-dsRNA antibody (J2) for detection. Two methods for isolating the EVs and two methods for isolating the RNA from these EVs are described. Together, these methods effectively produce, isolate, and detect long dsRNA from EVs.


Subject(s)
Extracellular Vesicles , Poly I-C , Humans , Extracellular Vesicles/metabolism , Poly I-C/pharmacology , U937 Cells , RNA, Double-Stranded/metabolism
14.
Exp Biol Med (Maywood) ; 249: 10122, 2024.
Article in English | MEDLINE | ID: mdl-38881847

ABSTRACT

Rheumatoid fibroblast-like synoviocytes (RFLS) have an important role in the inflammatory pathogenesis of rheumatoid arthritis (RA). Toll-like receptor 3 (TLR3) is upregulated in RFLS; its activation leads to the production of interferon-ß (IFN-ß), a type I IFN. IFN-stimulated gene 56 (ISG56) is induced by IFN and is involved in innate immune responses; however, its role in RA remains unknown. Therefore, the purpose of this study was to investigate the role of TLR3-induced ISG56 in human RFLS. RFLS were treated with polyinosinic-polycytidylic acid (poly I:C), which served as a TLR3 ligand. ISG56, melanoma differentiation-associated gene 5 (MDA5), and C-X-C motif chemokine ligand 10 (CXCL10) expression were measured using quantitative reverse transcription-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. Using immunohistochemistry, we found that ISG56 was expressed in synovial tissues of patients with RA and osteoarthritis. Under poly I:C treatment, ISG56 was upregulated in RFLS. In addition, we found that the type I IFN-neutralizing antibody mixture suppressed ISG56 expression. ISG56 knockdown decreased CXCL10 expression and MDA5 knockdown decreased ISG56 expression. In addition, we found that ISG56 was strongly expressed in the synovial cells of patients with RA. TLR3 signaling induced ISG56 expression in RFLS and type I IFN was involved in ISG56 expression. ISG56 was also found to be associated with CXCL10 expression, suggesting that ISG56 may be involved in TLR3/type I IFN/CXCL10 axis, and play a role in RA synovial inflammation.


Subject(s)
Arthritis, Rheumatoid , Chemokine CXCL10 , Poly I-C , Signal Transduction , Synoviocytes , Toll-Like Receptor 3 , Humans , Toll-Like Receptor 3/metabolism , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Poly I-C/pharmacology , Synoviocytes/metabolism , Chemokine CXCL10/metabolism , Interferon-Induced Helicase, IFIH1/metabolism , Interferon-Induced Helicase, IFIH1/genetics , Cells, Cultured , Synovial Membrane/metabolism , Synovial Membrane/pathology , Adaptor Proteins, Vesicular Transport/metabolism , Adaptor Proteins, Vesicular Transport/genetics , RNA-Binding Proteins , Adaptor Proteins, Signal Transducing , Apoptosis Regulatory Proteins
15.
Fish Shellfish Immunol ; 151: 109694, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38871143

ABSTRACT

Type I interferons (IFN-I) play a pivotal role in vertebrate innate immunity against viruses. This study is an analysis of IFN-I genes in an updated version of the Atlantic salmon genome published in 2021 (version Ssal_v3.1), revealing 47 IFN-I genes in the Atlantic salmon genome. The GH1 locus of chromosome (Chr) 3 harbors 9 IFNa genes, 5 IFNb genes, 6 IFNc genes, 11 IFNe genes and 1 IFNf gene. The GH2 locus on Chr6 contains 1 IFNa gene, 12 IFNc genes and 1 IFNf gene while Chr19 carries a single IFNd gene. Intraperitoneal injection of Atlantic salmon presmolts with poly I:C, a mimic of virus double-stranded RNA, significantly up-regulated IFNc genes from both Chr3 and Chr6 in heart, with lower expression in head kidney. IFNe expression increased in the heart, but not in the head kidney while IFNf was strongly up-regulated in both tissues. Antiviral activity of selected IFNs was assessed by transfection of salmon cells with IFN-expressing plasmids followed by infectious pancreatic necrosis virus infection, and by injection of fish with IFN-plasmids followed by measuring expression of the antiviral Mx1 gene. The results demonstrated that IFNc from both Chr3 and Chr6 provided full protection of cells against virus infection, whereas IFNe and IFNf showed lesser protection. IFNc from Chr3 and Chr6 along with IFNe and IFNf, up-regulated the Mx1 gene in the muscle, while only the IFNcs caused induction of Mx1 in liver. Overall, this study reveals that Atlantic salmon possesses an even more potent innate immune defense against viruses than previously understood.


Subject(s)
Fish Diseases , Fish Proteins , Immunity, Innate , Interferon Type I , Poly I-C , Salmo salar , Animals , Salmo salar/genetics , Salmo salar/immunology , Fish Diseases/immunology , Interferon Type I/genetics , Interferon Type I/immunology , Fish Proteins/genetics , Fish Proteins/immunology , Immunity, Innate/genetics , Poly I-C/pharmacology , Birnaviridae Infections/immunology , Birnaviridae Infections/veterinary , Birnaviridae Infections/genetics , Infectious pancreatic necrosis virus/physiology , Gene Expression Regulation/immunology , Gene Expression Profiling/veterinary
16.
Mol Immunol ; 172: 47-55, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38875755

ABSTRACT

3-phosphoinositide-dependent protein kinase-1 (PDK-1) is a key kinase regulating the activity of the PI3K/AKT pathway and a major regulator of the AGC protein kinase family. It is essential in the physiological activities of cells, embryonic development, individual development and immune response. In this study, we have identified for the first time an analogue of PDK-1 in the most primitive vertebrate, lamprey, and named it PDK-1-like. The protein sequence similarity of lamprey PDK-1-like to human, mouse, chicken, African xenopus and zebrafish PDK-1 were 64.4 %, 64.5 %, 65.0 %, 61.3 % and 63.2 %, respectively. The phylogenetic tree showed that PDK-1-like of lamprey were located at the base of the vertebrate branch, in line with the trend of biological evolution. Meanwhile, homology analysis showed that PDK-1 proteins across species shared a conserved kinase structural domain and a Pleckstrin Homology (PH) domain. Genomic synteny analysis revealed that the large-scale duplication blocks were not found in lamprey genome and neighbor genes of lamprey PDK-1-like presented dramatic differences compared with jawed vertebrates. More importantly, qPCR analysis showed that PDK-1-like was widely expressed in lamprey. Its mRNA expression levels varied in response to different pathogenic stimuli, and its expression was generally up-regulated under Polyinosinic-Polycytidylic acid (Poly(I:C)) stimulation. Pearson's correlation analysis showed that PDK-1-like was involved in co-expressed with MyD88-independent TLR-3 pathway during the immune response of lamprey, instead of MyD88-dependent TLR-3 pathway. In summary, our composite results offer valuable clues to the origin and evolution of PDK-1, and imply that PDK-1 s are among the most ancestral immune regulators in vertebrates.


Subject(s)
Evolution, Molecular , Immunity, Innate , Lampreys , Phylogeny , Animals , Lampreys/immunology , Lampreys/genetics , Immunity, Innate/genetics , Immunity, Innate/immunology , Humans , 3-Phosphoinositide-Dependent Protein Kinases/genetics , 3-Phosphoinositide-Dependent Protein Kinases/metabolism , Amino Acid Sequence , Poly I-C/immunology , Fish Proteins/genetics , Fish Proteins/immunology
17.
Neuroscience ; 551: 205-216, 2024 Jul 23.
Article in English | MEDLINE | ID: mdl-38843988

ABSTRACT

Here, we explored the impact of prolonged environmental enrichment (EE) on behavioral, neurochemical, and epigenetic changes in the serotonin transporter gene in mice subjected to a two-hit schizophrenia model. The methodology involved administering the viral mimetic PolyI:C to neonatal Swiss mice as a first hit during postnatal days (PND) 5-7, or a sterile saline solution as a control. At PND21, mice were randomly assigned either to standard environment (SE) or EE housing conditions. Between PND35-44, the PolyI:C-treated group was submitted to various unpredictable stressors, constituting the second hit. Behavioral assessments were conducted on PND70, immediately after the final EE exposure. Following the completion of behavioral assessments, we evaluated the expression of proteins in the hippocampus that are indicative of microglial activation, such as Iba-1, as well as related to neurogenesis, including doublecortin (Dcx). We also performed methylation analysis on the serotonin transporter gene (Slc6a4) to investigate alterations in serotonin signaling. The findings revealed that EE for 50 days mitigated sensorimotor gating deficits and working memory impairments in two-hit mice and enhanced their locomotor and exploratory behaviors. EE also normalized the overexpression of hippocampal Iba-1 and increased the expression of hippocampal Dcx. Additionally, we observed hippocampal demethylation of the Slc6a4 gene in the EE-exposed two-hit group, indicating epigenetic reprogramming. These results contribute to the growing body of evidence supporting the protective effects of long-term EE in counteracting behavioral disruptions caused by the two-hit schizophrenia model, pointing to enhanced neurogenesis, diminished microglial activation, and epigenetic modifications of serotonergic pathways as underlying mechanisms.


Subject(s)
Disease Models, Animal , Environment , Hippocampus , Schizophrenia , Serotonin Plasma Membrane Transport Proteins , Animals , Serotonin Plasma Membrane Transport Proteins/genetics , Serotonin Plasma Membrane Transport Proteins/metabolism , Hippocampus/metabolism , Schizophrenia/metabolism , Schizophrenia/genetics , Mice , Male , Doublecortin Protein , Promoter Regions, Genetic , DNA Methylation , Poly I-C , Neurogenesis/physiology , Sensory Gating/physiology
18.
Front Immunol ; 15: 1374541, 2024.
Article in English | MEDLINE | ID: mdl-38807598

ABSTRACT

Objective: The coronavirus disease 2019 (COVID-19) spread rapidly and claimed millions of lives worldwide. Acute respiratory distress syndrome (ARDS) is the major cause of COVID-19-associated deaths. Due to the limitations of current drugs, developing effective therapeutic options that can be used rapidly and safely in clinics for treating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections is necessary. This study aims to investigate the effects of two food-extracted immunomodulatory agents, ajoene-enriched garlic extract (AGE) and cruciferous vegetables-extracted sulforaphane (SFN), on anti-inflammatory and immune responses in a SARS-CoV-2 acute lung injury mouse model. Methods: In this study, we established a mouse model to mimic the SARS-CoV-2 infection acute lung injury model via intratracheal injection of polyinosinic:polycytidylic acid (poly[I:C]) and SARS-CoV-2 recombinant spike protein (SP). After the different agents treatment, lung sections, bronchoalveolar lavage fluid (BALF) and fresh faeces were harvested. Then, H&E staining was used to examine symptoms of interstitial pneumonia. Flow cytometry was used to examine the change of immune cell populations. Multiplex cytokines assay was used to examine the inflammatory cytokines.16S rDNA high-throughput sequencing was used to examine the change of gut microbiome. Results: Our results showed that AGE and SFN significantly suppressed the symptoms of interstitial pneumonia, effectively inhibited the production of inflammatory cytokines, decreased the percentage of inflammatory cell populations, and elevated T cell populations in the mouse model. Furthermore, we also observed that the gut microbiome of genus Paramuribaculum were enriched in the AGE-treated group. Conclusion: Here, for the first time, we observed that these two novel, safe, and relatively inexpensive immunomodulatory agents exhibited the same effects on anti-inflammatory and immune responses as neutralizing monoclonal antibodies (mAbs) against interleukin 6 receptor (IL-6R), which have been suggested for treating COVID-19 patients. Our results revealed the therapeutic ability of these two immunomodulatory agents in a mouse model of SARS-CoV-2 acute lung injury by promoting anti-inflammatory and immune responses. These results suggest that AGE and SFN are promising candidates for the COVID-19 treatment.


Subject(s)
Acute Lung Injury , Angiotensin-Converting Enzyme 2 , Anti-Inflammatory Agents , COVID-19 Drug Treatment , COVID-19 , Disease Models, Animal , Immunomodulating Agents , SARS-CoV-2 , Animals , Mice , Acute Lung Injury/immunology , Acute Lung Injury/drug therapy , Acute Lung Injury/etiology , COVID-19/immunology , SARS-CoV-2/immunology , Immunomodulating Agents/pharmacology , Immunomodulating Agents/therapeutic use , Anti-Inflammatory Agents/therapeutic use , Anti-Inflammatory Agents/pharmacology , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Isothiocyanates/pharmacology , Isothiocyanates/therapeutic use , Sulfoxides , Humans , Cytokines/metabolism , Spike Glycoprotein, Coronavirus/immunology , Lung/immunology , Lung/pathology , Lung/virology , Lung/drug effects , Male , Poly I-C , Plant Extracts/pharmacology , Plant Extracts/therapeutic use
19.
J Neuroinflammation ; 21(1): 118, 2024 May 07.
Article in English | MEDLINE | ID: mdl-38715090

ABSTRACT

Maternal inflammation during gestation is associated with a later diagnosis of neurodevelopmental disorders including autism spectrum disorder (ASD). However, the specific impact of maternal immune activation (MIA) on placental and fetal brain development remains insufficiently understood. This study aimed to investigate the effects of MIA by analyzing placental and brain tissues obtained from the offspring of pregnant C57BL/6 dams exposed to polyinosinic: polycytidylic acid (poly I: C) on embryonic day 12.5. Cytokine and mRNA content in the placenta and brain tissues were assessed using multiplex cytokine assays and bulk-RNA sequencing on embryonic day 17.5. In the placenta, male MIA offspring exhibited higher levels of GM-CSF, IL-6, TNFα, and LT-α, but there were no differences in female MIA offspring. Furthermore, differentially expressed genes (DEG) in the placental tissues of MIA offspring were found to be enriched in processes related to synaptic vesicles and neuronal development. Placental mRNA from male and female MIA offspring were both enriched in synaptic and neuronal development terms, whereas females were also enriched for terms related to excitatory and inhibitory signaling. In the fetal brain of MIA offspring, increased levels of IL-28B and IL-25 were observed with male MIA offspring and increased levels of LT-α were observed in the female offspring. Notably, we identified few stable MIA fetal brain DEG, with no male specific difference whereas females had DEG related to immune cytokine signaling. Overall, these findings support the hypothesis that MIA contributes to the sex- specific abnormalities observed in ASD, possibly through altered neuron developed from exposure to inflammatory cytokines. Future research should aim to investigate how interactions between the placenta and fetal brain contribute to altered neuronal development in the context of MIA.


Subject(s)
Brain , Cytokines , Mice, Inbred C57BL , Neurodevelopmental Disorders , Placenta , Prenatal Exposure Delayed Effects , Sex Characteristics , Female , Animals , Pregnancy , Male , Cytokines/metabolism , Cytokines/genetics , Mice , Brain/metabolism , Brain/immunology , Brain/embryology , Placenta/metabolism , Placenta/immunology , Prenatal Exposure Delayed Effects/immunology , Prenatal Exposure Delayed Effects/metabolism , Prenatal Exposure Delayed Effects/chemically induced , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/immunology , Neurodevelopmental Disorders/metabolism , Poly I-C/toxicity , Transcriptome , Disease Models, Animal , Fetus/metabolism
20.
Biol Pharm Bull ; 47(5): 946-954, 2024.
Article in English | MEDLINE | ID: mdl-38735732

ABSTRACT

There is accumulating evidence that selective serotonin reuptake inhibitors (SSRIs), clinically used as antidepressants, have a beneficial effect on inflammatory diseases such as coronavirus disease 2019 (COVID-19). We previously compared the inhibitory effects of five U.S. Food and Drug Administration (FDA)-approved SSRIs on the production of an inflammatory cytokine, interleukin-6 (IL-6), and concluded that fluoxetine (FLX) showed the most potent anti-inflammatory activity. Here, we investigated the structure-activity relationship of FLX for anti-inflammatory activity towards J774.1 murine macrophages. FLX suppressed IL-6 production induced by the TLR3 agonist polyinosinic-polycytidylic acid (poly(I : C)) with an IC50 of 4.76 µM. A derivative of FLX containing chlorine instead of the methylamino group lacked activity, suggesting that the methylamino group is important for the anti-inflammatory activity. FLX derivatives bearing an N-propyl or N-(pyridin-3-yl)methyl group in place of the N-methyl group exhibited almost the same activity as FLX. Other derivatives showed weaker activity, and the N-phenyl and N-(4-trifluoromethyl)benzyl derivatives were inactive. The chlorine-containing derivative also lacked inhibitory activity against TLR9- or TLR4-mediated IL-6 production. These derivatives showed similar structure-activity relationships for TLR3- and TLR9-mediated inflammatory responses. However, the activities of all amino group-containing derivatives against the TLR4-mediated inflammatory response were equal to or higher than the activity of FLX. These results indicate that the substituent at the nitrogen atom in FLX strongly influences the anti-inflammatory effect.


Subject(s)
Anti-Inflammatory Agents , Fluoxetine , Interleukin-6 , Structure-Activity Relationship , Animals , Fluoxetine/pharmacology , Mice , Interleukin-6/metabolism , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/chemistry , Cell Line , Macrophages/drug effects , Macrophages/metabolism , Cytokines/metabolism , Toll-Like Receptor 3/metabolism , Poly I-C/pharmacology , Selective Serotonin Reuptake Inhibitors/pharmacology , Selective Serotonin Reuptake Inhibitors/chemistry , Inflammation/drug therapy
SELECTION OF CITATIONS
SEARCH DETAIL